Search

The biodiversity of Madagascar is extraordinarily distinctive, diverse, and endangered. It is therefore urgent that steps be taken to document, describe, interpret, and protect this exceptional biota. As a collaborative group of field and laboratory biologists, we employ a suite of methodological and analytical tools to investigate the vertebrate portion of Madagascar's fauna. Given that species are the fundamental unit of evolution, where micro- and macroevolutionary forces converge to generate biological diversity, a thorough understanding of species distribution and abundance is critical for understanding the evolutionary, ecological, and biogeographic forces that have shaped Malagasy vertebrate diversity. We illustrate the means by which we apply Mayr's “three basic tasks” of the systematist [Mayr, E. (1942) Systematics and the Origin of Species from the Viewpoint of a Zoologist (Harvard Univ. Press, Cambridge, MA)] to identify, classify, and study the organisms that together constitute Madagascar's vertebrate community. Using field inventory methods, specimen-based studies, and morphological and molecular analyses, we formulate hypotheses of species identity that then serve as the foundation for subsequent studies of biology and history. Our experience, as well as that of other investigators, has shown that much of the vertebrate species diversity in Madagascar is “cryptic” for both biological and practical reasons. Beyond issues of cryptic biological diversity, the resolution of species identity in Madagascar has been hampered because of a lack of vouchered comparative material at the population level. Through our activities, we are attempting to remedy these limitations while simultaneously enhancing research capacity in Madagascar.

Figs ( Ficus spp., Moraceae) and their pollinating wasps (Agaonidae, Chalcidoidea) constitute perhaps the most tightly integrated pollination mutualism that is known. Figs are characterized by extraordinarily high global and local species diversity. It has been proposed that the diversification of this mutualism has occurred through strict-sense coadaptation and cospeciation between pairs of fig and wasp species that are associated in highly specific one-to-one relationships. However, existing studies cast doubt on the generality of this proposition. Here, we review our current knowledge of the evolutionary history of the fig/fig-wasp mutualism. We critically examine the idea that codivergence between figs and their pollinators has been dominated by strict-sense cospeciation. We present phylogenetic and population genetic data from neotropical fig and fig wasp species that suggest that a more accurate model for diversification in this mutualism is that of groups of genetically well defined wasp species coevolving with genetically less well defined (frequently hybridizing) groups of figs. Last, we use our results to assess previously proposed hypotheses on models of speciation in this mutualism.

The first steps of animal speciation are thought to be the development of sexual isolating mechanisms. In contrast to recent progress in understanding the genetic basis of postzygotic isolating mechanisms, little is known about the genetic architecture of sexual isolation. Here, we have subjected Drosophila melanogaster to 29 generations of replicated divergent artificial selection for mating speed. The phenotypic response to selection was highly asymmetrical in the direction of reduced mating speed, with estimates of realized heritability averaging 7%. The selection response was largely attributable to a reduction in female receptivity. We assessed the whole genome transcriptional response to selection for mating speed using Affymetrix GeneChips and a rigorous statistical analysis. Remarkably, >3,700 probe sets (21% of the array elements) exhibited a divergence in message levels between the Fast and Slow replicate lines. Genes with altered transcriptional abundance in response to selection fell into many different biological process and molecular function Gene Ontology categories, indicating substantial pleiotropy for this complex behavior. Future functional studies are necessary to test the extent to which transcript profiling of divergent selection lines accurately predicts genes that directly affect the selected trait.

Specific antigen vaccination by administration of the target antigen in aqueous solution has resulted in significant decreases of disease severity in animal models of experimental allergic encephalomyelitis, type I diabetes, and several forms of antigen-induced arthritis, even if administered after the initiation of symptoms. However, in experimental autoimmune encephalomyelitis (EAE) and type I diabetes in nonobese diabetic (NOD) mice, repeated administration of peptide fragments of target antigens in incomplete Freund's adjuvant has resulted in severe anaphylactic reactions. Although these methods of administration are known to potentiate CD4 T helper 2 (Th2) responses, which is the goal of specific antigen vaccination, the risk of anaphylaxis raises a red flag concerning use of this therapy for diseases such as type I diabetes, where the survival time after onset is quite long. It is clear that specific antigen vaccination is effective in preventing several animal models of autoimmune disease, and in treating these diseases once the symptoms are overt. However, the risks of this therapy require serious consideration of alternative methods for down-regulation of the autoimmune process.

Retroelements constitute a large portion of our genomes. One class of these elements, the human endogenous retroviruses (HERVs), is comprised of remnants of ancient exogenous retroviruses that have gained access to the germ line. After integration, most proviruses have been the subject of numerous amplifications and have suffered extensive deletions and mutations. Nevertheless, HERV-derived transcripts and proteins have been detected in healthy and diseased human tissues, and HERV-K, the youngest, most conserved family, is able to form virus-like particles. Although it is generally accepted that the integration of retroelements can cause significant harm by disrupting or disregulating essential genes, the role of HERV expression in the etiology of malignancies and autoimmune and neurologic diseases remains controversial. In recent years, striking evidence has accumulated indicating that some proviral sequences and HERV proteins might even serve the needs of the host and are therefore under positive selection. The remarkable progress in the analysis of host genomes has brought to light the significant impact of HERVs and other retroelements on genetic variation, genome evolution, and gene regulation.

A decade ago, it seemed rational that our rapidly increasing knowledge of the molecular identities of tumor antigens and a deeper understanding of basic immunology would point the way to an effective therapeutic cancer vaccine. Significant progress has been made, but we do not yet have a cancer vaccine that can reliably and consistently induce tumor destruction or improve patient survival. Random mutations in cancer cells generate unique antigens in each individual, and this may be important in terms of generating a therapeutic immune response. Autologous heat shock protein–peptide complexes produced from each patient's tumor is a logical personalized approach that may obviate the need to identify the unique antigens contained in the individual vaccine. Heat shock proteins elicit adaptive and innate immune responses and have been tested in a variety of animal models and different human cancers. Activity has been seen in several animal studies. Early-phase human studies have also suggested some activity in certain cancers. Large, randomized phase 3 studies are ongoing, and these will effectively answer the question of efficacy regarding this approach to therapeutic vaccination. There are sufficient data to support the notion that cancer vaccines can induce anti-tumor immune responses in humans with cancer. How best to translate this increase in immune responsiveness to consistently and reproducibly induce objective cancer regression or increased survival remains unclear at this time.

Persistent viral infections causing serious diseases derive, primarily, from altered function of the immune system. Knowledge of the very complex composition and function of the innate and adaptive branches of the immune system is essential to understanding persistent infection. The best solution to the problem of persistent infection is by prevention using prophylactic vaccines. Hit and run viruses evade immune destruction by infecting new hosts and rarely persist. Hit and stay viruses evade immune control by sequestration, blockade of antigen presentation, cytokine escape, evasion of natural killer cell activities, escape from apoptosis, and antigenic change. Twelve prophylactic vaccines against hit and run agents exist, and there are only three vaccines against hit and stay viruses, all of which are of DNA composition. Several new vaccines against hit and stay viruses are feasible, but protective vaccines against RNA HIV and hepatitis C agents are highly unlikely, short of a major breakthrough. Therapeutic vaccines are very improbable without a magnitude of favorable new discoveries. In the meantime, antiviral chemotherapy, chemotherapy/prophylactic vaccination, and short interfering RNA silencing are worthy of intense investigation.

Delivery of antigens by injection of the encoding DNA allows access to multiple antigen-presenting pathways. Knowledge of immunological processes can therefore be used to modify construct design to induce selected effector functions. Expression can be directed to specific intracellular sites, and additional genes can be fused or codelivered to amplify responses. Therapeutic vaccination against cancer adds a requirement to overcome tolerance and to activate a weakened immune repertoire. Induction of CD4+T helper cells is critical for both antibody and T cell effector responses. To activate immunity against tumor antigens, we fused the tumor-derived sequences to genes encoding microbial proteins. This strategy engages T helper cells from the large antimicrobial repertoire for linked help for inducing antibody against cell-surface tumor antigens. The principle of linked T cell help also holds for induction of epitope-specific antitumor CD8+T cells, but the microbial sequence has to be minimized to avoid competition with tumor antigens. Epitope-specific DNA vaccination leads to powerful antitumor attack and can activate immunity from a profoundly tolerized repertoire. Vaccine designs validated in preclinical models are now in clinical trial with immune responses detected against both tumor antigens and fused microbial antigens. DNA priming is highly efficient, but boosting may benefit from increased antigen expression. Physical methods including electroporation provide increased expression without introducing additional competing antigens. A wide range of cancers can be targeted, and objective assays of response will determine efficacy.

PNAS is one of world's most cited multidisciplinary scientific journals. The PNAS official classification structure of subjects is reflected in topic labels submitted by the authors of articles, largely related to traditionally established disciplines. These include broad field classifications into physical sciences, biological sciences, social sciences, and further subtopic classifications within the fields. Focusing on biological sciences, we explore an internal soft-classification structure of articles based only on semantic decompositions of abstracts and bibliographies and compare it with the formal discipline classifications. Our model assumes that there is a fixed number of internal categories, each characterized by multinomial distributions over words (in abstracts) and references (in bibliographies). Soft classification for each article is based on proportions of the article's content coming from each category. We discuss the appropriateness of the model for the PNAS database as well as other features of the data relevant to soft classification.

How does a network of documents grow without centralized control? This question is becoming crucial as we try to explain the emergent scale-free topology of the World Wide Web and use link analysis to identify important information resources. Existing models of growing information networks have focused on the structure of links but neglected the content of nodes. Here I show that the current models fail to reproduce a critical characteristic of information networks, namely the distribution of textual similarity among linked documents. I propose a more realistic model that generates links by using both popularity and content. This model yields remarkably accurate predictions of both degree and similarity distributions in networks of web pages and scientific literature.

(9-ODA), (R,E)-()- and (S,E)-()-9-hydroxy-2-decenoic acid (9HDA), methyl p-hydroxybenzoate (HOB), and 4-hydroxy-3methyoxyphenylethanol (HVA) (7). QMP plays many roles in social regulation (7). It prevents reproduction both by inhibiting worker ovary development (10) and the rearing of new queens (11). It may control brain development in the olfactory system of young bees (12). QMP also influences age-related division of labor among worker bees. Bees perform in-hive tasks such as brood care (nursing) for the first 2–3 weeks of their adult life and switch to foraging for food outside the hive in the last weeks of their life. Honey bees show many changes in physiology in association with this behavioral maturation, including structural changes in the mushroom bodies of the brain (which are sites of multimodal sensory integration and higher-order functions such as learning and memory), and differences in brain gene expression in nurses compared with foragers (13, 14). QMP delays honey bee behavioral maturation (15). In this article we report on experiments that test two hypotheses: exposure to QMP causes changes in gene expression in the brain, and these changes correlate with the downstream behavioral effects of the pheromone. We used a recently developed honey bee brain microarray (16) to profile pheromone-induced changes in brain gene expression. Experiments were performed both with cage studies under controlled conditions in the laboratory and natural colony conditions in the field, to determine the robustness of the QMP effects. Because differences in gene expression associated with nursing and foraging behaviors have been analyzed extensively with microarrays (14), we used this information to explore our second hypothesis, that pheromoneregulated changes in gene expression are correlated with pheromone-induced changes in behavior. Finally, we began to characterize the effects of QMP on an ortholog of the Drosophila transcription factor kruppel homolog 1 (Kr-h1) (17), a particularly promising candidate gene that emerged from our microarray analyses.

Glucosinolate profiles differ among Arabidopsis thaliana ecotypes, caused by the composition of alleles at several glucosinolate biosynthetic loci. One of these, GS-Elong , harbors a family of methylthioalkylmalate synthase (MAM) genes that determine the side chain length of aliphatic glucosinolate structures. Fine mapping reveals that GS-Elong constitutes an insect resistance quantitative trait locus, caused by variation in glucosinolate profiles conferred by polymorphism of MAM alleles in this region. A sequence survey of randomly chosen ecotypes indicates that GS-Elong is highly variable among A. thaliana ecotypes: indel polymorphisms are frequent, as well as gene conversion events between gene copies arranged in tandem. Furthermore, statistical methods of molecular population genetics suggest that one of the genes, MAM2 , is subject to balancing selection. This may be caused by ecological tradeoffs, i.e., by contrasting physiological effects of glucosinolates on generalist vs. specialist insects.

Most recent evidence suggests that the process of tissue repair is driven by stem-like cells that reside in multiple tissues but are replenished by precursor cells from bone marrow. Among the candidates for the reparative cells are the adult stem cells from bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (MSCs). We recently found that after MSCs were replated at very low densities to generate single-cell-derived colonies, they did not exit a prolonged lag period until they synthesized and secreted considerable quantities of Dickkopf-1, an inhibitor of the canonical Wnt signaling pathway. We also found that when the cells were cocultured with heat-shocked pulmonary epithelial cells, they differentiated into epithelial cells. Most of the MSCs differentiated without evidence of cell fusion but up to one-quarter underwent cell fusion with the epithelial cells. A few also underwent nuclear fusion. The results are consistent with the interesting possibility that MSCs and similar cells repair tissue injury by three different mechanisms: creation of a milieu that enhances regeneration of endogenous cells, transdifferentiation, and perhaps cell fusion.

In this article we briefly review theories about the ecological roles of microbial secondary metabolites and discuss the prevalence of multiple secondary metabolite production by strains of Streptomyces , highlighting results from analysis of the recently sequenced Streptomyces coelicolor and Streptomyces avermitilis genomes. We address this question: Why is multiple secondary metabolite production in Streptomyces species so commonplace? We argue that synergy or contingency in the action of individual metabolites against biological competitors may, in some cases, be a powerful driving force for the evolution of multiple secondary metabolite production. This argument is illustrated with examples of the coproduction of synergistically acting antibiotics and contingently acting siderophores: two well-known classes of secondary metabolite. We focus, in particular, on the coproduction of β-lactam antibiotics and β-lactamase inhibitors, the coproduction of type A and type B streptogramins, and the coregulated production and independent uptake of structurally distinct siderophores by species of Streptomyces . Possible mechanisms for the evolution of multiple synergistic and contingent metabolite production in Streptomyces species are discussed. It is concluded that the production by Streptomyces species of two or more secondary metabolites that act synergistically or contingently against biological competitors may be far more common than has previously been recognized, and that synergy and contingency may be common driving forces for the evolution of multiple secondary metabolite production by these sessile saprophytes.

Cell–cell communication in bacteria is accomplished through the exchange of chemical signal molecules called autoinducers. This process, called quorum sensing, allows bacteria to monitor their environment for the presence of other bacteria and to respond to fluctuations in the number and/or species present by altering particular behaviors. Most quorum-sensing systems are species- or group-specific, which presumably prevents confusion in mixed-species environments. However, some quorum-sensing circuits control behaviors that involve interactions among bacterial species. These quorum-sensing circuits can involve both intra- and interspecies communication mechanisms. Finally, anti-quorumsensing strategies are present in both bacteria and eukaryotes, and these are apparently designed to combat bacteria that rely on cell–cell communication for the successful adaptation to particular niches.

Flowering plants have the unique ability to produce new organs continuously, for hundreds of years in some species, from stem cell populations maintained at their actively growing tips. The shoot tip is called the shoot apical meristem, and it acts as a self-renewing source of undifferentiated, pluripotent stem cells whose descendents become incorporated into organ and tissue primordia and acquire different fates. Stem cell maintenance is an active process, requiring constant communication between different regions of the shoot apical meristem to coordinate loss of stem cells from the meristem through differentiation with their replacement through cell division. Stem cell research in model plant systems is facilitated by the fact that mutants with altered meristem cell identity or accumulation are viable, allowing dissection of stem cell behavior by using genetic, molecular, and biochemical methods. Such studies have determined that in the model plant Arabidopsis thaliana stem cell maintenance information flows via a signal transduction pathway that is established during embryogenesis and maintained throughout the life cycle. Signaling through this pathway results in the generation of a spatial feedback loop, involving both positive and negative interactions, that maintains stem cell homeostasis. Stem cell activity during reproductive development is terminated by a temporal feedback loop involving both stem cell maintenance genes and a phase-specific flower patterning gene. Our current investigations provide additional insights into the molecular mechanisms that regulate stem cell activity in higher plants.

Stem cells (SCs) are functionally defined by their abilities to self-renew and generate differentiated cells. Although much effort has been focused on defining the common characteristics among various types of SCs, the genetic and functional differences between multipotent and pluripotent SCs have garnered less attention. We report a direct genetic and functional comparison of molecularly defined and clonally related populations of neural SCs (NSCs) and embryonic SCs (ESCs), using the Sox2 promoter for isolation of purified populations by fluorescence-activated cell sorting. A stringent expression profile comparison of promoter-defined NSCs and ESCs revealed a striking dissimilarity, and subsequent chimera analyses confirmed the fundamental differences in cellular potency between these populations. This direct comparison elucidates the molecular basis for the functional differences in pluripotent ESCs and multipotent NSCs.

The transplantation of a somatic cell nucleus to an enucleated egg results in a major reprogramming of gene expression and switch in cell fate. We review the efficiency of nuclear reprogramming by nuclear transfer. The serial transplantation of nuclei from defective first-transfer embryos and the grafting of cells from such embryos to normal host embryos greatly increases the proportion of nuclei that can be seen to have been reprogrammed. We discuss possible reasons for the early failure of most nuclear transfers from differentiated cells and describe the potential value of growing oocytes, rather than unfertilized eggs, as a source of nuclear reprogramming molecules and for the eventual identification of these molecules. Nuclear transfer provides a possible route for the creation of stem cells from adult somatic cells.

Freshwater planarian flatworms are capable of regenerating complete organisms from tiny fragments of their bodies; the basis for this regenerative prowess is an experimentally accessible stem cell population that is present in the adult planarian. The study of these organisms, classic experimental models for investigating metazoan regeneration, has been revitalized by the application of modern molecular biological approaches. The identification of thousands of unique planarian ESTs, coupled with large-scale whole-mount in situ hybridization screens, and the ability to inhibit planarian gene expression through double-stranded RNA-mediated genetic interference, provide a wealth of tools for studying the molecular mechanisms that regulate tissue regeneration and stem cell biology in these organisms. Here we show that, as in Caenorhabditis elegans , ingestion of bacterially expressed double-stranded RNA can inhibit gene expression in planarians. This inhibition persists throughout the process of regeneration, allowing phenotypes with disrupted regenerative patterning to be identified. These results pave the way for large-scale screens for genes involved in regenerative processes.

Several vertebrates display the ability to regenerate parts of their body after amputation. During this process, differentiated cells reenter the cell cycle and proliferate to generate a mass of undifferentiated cells. Repatterning mechanisms act on these cells to eventually shape a regenerated tissue or organ that replaces the amputated one. Experiments with regenerating limbs/fins in newts and zebrafish have shown that members of the Msx family of homeodomain-containing transcription factors play key roles during blastema formation and patterning. Here we show that adult zebrafish have a remarkable capacity to regenerate the heart in a process that involves up-regulation of msxB and msxC genes. We present evidence indicating that heart regeneration involves the execution of a specific genetic program, rather than redeployment of a cardiac development program. Preceding Msx activation, there is a marked increase in the expression of notch1b and deltaC , which we show are also up-regulated during fin regeneration. These data suggest a role for the Notch pathway in the activation of the regenerative response. Taken together, our results underscore the use of zebrafish as a model for investigating the process of regeneration in particular and the biology of stem cells in general. Advances in these fields will undoubtedly aid in the implementation of strategies for regenerative medicine.

The existence of a postnatal prostate stem cell is supported by several types of evidence. Withdrawal of androgen leads to involution of the gland, but readdition can rapidly stimulate regeneration. Tissue fragments derived from mouse or rat prostatic epithelia from midgestation embryos or adult mice, when combined with tissue fragments from urogenital sinus mesenchyme and grafted under the kidney capsule, can regenerate prostatic structures. Indirect evidence supports that the stem cell population is contained within the basal layer. Purified prostatic stem cell preparations would be useful to define the physical and functional properties required for regeneration and to compare with cells that accumulate during abnormal growth states, like prostate cancer. We have developed a regeneration system using dissociated cell populations of postnatal prostate epithelia and embryonic urogenital sinus mesenchyme. Efficient in vivo regeneration of prostatic structures in the subcapsular space of the kidney was observed within 4-8 wk with as few as 10 3 epithelial cells from prostates derived from donors 10 d to 6 wk of age. The regenerated structures show a branching tubular epithelial morphology, with expression of a panel of markers consistent with prostate development. Donor epithelial populations can be readily infected with GFP expressing lentiviral vectors to provide integration markers and easy visualization. The cell preparations of urogenital sinus mesenchyme can be expanded in short-term in vitro culture while their inductive capabilities are retained. Further definition of the subpopulation of prostate epithelial cells containing the regeneration activity should be possible with such technologies.

The appearance of bipotential oval cells in chronic liver injury suggests the existence of hepatocyte progenitor/stem cells. To study the origin and properties of this cell population, oval cell proliferation was induced in adult mouse liver by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and a method for their isolation was developed. Transplantation into fumarylacetoacetate hydrolase (Fah) deficient mice was used to determine their capacity for liver repopulation. In competitive repopulation experiments, hepatic oval cells were at least as efficient as mature hepatocytes in repopulating the liver. In mice with chimeric livers, the oval cells were not derived from hepatocytes but from liver nonparenchymal cells. This finding supports a model in which intrahepatic progenitors differentiate into hepatocytes irreversibly. To determine whether oval cells originated from stem cells residing in the bone marrow, bone marrow transplanted wild-type mice were treated with DDC for 8 months and oval cells were then serially transferred into Fah mutants. The liver repopulating cells in these secondary transplant recipients lacked the genetic markers of the original bone marrow donor. We conclude that hepatic oval cells do not originate in bone marrow but in the liver itself, and that they have valuable properties for therapeutic liver repopulation.

The energetics of the TiO 2 polymorphs (rutile, anatase, and brookite) were studied by high temperature oxide melt drop solution calorimetry. Relative to bulk rutile, bulk brookite is 0.71 ± 0.38 kJ/mol (6) and bulk anatase is 2.61 ± 0.41 kJ/mol higher in enthalpy. The surface enthalpies of rutile, brookite, and anatase are 2.2 ± 0.2 J/m 2 , 1.0 ± 0.2 J/m 2 , and 0.4 ± 0.1 J/m 2 , respectively. The closely balanced energetics directly confirm the crossover in stability of nanophase polymorphs inferred by Zhang and Banfield (7). An amorphous sample with surface area of 34,600 m 2 /mol is 24.25 ± 0.88 kJ/mol higher in enthalpy than bulk rutile.