Identification of an inhibitory domain in RAG-2 that gates access of the V(D)J recombinase to chromatin

V(D)J recombination is initiated by a specialized transposase consisting of the subunits RAG-1 and RAG-2. The susceptibility of gene segments to DNA cleavage by the V(D)J recombinase is correlated with epigenetic modifications characteristic of active chromatin, including trimethylation of histone H3 on lysine 4 (H3K4me3). Engagement of H3K4me3 by a plant homeodomain (PHD) in RAG-2 promotes recombination in vivo and stimulates DNA cleavage by RAG in vitro. We characterized features of this PHD-mediated inhibitory domain and identified a second, independent, inhibitory domain. Mutation of this second inhibitory domain allows bypass of the requirement for engagement of H3K4me3 by the RAG-2 PHD. Disruption of this inhibitory domain was associated with constitutive increases in recombination frequency, DNA cleavage activity, substrate binding affinity, and catalytic rate. We further characterized this domain genetically and determined that the inhibitory function is imposed by acidic residues between residues 352 and 405. Further, we were able to demonstrate that the inhibitory domain mutation is refractory to removal of the entire noncore portion of RAG-2. Therefore, this inhibitory domain acts independently of the PHD. Inactivation of the inhibitory domain confers a gain-of-function recombination phenotype and permits rearrangement at endogenous IgH and Igκ loci in the absence of H3K4me3 binding. In B progenitor cells, localization of wild-type RAG-2 to the IgH locus and actively transcribed loci is abolished by the W453A mutation, indicating that this pattern of chromatin localization is dependent on recognition of H3K4me3. This same mutation also abolishes association of RAG-1 with the IgH locus. Strikingly, disruption of the inhibitory domain permits association of RAG-2 and RAG-1 with the IgH locus even in the absence of H3K4me3 engagement by RAG-2. Thus, the RAG-2 inhibitory domain serves as a binary gate that permits the association of RAG-1 and RAG-2 with chromatin only if H3K4me3 is engaged by the RAG-2 PHD finger

An Autoregulatory Mechanism Imposes Allosteric Control on the V(D)J Recombinase by Histone H3 Methylation

SummaryV(D)J recombination is initiated by a specialized transposase consisting of the subunits RAG-1 and RAG-2. The susceptibility of gene segments to DNA cleavage by the V(D)J recombinase is correlated with epigenetic modifications characteristic of active chromatin, including trimethylation of histone H3 on lysine 4 (H3K4me3). Engagement of H3K4me3 by a plant homeodomain (PHD) in RAG-2 promotes recombination in vivo and stimulates DNA cleavage by RAG in vitro. We now show that H3K4me3 acts allosterically at the PHD finger to relieve autoinhibition imposed by a separate domain within RAG-2. Disruption of this autoinhibitory domain was associated with constitutive increases in recombination frequency, DNA cleavage activity, substrate binding affinity, and catalytic rate, thus mimicking the stimulatory effects of H3K4me3. Our observations support a model in which allosteric control of RAG is enforced by an autoinhibitory domain whose action is relieved by engagement of active chromatin

Interactions among chronic and acute impacts on coral recruits: the importance of size-escape thresholds

Newly settled recruits typically suffer high mortality from disturbances, but rapid growth reduces their mortality once size-escape thresholds are attained. Ocean acidification (OA) reduces the growth of recruiting benthic invertebrates, yet no direct effects on survivorship have been demonstrated. We tested whether the reduced growth of coral recruits caused by OA would increase their mortality by prolonging their vulnerability to an acute disturbance: fish herbivory on surrounding algal turf. After two months' growth in ambient or elevated CO levels, the linear extension and calcification of coral (Acropora millepora) recruits decreased as CO partial pressure (pCO) increased. When recruits were subjected to incidental fish grazing, their mortality was inversely size dependent. However, we also found an additive effect of pCO such that recruit mortality was higher under elevated pCO irrespective of size. Compared to ambient conditions, coral recruits needed to double their size at the highest pCOto escape incidental grazing mortality. This general trend was observed with three groups of predators (blenny, surgeonfish, and parrotfish), although the magnitude of the fish treatment varied among species. Our study demonstrates the importance of size-escape thresholds in early recruit survival and how OA can shift these thresholds, potentially intensifying population bottlenecks in benthic invertebrate recruitment

Insulin-like Growth Factor 1 and Transforming Growth Factor-β Stimulate Cystine/Glutamate Exchange Activity in Dental Pulp Cells

Introduction The growth factors insulin-like growth factor (IGF-1) and transforming growth factor-β (TGF-β) are protective to dental pulp cells in culture against the toxicity of the composite materials Durafill VS and Flow Line (Henry Schein Inc, New York, NY). Because the toxicity of these materials is mediated by oxidative stress, it seemed possible that the protective effects of IGF-1 and TGF-β were through the enhancement of an endogenous antioxidant mechanism. Methods We used cultured dental pulp cells to determine the mechanism of the protective effects of IGF-1 and TGF-β, focusing on the glutathione system and the role of cystine/glutamate exchange (system xc-). Results We found that the toxicity of Durafill VS and Flow Line was attenuated by the addition of glutathione monoethylester, suggesting a specific role for the cellular antioxidant glutathione. Supporting this hypothesis, we found that IGF-1 and TGF-β were protective against the toxicity of the glutathione synthesis inhibitor buthionine sulfoximine. Because levels of cellular cystine are the limiting factor in the production of glutathione, we tested the effects of IGF-1 and TGF-β on cystine uptake. Both growth factors stimulated system xc–mediated cystine uptake. Furthermore, they attenuated the glutathione depletion induced by Durafill VS and Flow Line. Conclusions The results suggest that IGF-1 and TGF-β are protective through the stimulation of system xc–mediated cystine uptake, leading to maintenance of cellular glutathione. This novel action of growth factors on dental pulp cells has implications not only for preventing toxicity of dental materials but also for the general function of these cells

Anion-π Catalysis of Enolate Chemistry: Rigidified Leonard Turns as a General Tool to Run Reactions on Aromatic Surfaces

To integrate anion–π, cation–π, and ion pair–π interactions in catalysis, the fundamental challenge is to run reactions reliably on aromatic surfaces. Addressing a specific question concerning enolate addition to nitroolefins, this study elaborates on Leonard turns to tackle this problem in a general manner. Increasingly refined turns are constructed to position malonate half thioesters as close as possible on π-acidic surfaces. The resulting preorganization of reactive intermediates is shown to support the disfavored addition to enolate acceptors to an absolutely unexpected extent. This decisive impact on anion–π catalysis increases with the rigidity of the turns. The new, rigidified Leonard turns are most effective with weak anion–π interactions, whereas stronger interactions do not require such ideal substrate positioning to operate well. The stunning simplicity of the motif and its surprisingly strong relevance for function should render the introduced approach generally useful

The marine fish food web is globally connected

The productivity of marine ecosystems and the services they provide to humans are largely dependent on complex interactions between prey and predators. These are embedded in a diverse network of trophic interactions, resulting in a cascade of events following perturbations such as species extinction. The sheer scale of oceans, however, precludes the characterization of marine feeding networks through de novo sampling. This effort ought instead to rely on a combination of extensive data and inference. Here we investigate how the distribution of trophic interactions at the global scale shapes the marine fish food web structure. We hypothesize that the heterogeneous distribution of species ranges in biogeographic regions should concentrate interactions in the warmest areas and within species groups. We find that the inferred global metaweb of marine fish—that is, all possible potential feeding links between co-occurring species—is highly connected geographically with a low degree of spatial modularity. Metrics of network structure correlate with sea surface temperature and tend to peak towards the tropics. In contrast to open-water communities, coastal food webs have greater interaction redundancy, which may confer robustness to species extinction. Our results suggest that marine ecosystems are connected yet display some resistance to perturbations because of high robustness at most locations.Using a global interaction dataset, the authors quantify the distribution of trophic interactions among marine fish, finding a high degree of geographic connectivity but low spatial modularity.C.A. was supported by a MELS-FQRNT Postdoctoral Fellowship and a Ressources Aquatique Québec (RAQ) fellowship during the conception and writing of this manuscript. T.P., D.G. and D.B.S. acknowledge financial support by the CIEE through their working group programme. M.B.A. is funded through FCT project No. PTDC/AAG-MAA/3764/2014. A.R.C. is funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) PGS-D scholarship. D.G., T.P., M.-J.F., P.A. and S.J.L. are supported by NSERC Discovery Grants. T.P. also acknowledges a FRQNT New Investigator award and a Université de Montréal starting grant. D.B.S. acknowledges support from the Royal Society of New Zealand (via Marsden Fast-Start No. UOC-1101 and a Rutherford Discovery Fellowship)

Refinement of a 400-kb Critical Region Allows Genotypic Differentiation between Isolated Lissencephaly, Miller-Dieker Syndrome, and Other Phenotypes Secondary to Deletions of 17p13.3

Deletions of 17p13.3, including the LIS1 gene, result in the brain malformation lissencephaly, which is characterized by reduced gyration and cortical thickening; however, the phenotype can vary from isolated lissencephaly sequence (ILS) to Miller-Dieker syndrome (MDS). At the clinical level, these two phenotypes can be differentiated by the presence of significant dysmorphic facial features and a more severe grade of lissencephaly in MDS. Previous work has suggested that children with MDS have a larger deletion than those with ILS, but the precise boundaries of the MDS critical region and causative genes other than LIS1 have never been fully determined. We have completed a physical and transcriptional map of the 17p13.3 region from LIS1 to the telomere. Using fluorescence in situ hybridization, we have mapped the deletion size in 19 children with ILS, 11 children with MDS, and 4 children with 17p13.3 deletions not involving LIS1. We show that the critical region that differentiates ILS from MDS at the molecular level can be reduced to 400 kb. Using somatic cell hybrids from selected patients, we have identified eight genes that are consistently deleted in patients classified as having MDS. In addition, deletion of the genes CRK and 14-3-3ɛ delineates patients with the most severe lissencephaly grade. On the basis of recent functional data and the creation of a mouse model suggesting a role for 14-3-3ɛ in cortical development, we suggest that deletion of one or both of these genes in combination with deletion of LIS1 may contribute to the more severe form of lissencephaly seen only in patients with MDS

Toward a Continuous Intravascular Glucose Monitoring System

Proof-of-concept studies that display the potential of using a glucose-sensitive hydrogel as a continuous glucose sensor are presented. The swelling ratio, porosity, and diffusivity of the hydrogel increased with glucose concentration. In glucose solutions of 50, 100, 200, and 300 mg/dL, the hydrogel swelling ratios were 4.9, 12.3, 15.9, and 21.7, respectively, and the swelling was reversible. The impedance across the hydrogel depended solely on the thickness and had an average increase of 47 Ω/mm. The hydrogels exposed to a hyperglycemic solution were more porous than the hydrogels exposed to a normal glycemic solution. The diffusivity of 390 Da MW fluorescein isothiocyanate in hydrogels exposed to normal and hyperglycemic solutions was examined using fluorescence recovery after photobleaching and was found to be 9.3 × 10−14 and 41.4 × 10−14 m2/s, respectively, compared to 6.2 × 10−10 m2/s in glucose solution. There was no significant difference between the permeability of hydrogels in normal and hyperglycemic glucose solutions with averages being 5.26 × 10−17 m2 and 5.80 × 10−17 m2, respectively, which resembles 2–4% agarose gels. A prototype design is presented for continuous intravascular glucose monitoring by attaching a glucose sensor to an FDA-approved stent