Stephen J. Bent

Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor‐β1 in monolayer and insulin‐like growth factor‐I in a three‐dimensional matrix

This study evaluated chondrogenesis of mesenchymal progenitor stem cells (MSCs) cultured initially under pre‐confluent monolayer conditions exposed to transforming growth factor‐β (TGF‐β), 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‐β1 per ml of medium for 6 days. TGF‐β1 treated and untreated cultures were distributed to three‐dimensional 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 II in situ hybridization and immunohistochemistry, proteoglycan production by [Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor‐β1 in monolayer and insulin‐like growth factor‐I in a three‐dimensional matrix35S]‐sulfate incorporation, and disk DNA content by fluorometry. Mesenchymal cells in monolayer cultures treated with TGF‐β1 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‐β1 followed by exposure of cells to IGF‐I in three‐dimensional culture significantly increased the formation of markers of chondrocytic function including disk proteoglycan content and procollagen type II mRNA production. However, proteoglycan and procollagen type II production by MSCs remained lower than parallel chondrocyte cultures. MSC pretreatment with TGF‐β1 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‐β1 pretreatment.

The tragedy of the uncommon: understanding limitations in the analysis of microbial diversity

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.

MiCA: A Web-Based Tool for the Analysis of Microbial Communities Based on Terminal-Restriction Fragment Length Polymorphisms of 16S and 18S rRNA Genes

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/.

Measuring species richness based on microbial community fingerprints: the emperor has no clothes

Danovaro and colleagues ([3][1]) recently compared microbial community diversity and richness estimates obtained using automated ribosomal intergenic spacer analysis (ARISA) with those obtained using analysis of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes. While

Insulinlike growth factor-I gene therapy applications for cartilage repair

Cartilage function after resurfacing with cell-based transplantation procedures or during the early stages of arthritic disease may be bolstered by the addition of growth factor genes to the transplanted tissue. Insulinlike growth factor-I maintains chondrocyte metabolism in normal cartilage homeostasis and has been shown to improve cartilage healing in vivo. Given the relatively short half-life of insulinlike growth factor-I in biologic systems, however, maintenance of effective concentrations of this peptide has necessitated either very high initial doses or repeated treatment. Delivery of the insulinlike growth factor-I gene, using a deleted adenovirus vector, specifically targeting graftable articular chondrocytes, bone marrow-derived chondroprogenitor cells, or synovial lining cells, may provide more durable insulinlike growth factor-I fluxes to articular tissues. Cultured equine articular chondrocytes, mesenchymal stem cells, synovial explants, and synovial intimal cells were readily transfected with an E1-deleted adenoviral vector containing equine insulinlike growth factor-I coding sequence. Optimal viral concentrations for effective transduction were 100 multiplicities of infection in synoviocytes, 500 multiplicities of infection in chondrocytes, and 1000 multiplicities of infection in mesenchymal stem cells. Production of insulinlike growth factor-I ligand varied from 65 ng/mL to 246 ng/mL in medium from chondrocytes and synovial explants, respectively. For chondrocytes, these concentrations were sufficient to produce significant stimulation of cartilage matrix gene expression and subsequent proteoglycan production. Moreover, cells in infected cultures maintained a chondrocytic phenotype and continued to express elevated insulinlike growth factor-I levels during 28 days of monolayer culture. Minimal synthetic activity, other than insulinlike growth factor-I ligand synthesis, was evident in synovial cultures. These experiments suggest several avenues for insulinlike growth factor-I supplementation of articular cartilage, including preimplantation adenoviral-insulinlike growth factor gene transfer to chondrocytes or chondroprogenitor cells, and direct injection of adenoviral-insulinlike growth factor to transfect the synovial structures in situ.

Phylogeography of Borrelia burgdorferi in the eastern United States reflects multiple independent Lyme disease emergence events

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.

Bacterial succession in a glacier foreland of the high arctic

Succession is defined as changes in biological communities over time. It has been extensively studied in plant communities, but little is known about bacterial succession, in particular in environments such as High Arctic glacier forelands. Bacteria carry out key processes in the development of soil, biogeochemical cycling and facilitating plant colonization. In this study we sampled two roughly parallel chronosequences in the foreland of Midre Lovén glacier on Svalbard, Norway and tested whether any of several factors were associated with changes in the structure of bacterial communities, including time after glacier retreat, horizontal variation caused by the distance between chronosequences and vertical variation at two soil depths. The structures of soil bacterial communities at different locations were compared using terminal restriction fragment length polymorphisms of 16S rRNA genes, and the data were analyzed by sequential analysis of log-linear statistical models. Although no significant differences in community structure were detected between the two chronosequences, statistically significant differences between sampling locations in the surface and mineral soils could be demonstrated even though glacier forelands are patchy and dynamic environments. These findings suggest that bacterial succession occurs in High Arctic glacier forelands but may differ in different soil depths.

Integrative transcriptome meta-analysis reveals widespread sex-biased gene expression at the human fetal–maternal interface

As males and females share highly similar genomes, the regulation of many sexually dimorphic traits is constrained to occur through sex-biased gene regulation. There is strong evidence that human males and females differ in terms of growth and development in utero and that these divergent growth strategies appear to place males at increased risk when in sub-optimal conditions. Since the placenta is the interface of maternal–fetal exchange throughout pregnancy, these developmental differences are most likely orchestrated by differential placental function. To date, progress in this field has been hampered by a lack of genome-wide information on sex differences in placental gene expression. Therefore, our motivation in this study was to characterize sex-biased gene expression in the human placenta. We obtained gene expression data for >300 non-pathological placenta samples from 11 microarray datasets and applied mapping-based array probe re-annotation and inverse-variance meta-analysis methods which showed that >140 genes (false discovery rate (FDR) <0.05) are differentially expressed between male and female placentae. A majority of these genes (>60%) are autosomal, many of which are involved in high-level regulatory processes such as gene transcription, cell growth and proliferation and hormonal function. Of particular interest, we detected higher female expression from all seven genes in the LHB-CGB cluster, which includes genes involved in placental development, the maintenance of pregnancy and maternal immune tolerance of the conceptus. These results demonstrate that sex-biased gene expression in the normal human placenta occurs across the genome and includes genes that are central to growth, development and the maintenance of pregnancy.

One Health approach to identify research needs in bovine and human babesioses: workshop report

BackgroundBabesia are emerging health threats to humans and animals in the United States. A collaborative effort of multiple disciplines to attain optimal health for people, animals and our environment, otherwise known as the One Health concept, was taken during a research workshop held in April 2009 to identify gaps in scientific knowledge regarding babesioses. The impetus for this analysis was the increased risk for outbreaks of bovine babesiosis, also known as Texas cattle fever, associated with the re-infestation of the U.S. by cattle fever ticks.ResultsThe involvement of wildlife in the ecology of cattle fever ticks jeopardizes the ability of state and federal agencies to keep the national herd free of Texas cattle fever. Similarly, there has been a progressive increase in the number of cases of human babesiosis over the past 25 years due to an increase in the white-tailed deer population. Human babesiosis due to cattle-associated Babesia divergens and Babesia divergens-like organisms have begun to appear in residents of the United States. Research needs for human and bovine babesioses were identified and are presented herein.ConclusionsThe translation of this research is expected to provide veterinary and public health systems with the tools to mitigate the impact of bovine and human babesioses. However, economic, political, and social commitments are urgently required, including increased national funding for animal and human Babesia research, to prevent the re-establishment of cattle fever ticks and the increasing problem of human babesiosis in the United States.

Phylogeny of beak and feather disease virus in cockatoos demonstrates host generalism and multiple-variant infections within Psittaciformes

Phylogenetic analyses of the highly genetically diverse but antigenically conserved, single-stranded circular, DNA genome of the avian circovirus, beak and feather disease virus (BFDV) from cockatoo species throughout Australia demonstrated a high mutation rate for BFDV (orders of magnitude fall in the range of 10(-4) substitutions/site/year) along with strong support for recombination indicating active cross-species transmission in various subpopulations. Multiple variants of BFDV were demonstrated with at least 30 genotypic variants identified within nine individual birds, with one containing up to 7 variants. Single genetic variants were detected in feathers from 2 birds but splenic tissue provided further variants. The rich BFDV genetic diversity points to Australasia as the most likely geographical origin of this virus and supports flexible host switching. We propose this as evidence of Order-wide host generalism in the Psittaciformes characterised by high mutability that is buffered by frequent recombination and slow replication strategy.