Acoel and platyhelminth models for stem-cell research

Acoel and platyhelminth worms are particularly attractive invertebrate models for stem-cell research because their bodies are continually renewed from large pools of somatic stem cells. Several recent studies, including one in BMC Developmental Biology, are beginning to reveal the cellular dynamics and molecular basis of stem-cell function in these animals

Postembryonic Polarity Modification in the Acoel Convolutriloba longifissura

Metazoans establish the bilateral body plan early in embryogenesis by patterning orthogonal body axes with polarity that is unaltered during the lifetime of most animals. While some organisms re-establish/modify body axes during regeneration and asexual reproduction, the acoel flatworm Convolutriloba longifissura is unusual in its ability to modify left-right (L-R) axis polarity during longitudinal fission. We have developed C. longifissura as a model for studying the mechanisms of L-R polarity modification during postembryonic development. Regeneration experiments have elucidated the temporal dynamics of midline re-specification, suggesting that parallel L-R axes replace the pre-existing midline prior to longitudinal fission. We have characterized the spatiotemporal expression of genes encoding ligands and receptors of signaling pathways with conserved functions in polarity specification and axial patterning. Expression domains of BMP, Notch, and Slit/Robo signaling components are dynamic prior to and during longitudinal fission. RNAi-mediated gene knockdown of Notch and Slit/Robo signals disrupt longitudinal fission while BMP disrupted normal midline patterning suggesting a role in modulating changes in L-R axis polarity

Mechanisms of axial polarity modification during postembryonic development of the basal bilaterian Convolutriloba macropyga

Acoel flatworms have varied modes of asexual reproduction that involve dramatic postembryonic modification of their anterior-posterior (AP) axis. The acoel species Convolutriloba macropyga reproduces through a reversed polarity budding process in which offspring develop from two posterior budding sites with a complete reversal of the AP axis compared to the parent. Reversed polarity budding is preceded by the development of a zone of tissue with disorganized musculature that is incapable of regeneration, suggesting a transient loss of axis polarity at each budding site. For this reason, these tissues are titled the polarity transition zone (PTZ). While this alteration of existing axial polarity seems to be required for subsequent reversal of the AP axis in the budding progeny, the mechanisms that allow for temporary axis modification and reversal are not known. Wnt, Hedgehog, and other signal transduction pathways have conserved roles in AP axis development and reestablishment during both metazoan embryogenesis and regeneration, suggesting these signals may function in mediating changes in axis polarity during budding in C. macropyga. Here, we have used a pharmacological screen to perturb conserved signaling pathways in Convolutriloba tissues and observed loss of axial polarity in tissues exposed to inhibitors of the Hedgehog pathway. Given the putative role of Hedgehog signal transduction in mediating alterations in axial polarity during budding, we have quantified changes in the expression of Hedgehog signaling components and regulators in budding tissues using qPCR. Hedgehog signaling was downregulated within the PTZ when compared to neighboring polarized tissues. RNAi mediated knockdown of Hedgehog gene products resulted in phenotypes of delayed bud detachment and failure to initiate future budding events. These data support Hedgehog as a key signaling pathway involved in the modification of AP axis polarity during asexual reproduction and may provide key insights towards better understanding the evolution of asexual reproduction strategies in other taxa

Acoel and Platyhelminth Models for Stem-cell Research

Acoel and platyhelminth worms are particularly attractive invertebrate models for stem-cell research because their bodies are continually renewed from large pools of somatic stem cells. Several recent studies, including one in BMC Developmental Biology, are beginning to reveal the cellular dynamics and molecular basis of stem-cell function in these animals. See research article http://www.biomedcentral.com/1471-213X/9/69. Adult somatic stem cells can play critical roles in postembryonic developmental processes such as tissue renewal, growth, repair, and regeneration [1]. Understanding how such cells are maintained and produce differentiated progeny is thus of general interest in developmental biology, in addition to being of clear biomedical relevance. Invertebrate models have great potential for elucidating the cellular and molecular basis of stem-cell function. However, in the main invertebrate models used for dissecting the details of animal development, including Drosophila and Caenorhabditis, adult somatic tissues are primarily post-mitotic and are largely or entirely devoid of adult stem cells, which limits the use of these established models for stem-cell research. Representatives of two groups of soft-bodied worms, the Acoela and the Platyhelminthes, possess large pools of adult somatic stem cells, making them useful invertebrate models for stem-cell biology. These organisms are now beginning to provide new insights into the cellular and molecular basis of adult stem-cell function

Breaking the A-P axis: Evolution of diverse asexual reproduction strategies in Convolutriloba acoels

The defining characteristic of the Bilateria is the presence of a distinct head end and tail end, which defines the anterior-posterior (A-P) axis, a feature that is established during embryogenesis and generally remains unaltered during the lifetime of an organism. While a few bilaterians have evolved asexual reproduction strategies that allow them to subdivide the A-P axis, acoels in the genus Convolutriloba have an unparalleled ability to alter the A-P axis during modes of transverse fission, longitudinal fission, and reversed polarity budding. Convolutriloba acoels thus offer an exceptional opportunity to investigate the mechanisms that allow for the radical modification of an already established A-P body axis and to explore the evolution and development of diverse asexual reproduction strategies among related species. In this study, I reconstruct the evolutionary history of asexual reproduction in the Convolutriloba and compare the diverse modes of asexual reproduction at the level of body-wall musculature, nervous system development, and cell proliferation while also exploring the regenerative potentials of tissues across species with different modes of asexual reproduction. In addition, I further explore the unusual process of A-P axis reversal that occurs during reversed polarity budding in C. retrogemma through studies of body patterning and regeneration. The results of these analyses suggest that a rich developmental toolkit of regenerative abilities, including the ability to utilize both epimorphosis and morphallaxis, to regenerate all parts of its body even from a small fragment, and to produce bifurcated A-P axes were present in the ancestor of the Convolutriloba allowing for the evolution of A-P axis modifications unlike any other bilaterian group. This toolkit along with the evolution of a seemingly unpatterned zone of tissue within the body of C. retrogemma capable of generating new anterior axes appear to have allowed this species to evolve the ability to form reversed A-P axes during budding

New Insights into Fluoroquinolone Resistance in Mycobacterium tuberculosis: Functional Genetic Analysis of gyrA and gyrB Mutations

Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB), and resistance to this class of antibiotics is one criterion for defining extensively drug resistant tuberculosis (XDR TB). Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR) of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven) and gyrB (nineteen) to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies among the different mutations. The results from this study provide support for the inclusion of the QRDR of gyrB in molecular assays used to detect fluoroquinolone resistance in M. tuberculosis

Custom Integrated Circuits

Contains reports on twelve research projects.Analog Devices, Inc.International Business Machines, Inc.Joint Services Electronics Program (Contract DAAL03-86-K-0002)Joint Services Electronics Program (Contract DAAL03-89-C-0001)U.S. Air Force - Office of Scientific Research (Grant AFOSR 86-0164)Rockwell International CorporationOKI Semiconductor, Inc.U.S. Navy - Office of Naval Research (Contract N00014-81-K-0742)Charles Stark Draper LaboratoryNational Science Foundation (Grant MIP 84-07285)National Science Foundation (Grant MIP 87-14969)Battelle LaboratoriesNational Science Foundation (Grant MIP 88-14612)DuPont CorporationDefense Advanced Research Projects Agency/U.S. Navy - Office of Naval Research (Contract N00014-87-K-0825)American Telephone and TelegraphDigital Equipment CorporationNational Science Foundation (Grant MIP-88-58764

Empirical Legal Studies Before 1940: A Bibliographic Essay

The modern empirical legal studies movement has well-known antecedents in the law and society and law and economics traditions of the latter half of the 20th century. Less well known is the body of empirical research on legal phenomena from the period prior to World War II. This paper is an extensive bibliographic essay that surveys the English language empirical legal research from approximately 1940 and earlier. The essay is arranged around the themes in the research: criminal justice, civil justice (general studies of civil litigation, auto accident litigation and compensation, divorce, small claims, jurisdiction and procedure, civil juries), debt and bankruptcy, banking, appellate courts, legal needs, legal profession (including legal education), and judicial staffing and selection. Accompanying the essay is an extensive bibliography of research articles, books, and reports

Regeneration in Spiralians: Evolutionary Patterns and Developmental Processes

Animals differ markedly in their ability to regenerate, yet still little is known about how regeneration evolves. In recent years, important advances have been made in our understanding of animal phylogeny and these provide new insights into the phylogenetic distribution of regeneration. The developmental basis of regeneration is also being investigated in an increasing number of groups, allowing commonalities and differences across groups to become evident. Here, we focus on regeneration in the Spiralia, a group that includes several champions of animal regeneration, as well as many groups with more limited abilities. We review the phylogenetic distribution and developmental processes of regeneration in four major spiralian groups: annelids, nemerteans, platyhelminths, and molluscs. Although comparative data are still limited, this review highlights phylogenetic and developmental patterns that are emerging regarding regeneration in spiralians and identifies important avenues for future research