Using defined finger–finger interfaces as units of assembly for constructing zinc-finger nucleases

Zinc-finger nucleases (ZFNs) have been used for genome engineering in a wide variety of organisms; however, it remains challenging to design effective ZFNs for many genomic sequences using publicly available zinc-finger modules. This limitation is in part because of potential finger–finger incompatibility generated on assembly of modules into zinc-finger arrays (ZFAs). Herein, we describe the validation of a new set of two-finger modules that can be used for building ZFAs via conventional assembly methods or a new strategy—finger stitching—that increases the diversity of genomic sequences targetable by ZFNs. Instead of assembling ZFAs based on units of the zinc-finger structural domain, our finger stitching method uses units that span the finger–finger interface to ensure compatibility of neighbouring recognition helices. We tested this approach by generating and characterizing eight ZFAs, and we found their DNA-binding specificities reflected the specificities of the component modules used in their construction. Four pairs of ZFNs incorporating these ZFAs generated targeted lesions in vivo, demonstrating that stitching yields ZFAs with robust recognition properties.     

Characterization of the interaction between Robo1 and heparin and other glycosaminoglycans

Roundabout 1 (Robo1) is the cognate receptor for secreted axon guidance molecule, Slits, which function to direct cellular migration during neuronal development and angiogenesis. The Slit2–Robo1 signaling is modulated by heparan sulfate, a sulfated linear polysaccharide that is abundantly expressed on the cell surface and in the extracellular matrix. Biochemical studies have further shown that heparan sulfate binds to both Slit2 and Robo1 facilitating the ligand–receptor interaction. The structural requirements for heparan sulfate interaction with Robo1 remain unknown. In this report, surface plasmon resonance (SPR) spectroscopy was used to examine the interaction between Robo1 and heparin and other GAGs and determined that heparin binds to Robo1 with an affinity of ∼650 nM. SPR solution competition studies with chemically modified heparins further determined that although all sulfo groups on heparin are important for the Robo1–heparin interaction, the N-sulfo and 6-O-sulfo groups are essential for the Robo1–heparin binding. Examination of differently sized heparin oligosaccharides and different GAGs also demonstrated that Robo1 prefers to bind full-length heparin chains and that GAGs with higher sulfation levels show increased Robo1 binding affinities.     

Analysis of agonism by dopamine at the dopaminergic D 2 G-protein coupled receptor based on comparative modelling of rhodopsin

A number of theoretical dopaminergic D 2 models were constructed using comparative modelling techniques. The models constructed were based on the recently published crystal structure of rhodopsin. D 2 models were constructed without any ligands bound in the active site and also with the endogenous agonist, dopamine, bound in the active site. Comparison of the bound and unbound models revealed the importance of the chi angle of serine 197 and the effect this has on the bending of helix 5 when an agonist is present in the binding site.     

CHITIN — A promising biomaterial for tissue engineering and stem cell technologies

Chitin, after cellulose, is the second most abundant natural polymer. With a 200-year history of scientific research, chitin is beginning to see fruitful application in the fields of stem cell and tissue engineering. To date, however, research in chitin as a biomaterial appears to lag far behind that of its close relative, chitosan, due to the perceived difficulty in processing chitin. This review presents methods to improve the processability of chitin, and goes on further to discuss the unique physicochemical and biological characteristics of chitin that favor it as a biomaterial for regenerative medicine applications. Examples of the latter are presented, with special attention on the qualities of chitin that make it inherently suitable as scaffolds and matrices for tissue engineering, stem cell propagation and differentiation.     

A nitrogen fertilization field study of carbon-13 and nitrogen-15 transfers in ectomycorrhizas of Pinus sabiniana

Ectomycorrhizal (EM) fungi form relationships with higher plants; plants transfer C to fungi, and fungi transfer nutrients to their host. While evidence indicates that this interaction is largely mutualistic, less is known about how nutrient supply and EM associates may alter C and nutrient exchanges, especially in intact plant-soil-microbe systems in the field. In a dual-labeling experiment with N fertilization, we used C and N stable isotopes to examine in situ transfers in EM pine trees in a Pinus sabiniana woodland in northern California. We added ¹⁵NH₄SO₂ and ¹³CO₂ to track ¹³C transfer from pine needles to EM roots and ¹⁵N transfer from soil to EM roots and pine needles. Transfers of ¹³C and ¹⁵N differed with EM morphotype and with N fertilization. The brown morphotype received the least C per unit of N transferred (5:1); in contrast red and gold morphotypes gained more C and transferred less N (17:1 and 25:1, respectively). N fertilization increased N retention by ectomycorrhizas (EMs) but did not increase N transfer from EMs to pine needles. Therefore N fertilization positively affected both nutrient and C gains by EMs, increasing net C flows and N retention in EMs. Our work on intact and native trees/EM associations thereby extends earlier conclusions based on pot studies with young plants and culturable EM fungi; our results support the concept that EM-host relationships depend on species-level differences as well as responses to soil resources such as N.     

Soil nutrients trump intraspecific effects on understory plant communities

Understanding the links between intraspecific genetic variation and patterns of diversity in associated communities has been the primary focus of community genetics or ‘genes-to-ecosystem’ research in ecology. While other ecological factors, such as the abiotic environment, have well-documented influences on communities, the relative contributions of genetic variation versus the environment to species interactions remains poorly explored. In this study, we use a common garden experiment to study a coastal dune plant community dominated by the shrub, Baccharis pilularis, which displays a morphological dimorphism in plant architecture. We found the differences in the understory plant community between erect and prostrate morphs of Baccharis to be statistically significant, but small relative to the impacts of nutrient additions (NPK and C additions), for the richness, cover, and biomass of the understory plant community. There were no significant interactions between Baccharis morphology and nutrient-addition treatments, suggesting the influence of nutrient addition was consistent between erect and prostrate morphs. Moreover, we found no difference in overall plant community composition between Baccharis morphs, while NPK additions led to shifts in understory community composition compared to unfertilized shrubs. In sum, our results indicate that nutrients are the more important factor governing understory plant community structure in a coastal dunes ecosystem followed by intraspecific variation in dominant shrub architecture. Our results address a growing call to understand the extended consequences of intraspecific variation across heterogeneous environments in terrestrial ecosystems.