Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease

We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.     

Immunisation against a serine protease inhibitor reduces intensity of Plasmodium berghei infection in mosquitoes

The mosquito innate immune response is able to clear the majority of Plasmodium parasites. This immune clearance is controlled by a number of regulatory molecules including serine protease inhibitors (serpins). To determine whether such molecules could represent a novel target for a malaria transmission-blocking vaccine, we vaccinated mice with Anopheles gambiae serpin-2. Antibodies against Anopheles gambiae serpin-2 significantly reduced the infection of a heterologous Anopheles species (Anopheles stephensi) by Plasmodium berghei, however this effect was not observed with Plasmodium falciparum. Therefore, this approach of targeting regulatory molecules of the mosquito immune system may represent a novel approach to transmission-blocking malaria vaccines.     

In vitro structure–activity relationships of aplysinopsin analogs and their in vivo evaluation in the chick anxiety–depression model

Aplysinopsins are tryptophan-derived natural products that have been isolated from a variety of marine organisms and have been shown to possess a range of biological activities. In vitro receptor binding assays showed that of the 12 serotonin receptor subtypes, analogues showed a high affinity for the 5-HT_2B and 5-HT_2C receptor subtypes, with selectivity for 5-HT_2B over 5-HT_2C. While no conclusions could be drawn about the number and position of N-methylations, bromination at C-4 and C-5 of the indole ring resulted in greater binding affinities, with K_i’s as low as 35 nM. This data, combined with previous knowledge of the CNS activity of aplysinopsin analogs, suggested that these compounds may have potential as leads for antidepressant drugs. Compounds 3c, 3u, and 3x were evaluated in the chick anxiety–depression model to assess their in vivo efficacy. Compound 3c showed a modest antidepressant effect at a dose of 30 nM/kg in the animal model.     

Microsatellite and mtDNA analysis of lake trout,Salvelinus namaycush,from Great Bear Lake,Northwest Territories: impacts of historical and contemporary evolutionary forces on Arctic ecosystems

Resolving the genetic population structure of species inhabiting pristine, high latitude ecosystems can provide novel insights into the post‐glacial, evolutionary processes shaping the distribution of contemporary genetic variation. In this study, we assayed genetic variation in lake trout (Salvelinus namaycush) from Great Bear Lake (GBL), NT and one population outside of this lake (Sandy Lake, NT) at 11 microsatellite loci and the mtDNA control region (d‐loop). Overall, population subdivision was low, but significant (global F_ST θ = 0.025), and pairwise comparisons indicated that significance was heavily influenced by comparisons between GBL localities and Sandy Lake. Our data indicate that there is no obvious genetic structure among the various basins within GBL (global F_ST = 0.002) despite the large geographic distances between sampling areas. We found evidence of low levels of contemporary gene flow among arms within GBL, but not between Sandy Lake and GBL. Coalescent analyses suggested that some historical gene flow occurred among arms within GBL and between GBL and Sandy Lake. It appears, therefore, that contemporary (ongoing dispersal and gene flow) and historical (historical gene flow and large founding and present‐day effective population sizes) factors contribute to the lack of neutral genetic structure in GBL. Overall, our results illustrate the importance of history (e.g., post‐glacial colonization) and contemporary dispersal ecology in shaping genetic population structure of Arctic faunas and provide a better understanding of the evolutionary ecology of long‐lived salmonids in pristine, interconnected habitats.     

The Genome Sequences of Cellulomonas fimi and “Cellvibrio gilvus” Reveal the Cellulolytic Strategies of Two Facultative Anaerobes,Transfer of “Cellvibrio gilvus” to the Genus Cellulomonas,and Proposal of Cellulomonas gilvus sp. nov

Actinobacteria in the genus Cellulomonas are the only known and reported cellulolytic facultative anaerobes. To better understand the cellulolytic strategy employed by these bacteria, we sequenced the genome of the Cellulomonas fimi ATCC 484^T. For comparative purposes, we also sequenced the genome of the aerobic cellulolytic “Cellvibrio gilvus” ATCC 13127^T. An initial analysis of these genomes using phylogenetic and whole-genome comparison revealed that “Cellvibrio gilvus” belongs to the genus Cellulomonas. We thus propose to assign “Cellvibrio gilvus” to the genus Cellulomonas. A comparative genomics analysis between these two Cellulomonas genome sequences and the recently completed genome for Cellulomonas flavigena ATCC 482^T showed that these cellulomonads do not encode cellulosomes but appear to degrade cellulose by secreting multi-domain glycoside hydrolases. Despite the minimal number of carbohydrate-active enzymes encoded by these genomes, as compared to other known cellulolytic organisms, these bacteria were found to be proficient at degrading and utilizing a diverse set of carbohydrates, including crystalline cellulose. Moreover, they also encode for proteins required for the fermentation of hexose and xylose sugars into products such as ethanol. Finally, we found relatively few significant differences between the predicted carbohydrate-active enzymes encoded by these Cellulomonas genomes, in contrast to previous studies reporting differences in physiological approaches for carbohydrate degradation. Our sequencing and analysis of these genomes sheds light onto the mechanism through which these facultative anaerobes degrade cellulose, suggesting that the sequenced cellulomonads use secreted, multidomain enzymes to degrade cellulose in a way that is distinct from known anaerobic cellulolytic strategies.