The normal microbial flora of the vagina plays an important role in preventing genital and urinary tract infections in women. Thus an accurate understanding of the composition and ecology of the ecosystem is important to understanding the aetiology of these diseases. Common wisdom is that lactobacilli dominate the normal vaginal microflora of post-pubertal women. However, this conclusion is based on methods that require cultivation of microbial populations; an approach that is known to yield a biased and incomplete assessment of microbial community structure. In this study cultivation-independent methods were used to analyse samples collected from the mid-vagina of five normal healthy Caucasian women between the ages of 28 and 44. Total microbial community DNA was isolated following resuspension of microbial cells from vaginal swabs. To identify the constituent numerically dominant populations in each community 16S rRNA gene libraries were prepared following PCR amplification using the 8f and 926r primers. From each library, the DNA sequences of approximately 200 16S rRNA clones were determined and subjected to phylogenetic analyses. The diversity and kinds of organisms that comprise the vaginal microbial community varied among women. Species of Lactobacillus appeared to dominate the communities in four of the five women. However, the community of one woman was dominated by Atopobium sp., whereas a second woman had appreciable numbers of Megasphaera sp., Atopobium sp. and Leptotrichia sp., none of which have previously been shown to be common members of the vaginal ecosystem. Of the women whose communities were dominated by lactobacilli, there were two distinct clusters, each of which consisted of a single species. One class consisted of two women with genetically divergent clones that were related to Lactobacillus crispatus, whereas the second group of two women had clones of Lactobacillus iners that were highly related to a single phylotype. These surprising results suggest that culture-independent methods can provide new insights into the diversity of bacterial species found in the human vagina, and this information could prove to be pivotal in understanding risk factors for various infectious diseases. INTRODUCTIONPrevious studies on the microbial flora of the human vagina indicate that micro-organisms normally present in the human vagina play a key role in preventing successful colonization by 'undesirable' organisms, including those responsible for bacterial vaginosis, yeast infections, sexually transmitted diseases and urinary tract infections (Donders et al., 2000;Gupta et al., 1998;Sobel, 1999;van De Wijgert et al., 2000). Moreover, epidemiologic studies have clearly established that abnormal vaginal microbial communities and lower genital tract infections are significantly associated with an increased risk of HIV infection (Cohen et al., 1995;Martin et al., 1999;Sewankambo et al., 1997). Based on this, researchers have speculated that the normal vaginal microbial ecosystem may play a pivotal role in lo...

The analysis of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes has proven to be a facile means to compare microbial communities and presumptively identify abundant members. The method provides data that can be used to compare different communities based on similarity or distance measures. Once communities have been clustered into groups, clone libraries can be prepared from sample(s) that are representative of each group in order to determine the phylogeny of the numerically abundant populations in a community. In this paper methods are introduced for the statistical analysis of T-RFLP data that include objective methods for (i) determining a baseline so that 'true' peaks in electropherograms can be identified; (ii) a means to compare electropherograms and bin fragments of similar size; (iii) clustering algorithms that can be used to identify communities that are similar to one another; and (iv) a means to select samples that are representative of a cluster that can be used to construct 16S rRNA gene clone libraries. The methods for data analysis were tested using simulated data with assumptions and parameters that corresponded to actual data. The simulation results demonstrated the usefulness of these methods in their ability to recover the true microbial community structure generated under the assumptions made. Software for implementing these methods is available at http://www.ibest.uidaho.edu/tools/trflp_stats/index.php.

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Molecular microbial community analysis methods have revolutionized our understanding of the diversity and distribution of bacteria, archaea and microbial eukaryotes. The information obtained has adequately demonstrated that the analysis of microbial model systems can provide important insights into ecosystem function and stability. However, the terminology and metrics used in macroecology must be applied cautiously because the methods available to characterize microbial diversity are inherently limited in their ability to detect the many numerically minor constituents of microbial communities. In this review, we focus on the use of indices to quantify the diversity found in microbial communities, and on the methods used to generate the data from which those indices are calculated. Useful conclusions regarding diversity can only be deduced if the properties of the various methods used are well understood. The commonly used diversity metrics differ in the weight they give to organisms that differ in abundance, so understanding the properties of these metrics is essential. In this review, we illustrate important methodological and metric-dependent differences using simulated communities. We conclude that the assessment of richness in complex communities is futile without extensive sampling, and that some diversity indices can be estimated with reasonable accuracy through the analysis of clone libraries, but not from community fingerprint data.

Since its first description in coastal Connecticut in 1976, both the incidence of Lyme disease and the geographic extent of endemic areas in the US have increased dramatically. The rapid expansion of Lyme disease into its current distribution in the eastern half of the US has been due to the range expansion of the tick vector, Ixodes scapularis, upon which the causative agent, Borrelia burgdorferi is dependent for transmission to humans. In this study, we examined the phylogeographic population structure of B. burgdorferi throughout the range of I. scapularis-borne Lyme disease using multilocus sequence typing based on bacterial housekeeping genes. We show that B. burgdorferi populations from the Northeast and Midwest are genetically distinct, but phylogenetically related. Our findings provide strong evidence of prehistoric population size expansion and east-to-west radiation of descendent clones from founding sequence types in the Northeast. Estimates of the time scale of divergence of northeastern and midwestern populations suggest that B. burgdorferi was present in these regions of North America many thousands of years before European settlements. We conclude that B. burgdorferi populations have recently reemerged independently out of separate relict foci, where they have persisted since precolonial times.geography ͉ phylogeny ͉ ticks ͉ multilocus sequence typing

This study evaluated chondrogenesis of mesenchymal progenitor stem cells (MSCs) cultured initially under pre-confluent monolayer conditions exposed to transforming growth factor-81 (TGF-Pl ), and subsequently in three-dimensional cultures containing insulin-like growth factor I (IGF-I). Bone marrow aspirates and chondrocytes were obtained from horses and cultured in monolayer with 0 or 5 ng of TGF-P1 per ml of medium for 6 days. TGIF-81 treated and untreated cultures were distributed to threedimensional fibrin disks containing 0 or 100 ng of IGF-I per ml of medium to establish four treatment groups. After 13 days, cultures were assessed by toluidine blue staining, collagen types I and I1 in situ hybridization and immunohistochemistry, proteoglycan production by [35S]-s~ilfate incorporation, and disk DNA content by fluorometry. Mesenchymal cells in monolayer cultures treated with TGF-81 actively proliferated for the first 4 days, developed cellular rounding, and formed cell clusters. Treated MSC cultures had a two-fold increase in medium proteoglycan content. Pretreatment of MSCs with TGF-81 followed by exposure of cells to IGF-I in three-dimensional culture significantly increased the formation of markers of chondrocytic flmction including disk proteoglycan content and procollagen type I1 mRNA production. However, proteoglycan and procollagen type I1 production by MSC's remained lower than parallel chondrocyte cultures. MSC pretreatment with TGF-P1 without sequential IGF-I was less effective in initiating expression of markers of chondrogenesis. This study indicates that although MSC differentiation was less than complete when compared to mature chondrocytes, chondrogenesis was observed in IGF-I supplemented cultures, particularly when used in concert with TGF-81 pretreatment. 0 9001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

A web-based resource, Microbial Community Analysis (MiCA), has been developed to facilitate studies on microbial community ecology that use analyses of terminal-restriction fragment length polymorphisms (T-RFLP) of 16S and 18S rRNA genes. MiCA provides an intuitive web interface to access two specialized programs and a specially formatted database of 16S ribosomal RNA sequences. The first program performs virtual polymerase chain reaction (PCR) amplification of rRNA genes and restriction of the amplicons using primer sequences and restriction enzymes chosen by the user. This program, in silico PCR and Restriction (ISPaR), uses a binary encoding of DNA sequences to rapidly scan large numbers of sequences in databases searching for primer annealing and restriction sites while permitting the user to specify the number of mismatches in primer sequences. ISPaR supports multiple digests with up to three enzymes. The number of base pairs between the 5' and 3' primers and the proximal restriction sites can be reported, printed, or exported in various formats. The second program, APLAUS, infers a plausible community structure(s) based on T-RFLP data supplied by a user. APLAUS estimates the relative abundances of populations and reports a listing of phylotypes that are consistent with the empirical data. MiCA is accessible at http://mica.ibest.uidaho.edu/.

Mural cells of the vertebrate brain maintain vascular integrity and function, play roles in stroke and are involved in maintenance of neural stem cells. However, the origins, diversity and roles of mural cells remain to be fully understood. Using transgenic zebrafish, we identified a population of isolated mural lymphatic endothelial cells surrounding meningeal blood vessels. These meningeal mural lymphatic endothelial cells (muLECs) express lymphatic endothelial cell markers and form by sprouting from blood vessels. In larvae, muLECs develop from a lymphatic endothelial loop in the midbrain into a dispersed, nonlumenized mural lineage. muLEC development requires normal signaling through the Vegfc-Vegfd-Ccbe1-Vegfr3 pathway. Mature muLECs produce vascular growth factors and accumulate low-density lipoproteins from the bloodstream. We find that muLECs are essential for normal meningeal vascularization. Together, these data identify an unexpected lymphatic lineage and developmental mechanism necessary for establishing normal meningeal blood vasculature.