Genital morphology and fertilization success in the dung beetle Onthophagus taurus: an example of sexually selected male genitalia

In animals with internal fertilization and promiscuous mating, male genitalia show rapid and divergent evolution. Three hypotheses have been suggested to explain the evolutionary processes responsible for genital evolution: the lock-and-key hypothesis, the pleiotropy hypothesis and the sexual-selection hypothesis. Here, we determine whether variation in male genital morphology influences fertilization success in the dung beetle Onthophagus taurus, as predicted by the sexual-selection hypothesis. Variation in four out of five genital sclerites of the endophallus influenced a male's fertilization success, supporting the general hypothesis that male genitalia can evolve under sexual selection. Furthermore, different genital sclerites were found to enhance first versus second male paternity, indicating that different sclerites serve offensive and defensive roles. Genital-trait variability was comparable to that in other species but was less variable than a non-genital sexually selected trait (head horns). We suggest that directional selection for genital elaboration may be countered by natural selection, which should favour genitalia of a size and shape necessary for efficient coupling and sperm transfer.     

Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

Background  

PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology.     

The phylogeographic history of Megistostegium (Malvaceae) in the dry,spiny thickets of southwestern Madagascar using RAD‐seq data and ecological niche modeling

The spiny thicket of southwestern Madagascar represents an extreme and ancient landscape with extraordinary levels of biodiversity and endemism. Few hypotheses exist for explaining speciation in the region and few plant studies have explored hypotheses for species diversification. Here, we investigate three species in the endemic genus Megistostegium (Malvaceae) to evaluate phylogeographic structure and explore the roles of climate, soil, and paleoclimate oscillations on population divergence and speciation throughout the region. We combine phylogenetic and phylogeographic inference of RADseq data with ecological niche modeling across space and time. Population structure is concurrent with major rivers in the region and we identify a new, potentially important biogeographic break coincident with several landscape features. Our data further suggests that niches occupied by species and populations differ substantially across their distribution. Paleodistribution modeling provide evidence that past climatic change could be responsible for the current distribution, population structure, and maintenance of species in Megistostegium.     

Active immunization combined with cisplatin confers enhanced therapeutic protection and prevents relapses of HPV-induced tumors at different anatomical sites

The active immunotherapy concept relies on the use of vaccines that are capable of inducing antitumor immunity, reversion of the suppressive immunological environment, and long-term memory responses. Previously, antitumor vaccines based on a recombinant plasmid (pgDE7h) or a purified protein (gDE7) led to regression of early-established human papillomavirus (HPV)-associated tumors in a preclinical model. In this work, the anticancer vaccines were combined with cisplatin to treat HPV-induced tumors at advanced growth stages. The antitumor effects were evaluated in terms of tumor regression, induction of specific CD8⁺ T cells, and immune modulation of the tumor microenvironment. Acute toxicity induced by the treatment was measured by weight loss and histological alterations in the liver and kidneys. Our results revealed that the combination of cisplatin with either one of the tested immunotherapies (pgDE7h or gDE7) led to complete tumor regression in mice. Also, the combined treatment resulted in synergistic effects, particularly among mice immunized with gDE7, including activation of systemic and tumor-infiltrating E7-specific CD8⁺ T cells, tumor infiltration of macrophages and dendritic cells, and prevention of tumor relapses at different anatomical sites. Furthermore, the protocol allowed the reduction of cisplatin dosage and its intrinsic toxic effects, without reducing antitumor outcomes. These results expand our knowledge of active immunotherapy protocols and open perspectives for alternative treatments of HPV-associated tumors.     

Drosophila rab GDI mutants disrupt development but have normal rab membrane extraction

Summary: Rab GTPases are essential for vesicular transport. Rab GDP dissociation inhibitor (GDI) binds to GDP‐bound rabs, removes rabs from acceptor membranes and delivers rabs to donor membranes. We isolated lethal GDI mutations in Drosophila and analyzed their developmental phenotypes. To learn how these mutations affect GDI structure, the crystal structure of Drosophila GDI was determined by molecular replacement to a resolution of 3.0 Å. Two hypomorphic, missense mutations are located in domain II of GDI at highly conserved positions, but not in previously identified sequence conserved regions. The mutant GDIs were tested for ability to extract rabs from membranes and showed wild‐type levels of rab membrane extraction. The two missense alleles showed intragenic complementation, indicating that domain II of GDI may have two separable functions. This study indicates that GDI function is essential for development of a complex, multicellular organism and that puparium formation and pole cell formation are especially dependent on GDI function. genesis 31:17–29, 2001. © 2001 Wiley‐Liss, Inc.     

EST analysis of mRNAs expressed during embryogenesis in Gallus gallus

Chicken Expressed Sequence Tags (ESTs) were analyzed to identify genes associated with myogenesis during embryonic development. A total of 6,184 ESTs were generated from three cDNA libraries constructed from whole embryos (Stage 26), somites associated with neural tube (Stage 15), and limb buds (Stages 21, 24 and 26). Clustering and assembly of 4,998 valid ESTs resulted in 2,329 unique sequences with 902 clusters (38.7%) and 1,427 singletons (61.3%). There are more than 400,000 chicken ESTs available at GenBank and we were able to identify 143 novel sequences. From these, 45 sequences found either a human EST homolog or a match with conserved regions among proteins. Most of these sequences were found to be expressed in somites, an important tissue for muscle development and not characterized before. This study revealed the value of micro dissected embryonic libraries for describing gene expression profiles associated with myogenesis and gene discovery.     

Sorting of vesicular monoamine transporter 2 to the regulated secretory pathway confers the somatodendritic exocytosis of monoamines

The release of monoamine neurotransmitters from cell bodies and dendrites has an important role in behavior, but the mechanism (vesicular or non vesicular) has remained unclear. Because the location of vesicular monoamine transporter 2 (VMAT2) defines the secretory vesicles capable of monoamine release, we have studied its trafficking to assess the potential for monoamine release by exocytosis. In neuroendocrine PC12 cells, VMAT2 localizes exclusively to large dense-core vesicles (LDCVs), and we now show that cytoplasmic signals target VMAT2 directly to LDCVs within the biosynthetic pathway. In neurons, VMAT2 localizes to a population of vesicles that we now find undergo regulated exocytosis in dendrites. Although hippocampal neurons do not express typical LDCV proteins, transfected chromogranins A, B, and brain-derived neurotrophic factor (BDNF) colocalize with VMAT2. VMAT2 thus defines a population of secretory vesicles that mediate the activity-dependent somatodendritic release of multiple retrograde signals involved in synaptic function, growth, and plasticity.     

Candida albicans biofilm-defective mutants

Biofilm formation plays a key role in the life cycles and subsistence of many microorganisms. For the human fungal pathogen Candida albicans, biofilm development is arguably a virulence trait, because medical implants that serve as biofilm substrates are significant risk factors for infection. The development of C. albicans biofilms in vitro proceeds through an early phase, in which yeast cells populate a substrate, an intermediate phase, in which pseudohyphal and hyphal cell types are produced, and a maturation phase, in which continued cell growth is accompanied by accumulation of an extracellular matrix. Here we report the results of a screen for C. albicans biofilm-defective mutants, in which homozygous insertions in NUP85, MDS3, KEM1, and SUV3 were found to block biofilm development. Confocal microscopic examination suggests that nup85, suv3, and mds3 mutations cause early-phase arrest, whereas the kem1 mutation causes intermediate-phase arrest. All of the mutants are defective in hypha production in several media. Analysis of mixed-biofilm development indicates that all of the mutants are defective in the production of hyphae in the context of a biofilm. Because all of the mutants are defective in the retention of cells in the biofilm, we infer that hyphae provide an adherent scaffold that stabilizes the biofilm structure.     

Functional Mapping of Human Dynamin-1-Like GTPase Domain Based on X-ray Structure Analyses

Human dynamin-1-like protein (DNM1L) is a GTP-driven molecular machine that segregates mitochondria and peroxisomes. To obtain insights into its catalytic mechanism, we determined crystal structures of a construct comprising the GTPase domain and the bundle signaling element (BSE) in the nucleotide-free and GTP-analogue-bound states. The GTPase domain of DNM1L is structurally related to that of dynamin and binds the nucleotide 5′-Guanylyl-imidodiphosphate (GMP-PNP) via five highly conserved motifs, whereas the BSE folds into a pocket at the opposite side. Based on these structures, the GTPase center was systematically mapped by alanine mutagenesis and kinetic measurements. Thus, residues essential for the GTPase reaction were characterized, among them Lys38, Ser39 and Ser40 in the phosphate binding loop, Thr59 from switch I, Asp146 and Gly149 from switch II, Lys216 and Asp218 in the G4 element, as well as Asn246 in the G5 element. Also, mutated Glu81 and Glu82 in the unique 16-residue insertion of DNM1L influence the activity significantly. Mutations of Gln34, Ser35, and Asp190 in the predicted assembly interface interfered with dimerization of the GTPase domain induced by a transition state analogue and led to a loss of the lipid-stimulated GTPase activity. Our data point to related catalytic mechanisms of DNM1L and dynamin involving dimerization of their GTPase domains.