A truncated RHAMM protein for discovering novel therapeutic peptides

The receptor for hyaluronan mediated motility (RHAMM, gene name HMMR) belongs to a group of proteins that bind to hyaluronan (HA), a high-molecular weight anionic polysaccharide that has pro-angiogenic and inflammatory properties when fragmented. We propose to use a chemically synthesized, truncated version of the protein (706–767), 7?kDa RHAMM, as a target receptor in the screening of novel peptide-based therapeutic agents. Chemical synthesis by Fmoc-based solid-phase peptide synthesis, and optimization using pseudoprolines, results in RHAMM protein of higher purity and yield than synthesis by recombinant protein production. 7?kDa RHAMM was evaluated for its secondary structure, ability to bind the native ligand, HA, and its bioactivity. This 62-amino acid polypeptide replicates the HA binding properties of both native and recombinant RHAMM protein. Furthermore, tubulin-derived HA peptide analogues that bind to recombinant RHAMM and were previously reported to compete with HA for interactions with RHAMM, bind with a similar affinity and specificity to the 7?kDa RHAMM. Therefore, in terms of its key binding properties, the 7?kDa RHAMM mini-protein is a suitable replacement for the full-length recombinant protein.     

Plant diversity does not buffer drought effects on early‐stage litter mass loss rates and microbial properties

Human activities are decreasing biodiversity and changing the climate worldwide. Both global change drivers have been shown to affect ecosystem functioning, but they may also act in concert in a non‐additive way. We studied early‐stage litter mass loss rates and soil microbial properties (basal respiration and microbial biomass) during the summer season in response to plant species richness and summer drought in a large grassland biodiversity experiment, the Jena Experiment, Germany. In line with our expectations, decreasing plant diversity and summer drought decreased litter mass loss rates and soil microbial properties. In contrast to our hypotheses, however, this was only true for mass loss of standard litter (wheat straw) used in all plots, and not for plant community‐specific litter mass loss. We found no interactive effects between global change drivers, that is, drought reduced litter mass loss rates and soil microbial properties irrespective of plant diversity. High mass loss rates of plant community‐specific litter and low responsiveness to drought relative to the standard litter indicate that soil microbial communities were adapted to decomposing community‐specific plant litter material including lower susceptibility to dry conditions during summer months. Moreover, higher microbial enzymatic diversity at high plant diversity may have caused elevated mass loss of standard litter. Our results indicate that plant diversity loss and summer drought independently impede soil processes. However, soil decomposer communities may be highly adapted to decomposing plant community‐specific litter material, even in situations of environmental stress. Results of standard litter mass loss moreover suggest that decomposer communities under diverse plant communities are able to cope with a greater variety of plant inputs possibly making them less responsive to biotic changes.     

Midgut Microbiota of the Malaria Mosquito Vector Anopheles gambiae and Interactions with Plasmodium falciparum Infection

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.     

The Rb tumor suppressor is required for stress erythropoiesis

The retinoblastoma tumor suppressor gene plays important roles in cell cycle control, differentiation and survival during development and is functionally inactivated in most human cancers. Early studies using gene targeting in mice suggested a critical role for pRb in erythropoiesis, while more recent experiments have suggested that many of the abnormal embryonic phenotypes in the Rb null mouse result from a defective placenta. To address this controversy and determine whether Rb has cell intrinsic functions in erythropoiesis, we examined the effects of Rb loss on red cell production following acute deletion of pRb in vitro and under different stress conditions in vivo. Under stress conditions, pRb was required to regulate erythroblast expansion and promote red cell enucleation. Acute deletion of Rb in vitro induced erythroid cell cycle and differentiation defects similar to those observed in vivo. These results demonstrate a cell intrinsic role for pRb in stress erythropoiesis and hematopoietic homeostasis that has relevance for human diseases.     

Augmented Reticular Thalamic Bursting and Seizures in Scn1a-Dravet Syndrome

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Evidence for a major gene (RP10) for autosomal dominant retinitis pigmentosa on chromosome 7q: linkage mapping in a second,unrelated family

Retinitis pigmentosa is a genetically heterogeneous form of retinal degeneration, which has X-linked, autosomal recessive and autosomal dominant forms. The disease genes in families with autosomal dominant retinitis pigmentosa (adRP) have been linked to six loci, on 3q, 6p, 7p, 7q, 8q and 19q. In a large American family with late-onset adRP, microsatellite markers were used to test for linkage to the loci on 3q, 6p, 7p, 7q and 8q. Linkage was found to 7q using the marker D7S480. Additional microsatellite markers from 7q were then tested. In total, five markers, D7S480, D7S514, D7S633, D7S650 and D7S677, show statistically significant evidence for link-age in this family, with a maximum two-point lod score of 5.3 at 0% recombination from D7S514. These results confirm an earlier report of linkage to an adRP locus (RP10) in an unrelated family of Spanish origin and indicate that RP10 may be a significant gene for inherited retinal degeneration. In addition, we used recently reported microsatellite markers from 7q to refine the linkage map of the RP10 locus.     

Cultured subventricular zone progenitor cells transduced with neurogenin-2 become mature glutamatergic neurons and integrate into the dentate gyrus

We have previously shown that transplantation of immature DCX+/NeuN+/Prox1+ neurons (found in the neonatal DG), but not undifferentiated neuronal progenitor cells (NPCs) from ventral subventricular zone (SVZ), results in neuronal maturation in vivo within the dentate niche. Here we investigated whether we could enhance the integration of SVZ NPCs by forced expression of the proneural gene Neurogenin 2 (NEUROG2). NPCs cultured from neonatal GFP-transgenic rat SVZ for 7 days in a non-differentiating medium were transduced with a retrovirus encoding NEUROG2 and DsRed or the DsRed reporter gene alone (control). By 3 days post-transduction, the NEUROG2-transduced cells maintained in culture contained mostly immature neurons (91% DCX+; 76% NeuN+), whereas the control virus-transduced cells remained largely undifferentiated (30% DCX+; <1% NeuN+). At 6 weeks following transplantation into the DG of adult male rats, there were no neurons among the transplanted cells treated with the control virus but the majority of the NEUROG2-transduced DsRed+ SVZ cells became mature neurons (92% NeuN+; DCX-negative). Although the NEUROG2-transduced SVZ cells did not express the dentate granule neuron marker Prox1, most of the NEUROG2-transduced SVZ cells (78%) expressed the glutamatergic marker Tbr1, suggesting the acquisition of a glutamatergic phenotype. Moreover, some neurons extended dendrites into the molecular layer, grew axons containing Ankyrin G+ axonal initial segments, and projected into the CA3 region, thus resembling mature DG granule neurons. A proportion of NEUROG2 transduced cells also expressed c-Fos and P-CREB, two markers of neuronal activation. We conclude that NEUROG2-transduction is sufficient to promote neuronal maturation and integration of transplanted NPCs from SVZ into the DG.     

Specific regions within the embryonic midbrain and cerebellum require different levels of FGF signaling during development

Prospective midbrain and cerebellum formation are coordinated by FGF ligands produced by the isthmic organizer. Previous studies have suggested that midbrain and cerebellum development require different levels of FGF signaling. However, little is known about the extent to which specific regions within these two parts of the brain differ in their requirement for FGF signaling during embryogenesis. Here, we have explored the effects of inhibiting FGF signaling within the embryonic mouse midbrain (mesencephalon) and cerebellum (rhombomere 1) by misexpressing sprouty2 (Spry2) from an early stage. We show that such Spry2 misexpression moderately reduces FGF signaling, and that this reduction causes cell death in the anterior mesencephalon, the region furthest from the source of FGF ligands. Interestingly, the remaining mesencephalon cells develop into anterior midbrain, indicating that a low level of FGF signaling is sufficient to promote only anterior midbrain development. Spry2 misexpression also affects development of the vermis, the part of the cerebellum that spans the midline. We found that, whereas misexpression of Spry2 alone caused loss of the anterior vermis, reducing FGF signaling further, by decreasing Fgf8 gene dose, resulted in loss of the entire vermis. Our data suggest that cell death is not responsible for vermis loss, but rather that it fails to develop because reducing FGF signaling perturbs the balance between vermis and roof plate development in rhombomere 1. We suggest a molecular explanation for this phenomenon by providing evidence that FGF signaling functions to inhibit the BMP signaling that promotes roof plate development.