Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability

Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.     

Using ecological networks to answer questions in global biogeography and ecology

Ecological networks have classically been studied at site and landscape scales, yet recent efforts have been made to collate these data into global repositories. This offers an opportunity to integrate and upscale knowledge about ecological interactions from local to global scales to gain enhanced insights from the mechanistic information provided by these data. By drawing on existing research investigating patterns in ecological interactions at continental to global scales, we show how data on ecological networks, collected at appropriate scales, can be used to generate an improved understanding of many aspects of ecology and biogeography—for example, species distribution modelling, restoration ecology and conservation. We argue that by understanding the patterns in the structure and function of ecological networks across scales, it is possible to enhance our understanding of the natural world.     

A Fish Eye Out of Water: Ten Visual Opsins in the Four-Eyed Fish,Anableps anableps

The “four-eyed” fish Anableps anableps has numerous morphological adaptations that enable above and below-water vision. Here, as the first step in our efforts to identify molecular adaptations for aerial and aquatic vision in this species, we describe the A. anableps visual opsin repertoire. We used PCR, cloning, and sequencing to survey cDNA using unique primers designed to amplify eight sequences from five visual opsin gene subfamilies, SWS1, SWS2, RH1, RH2, and LWS. We also used Southern blotting to count opsin loci in genomic DNA digested with EcoR1 and BamH1. Phylogenetic analyses confirmed the identity of all opsin sequences and allowed us to map gene duplication and divergence events onto a tree of teleost fish. Each of the gene-specific primer sets produced an amplicon from cDNA, indicating that A. anableps possessed and expressed at least eight opsin genes. A second PCR-based survey of genomic and cDNA uncovered two additional LWS genes. Thus, A. anableps has at least ten visual opsins and all but one were expressed in the eyes of the single adult surveyed. Among these ten visual opsins, two have key site haplotypes not found in other fish. Of particular interest is the A. anableps-specific opsin in the LWS subfamily, S180γ, with a SHYAA five key site haplotype. Although A. anableps has a visual opsin gene repertoire similar to that found in other fishes in the suborder Cyprinodontoidei, the LWS opsin subfamily has two loci not found in close relatives, including one with a key site haplotype not found in any other fish species. A. anableps opsin sequence data will be used to design in situ probes allowing us to test the hypothesis that opsin gene expression differs in the distinct ventral and dorsal retinas found in this species.     

Capitalizing on multi-element interactions through bal-anced nutrition——A pathway to improve nitrogen use efficiency in China,India and North America

A viable option for increasing nitrogen (N) use efficiency and mitigation of negative impacts of N on the environment is to capitalize on multi-element interactions through implementation of nutrient management programs that provide balanced nutrition. Numerous studies have demonstrated the immediate efficacy of this approach in the developing regions like China and India as well as developed countries in North America. Based on 241 site-years of experiments in these countries, the first-year N recovery efficiency (RE) for the conventional or check treatments averaged 21% while the balanced treatments averaged 54% RE, for an average increase of 33% in RE due to balanced nutrition. Effective policies to promote adoption are most likely those that enable site-specific approaches to nutrient management decisions rather than sweeping, nation-wide incentives supporting one nutrient over another. Local farmers, advisers and officials need to be empowered with tools and information to help them define necessary changes in practices to create more balanced nutrient management.     

Screening of plant growth promotion ability among bacteria isolated from field-grown sorghum under different managements in Brazilian drylands

Sorghum [Sorghum bicolor (L.) Moench] is a multipurpose grass cultivated in drylands due to its adaptation to drought. However the characteristics of sorghum-associated bacteria are not known in the Brazilian drylands. The aim of this study was to isolate and evaluate the plant growth promotion potential bacteria from field-grown sorghum under two irrigation and manure application levels in a Brazilian semi-arid reagion. Sorghum was irrigated with 3 or 1 mm day^?1 and fertilized or not with liquid goat manure. Bacteria were obtained from surface-disinfected roots applying two nitrogen-free semi-solid media. The bacteria were evaluated for the presence of nifH gene, 16S rRNA sequences, calcium-phosphate solubilization, production of auxins and siderophores and for sorghum growth promotion. We obtained 20 out of 24 positive bacteria for nifH. The isolates were classified as in six different genera. All isolates produced auxins “in vitro”, six bacteria produced siderophores and three Enterobacteriaceae solubilized calcium-phosphate. At least ten bacteria resulted in the increased total N content in the sorghum shoots, comparable to fertilization with 50 mg N plant^?1 week^?1 and to inoculation with Azospirillum brasilense Ab-V5. Enterobacter sp. ESA 57 was the best sorghum plant-growth promoting bacteria isolated in this study.     

Primary photochemical processes in isolated reaction centers of Rhodopseudomonas viridis

Picosecond and nanosecond spectroscopic techniques have been used to study the primary electron transfer processes in reaction centers isolated from the photosynthetic bacterium Rhodopseudomonas viridis. Following flash excitation, the first excited singlet state (P1) of the bacteriochlorophyll complex (P) transfers an electron to an intermediate acceptor (I) in less than 20 ps. The radical pair state (P⁺I^?) subsequently transfers an electron to another acceptor (X) in about 230 ps. There is an additional step of unknown significance exhibiting 35 ps kinetics. P⁺ subsequently extracts an electron from a cytochrome, with a time constant of about 270 ns. At low redox potential (X reduced before the flash), the state P⁺I^? (or P^F) lives approx. 15 ns. It decays, in part, into a longer lived state (P^R), which appears to be a triplet state. State P^R decays with an exponential time of approx. 55 μs. After continuous illumination at low redox potential (I and X both reduced), excitation with an 8-ps flash produces absorption changes reflecting the formation of the first excited singlet state, P1. Most of P1 then decays with a time constant of 20 ps. The spectra of the absorbance changes associated with the conversion of P to P1 or P⁺ support the view that P involves two or more interacting bacteriochlorophylls. The absorbance changes associated with the reduction of I to I^? suggest that I is a bacteriopheophytin interacting strongly with one or more bacteriochlorophylls in the reaction center.     

Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction

MDM2 is an important negative regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-molecule inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.     

Ground wētā in vines of the Awatere Valley,Marlborough: biology,density and distribution

The endemic New Zealand ground wētā (Hemiandrus sp. ‘promontorius’) has a Naturally Uncommon conservation status. This is because of the paucity of information on its density and distribution. Here, the biology, density and distribution of a population of this wētā found in and around vineyards in the Awatere Valley, Marlborough was studied. Wētā density was assessed in vineyards, paddocks and shrublands in this valley. Soil moisture, penetration resistance, pH and organic matter were recorded at locations with and without wētā. Wētā density in vineyards was significantly higher than in either paddocks or shrub habitats. In vineyards, the density of this insect was significantly higher under-vines than in the inter-rows. Higher numbers of this wētā were found in moist soils that required lower force to burrow. Females laid an average of 55 eggs between March and April, which hatched in September. These findings highlight the intersection between agriculture and conservation.     

Purification and crystallization of a complex between human interferon γ receptor (extracellular domain) and human interferon γ

X-ray diffraction quality crystals have been obtained from a complex between interferon γ and the extracellular domain of its high-affinity cell surface receptor. The crystals were obtained from interferon γ/interferon γ receptor complexes purified by size exclusion chromatography. Diffraction quality crystals required analyzing these complex samples by isoelectric focusing gels to select purified complex fractions devoid of unbound interferon γ. These studies used interferon γ receptor engineered with an eight amino acid N-terminal deletion to eliminate heterogeneity generated due to proteolytic cleavage. In addition, the receptor was expressed in an E. coli secretion cell line which eliminated the need to refold the protein. Hexagonal crystals were grown from 1.6 M ammonium phosphate solutions and belong to a spacegroup of P6₅22 with unit cell dimensions a = 145.9 Å and c = 180.3 Å. These crystals diffract to at least 2.9 Å resolution when exposed to synchrotron radiation. SDS PAGE analysis of the crystals demonstrated that both interferon γ and the receptor were present. Analysis of the x-ray diffraction data revealed that the crystals contain complexes with a stoichiometry of 2:1 receptor: ligand within the crystallographic asymmetric unit and consist of approximately 55% solvent. © 1996 Wiley-Liss, Inc.     

Double-stranded DNA-induced localized unfolding of HCV NS3 helicase subdomain 2

The NS3 helicase of the hepatitis C virus (HCV) unwinds double-stranded (ds) nucleic acid (NA) in an NTP-dependent fashion. Mechanistic details of this process are, however, largely unknown for the HCV helicase. We have studied the binding of dsDNA to an engineered version of subdomain 2 of the HCV helicase (d(2Delta)NS3h) by NMR and circular dichroism. Binding of dsDNA to d(2Delta)NS3h induces a local unfolding of helix (alpha(3)), which includes residues of conserved helicase motif VI (Q(460)RxxRxxR(467)), and strands (beta(1) and beta(8)) from the central beta-sheet. This also occurs upon lowering the pH (4.4) and introducing an R461A point mutation, which disrupt salt bridges with Asp 412 and Asp 427 in the protein structure. NMR studies on d(2Delta)NS3h in the partially unfolded state at low pH map the dsDNA binding site to residues previously shown to be involved in single-stranded DNA binding. Sequence alignment and structural comparison suggest that these Arg-Asp interactions are highly conserved in SF2 DEx(D/H) proteins. Thus, modulation of these interactions by dsNA may allow SF2 helicases to switch between conformations required for helicase function.