Multiplexed CRISPR/CAS9‐mediated engineering of pre‐clinical mouse models bearing native human B cell receptors

B‐cell receptor (BCR) knock‐in (KI) mouse models play an important role in vaccine development and fundamental immunological studies. However, the time required to generate them poses a bottleneck. Here we report a one‐step CRISPR/Cas9 KI methodology to combine the insertion of human germline immunoglobulin heavy and light chains at their endogenous loci in mice. We validate this technology with the rapid generation of three BCR KI lines expressing native human precursors, instead of computationally inferred germline sequences, to HIV broadly neutralizing antibodies. We demonstrate that B cells from these mice are fully functional: upon transfer to congenic, wild type mice at controlled frequencies, such B cells can be primed by eOD‐GT8 60mer, a germline‐targeting immunogen currently in clinical trials, recruited to germinal centers, secrete class‐switched antibodies, undergo somatic hypermutation, and differentiate into memory B cells. KI mice expressing functional human BCRs promise to accelerate the development of vaccines for HIV and other infectious diseases.     

Influence of differently managed bush‐encroached sites on the large herbivore distribution in the Namibian Savannah

Bush encroachment is reported from savannah regions worldwide. Different management strategies are used to rehabilitate these areas. In this context, the mutual interaction between vegetation and large herbivore's distribution is evident. We studied effects of land management on vegetation structure in regard to encroaching species and the subsequent habitat use of two grazing (oryx, Oryx gazella L.; common warthog, Phacochoerus africanusGmelin ) and one browsing (greater kudu, Tragelaphus strepsicerosPallas ) herbivore species. We assumed that (i) cleared areas will be favoured by grazers and (ii) noncleared areas will be favoured by browsers. Specifically, we asked: Which factors determine the habitat use of these different feeding guilds? Consistently with our expectations, we found that warthog favoured sites with high grass cover. For oryx, surprisingly shrubs with a height of 80–150 cm influenced their distribution positively, whereas for kudu, only the interaction of site and grass cover was significant in our models. However, this was related to the occurrence of shrubs of 80–150 cm height. We conclude that the management of encroachers, resulting in differences in vegetation, did not influence herbivore distribution as expected. Other factors like human impact and vegetation cover among others are discussed as additional drivers of habitat use.     

Reduced food intake and body weight in mice deficient for the G protein-coupled receptor GPR82

G protein-coupled receptors (GPCR) are involved in the regulation of numerous physiological functions. Therefore, GPCR variants may have conferred important selective advantages during periods of human evolution. Indeed, several genomic loci with signatures of recent selection in humans contain GPCR genes among them the X-chromosomally located gene for GPR82. This gene encodes a so-called orphan GPCR with unknown function. To address the functional relevance of GPR82 gene-deficient mice were characterized. GPR82-deficient mice were viable, reproduced normally, and showed no gross anatomical abnormalities. However, GPR82-deficient mice have a reduced body weight and body fat content associated with a lower food intake. Moreover, GPR82-deficient mice showed decreased serum triacylglyceride levels, increased insulin sensitivity and glucose tolerance, most pronounced under Western diet. Because there were no differences in respiratory and metabolic rates between wild-type and GPR82-deficient mice our data suggest that GPR82 function influences food intake and, therefore, energy and body weight balance. GPR82 may represent a thrifty gene most probably representing an advantage during human expansion into new environments.     

Genetic analysis of functions involved in the late stages of biofilm development in Burkholderia cepacia H111

Burkholderia cepacia and Pseudomonas aeruginosa often co-exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here, we report the isolation of 13 random mini-Tn5 insertion mutants of B. cepacia H111 that are defective in biofilm formation on a polystyrene surface. We show that the screening procedure used in this study is biased towards mutants defective in the late stages of biofilm development. A detailed quantitative analysis of the biofilm structures formed by wild-type and mutant strains revealed that the isolated mutants are impaired in their abilities to develop a typical three-dimensional biofilm structure. Molecular investigations showed that the genes required for biofilm maturation fall into several classes: (i). genes encoding for surface proteins; (ii). genes involved in the biogenesis and maintenance of an integral outer membrane; and (iii). genes encoding regulatory factors. It is shown that three of the regulatory mutants produce greatly reduced amounts of N-octanoylhomoserine lactone (C8-HSL). This compound serves as the major signal molecule of the cep quorum-sensing system. As this density-dependent regulatory system is involved in the regulation of biofilm maturation, we investigated the interplay between the three regulatory genes and the quorum-sensing cascade. The results of these investigations show that the identified genes encode for regulatory elements that are positioned upstream of the cep system, indicating that the quorum-sensing system of B. cepacia is a major checkpoint for biofilm formation.     

Characterizing the socially transmitted foraging tactic “sponging” by bottlenose dolphins (Tursiops sp.) in the western gulf of Shark Bay,Western Australia

Individual foraging tactics are widespread in animals and have ecological and evolutionary implications. Indo‐Pacific bottlenose dolphins (Tursiops sp.) in Shark Bay, Western Australia, exhibit a foraging tactic involving tool use, called “sponging.” Sponging is vertically, socially transmitted through the matriline and, to date, has been described in detail in the eastern gulf of Shark Bay (ESB). Here, we characterize sponging in the western gulf of Shark Bay (WSB), in which a different matriline engages in the behavior. We identified 40 individual “spongers” in 9 mo of boat‐based surveys over three field seasons. As is the case in ESB, the majority of WSB spongers was female and engaged in sponging in deep channel habitats. In contrast to ESB, however, there was no difference in the number of associates between spongers and nonspongers in WSB, and activity budgets differed between spongers and deep‐water nonspongers; spongers foraged more frequently and rested less than nonspongers. Group sizes in deep channel habitat, where sponging was prevalent, were typically larger than those in shallow habitat, except for foraging, perhaps indicative of higher predator abundance and/or scattered prey distribution in deep‐water habitat. This research improves our understanding of within‐population foraging variations in bottlenose dolphins.     

Chloroplast DNA-relationship in palaeoaustralLopidium concinnum (Hypopterygiaceae, Musci). An example of stenoevolution in mosses Studies in austral temperate rain forest bryophytes 2

Evolutionary relationship between disjunct populations of the palaeoaustral moss taxonLopidium concinnum (Hypopterygiaceae) from New Zealand and southern South America were studied using non-coding chloroplast DNA sequences. No or only slight changes could be observed within the sequences oftrnT_UGU —trnL_UAA 5exon intergenic spacer,trnL_UAA intron andtrnL_UAA 3exon —trnF_GAA intergenic spacer. This indicates nearly no genetic divergence between extant New Zealand and Chilean populations, i.e. no significant differing pathways of evolution within the 80–60 million years of disrupted areas with interrupted gene flow. Molecular data support the idea of an old Gondwanan relict species of stenoevolutionary character. Ecological data on short-range dispersal strengthen this assessment.     

Effects of genetic variants in ADCY5, GIPR, GCKR and VPS13C on early impairment of glucose and insulin metabolism in children

Objective

Recent genome-wide association studies identified novel candidate genes for fasting and 2 h blood glucose and insulin levels in adults. We investigated the role of four of these loci (ADCY5, GIPR, GCKR and VPS13C) in early impairment of glucose and insulin metabolism in children.

Research Design and Methods

We genotyped four variants (rs2877716; rs1260326; rs10423928; rs17271305) in 638 Caucasian children with detailed metabolic testing including an oGTT and assessed associations with measures of glucose and insulin metabolism (including fasting blood glucose, insulin levels and insulin sensitivity/secretion indices) by linear regression analyses adjusted for age, sex, BMI-SDS and pubertal stage.

Results

The major allele (C) of rs2877716 (ADCY5) was nominally associated with decreased fasting plasma insulin (P = 0.008), peak insulin (P = 0.009) and increased QUICKI (P = 0.016) and Matsuda insulin sensitivity index (P = 0.013). rs17271305 (VPS13C) was nominally associated with 2 h blood glucose (P = 0.009), but not with any of the insulin or insulin sensitivity parameters. We found no association of the GIPR and GCKR variants with parameters of glucose and insulin metabolism. None of the variants correlated with anthropometric traits such as height, WHR or BMI-SDS, which excluded potential underlying associations with obesity.

Conclusions

Our data on obese children indicate effects of genetic variation within ADCY5 in early impairment of insulin metabolism and VPS13C in early impairment of blood glucose homeostasis.     

Langfristige Auswirkungen ehemaliger Bewirtschaftungsvarianten auf die Diversit?t von Blütenpflanzen, Moosen und Flechten eines Kalkmagerrasens

Jeschke M., Kiehl K., Pfadenhauer J. and Gigon A. 2008. Long-term effects of former management on the diversity of vascular plants, mosses and lichens in a calcareous grassland. Bot. Helv. 118: 95 – 109. Long-term effects of grassland management on the species richness of vascular plants, mosses and lichens were studied in a calcareous grassland near Schaffhausen (Switzerland). Experimental plots were treated for 22 years (1977 – 1999) with different mowing regimes, annual burning in late winter, or abandonment. From 2000 onwards, all plots were mown annually in autumn. Five years later (2004), we studied the after-effects of the previous treatments by recording the cover of vascular plants, mosses and lichens in nested plots with sizes from 0.01 m² to 16 m². Vascular plant species richness as well as the number of species characteristic for calcareous grasslands were significantly lower in formerly abandoned or burnt plots than in regularly mown plots, with larger differences on small subplots. There was no significant effect of the previous mowing time (July vs. October) or mowing frequency (annual or every two years) on vascular plants. Mosses and lichens had been eliminated by annual burning between 1977 and 1999, and only few moss species re-colonized the formerly burnt plots until 2004. The species richness of mosses did not differ between formerly abandoned and mown plots, but there were more mesophytic and fewer xerophytic species in the formerly abandoned plots. Mesophytic mosses were also more abundant in plots that used to be mown every second year than in those mown annually. Our data indicate that even after five years of similar management, i.e. annual mowing in autumn, the previous long-term management had strong after-effects on species richness and species composition of calcareous grasslands. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Eingereicht am 19. M?rz 2008, Angenommen am 30. August 2008, Redaktion: Sabine Güsewell