Engrailed2 modulates cerebellar granule neuron precursor proliferation, differentiation and insulin-like growth factor 1 signaling during postnatal development

BACKGROUND: The homeobox transcription factor Engrailed2 (En2) has been studied extensively in neurodevelopment, particularly in the midbrain/hindbrain region and cerebellum, where it exhibits dynamic patterns of expression and regulates cell patterning and morphogenesis. Because of its roles in regulating cerebellar development and evidence of cerebellar pathology in autism spectrum disorder (ASD), we previously examined an ENGRAILED2 association and found evidence to support EN2 as a susceptibility gene, a finding replicated by several other investigators. However, its functions at the cell biological level remain undefined. In the mouse, En2 gene is expressed in granule neuron precursors (GNPs) just as they exit the cell cycle and begin to differentiate, raising the possibility that En2 may modulate these developmental processes. METHODS: To define En2 functions, we examined proliferation, differentiation and signaling pathway activation in En2 knockout (KO) and wild-type (WT) GNPs in response to a variety of extracellular growth factors and following En2 cDNA overexpression in cell culture. In vivo analyses of cerebellar GNP proliferation as well as responses to insulin-like growth factor-1 (IGF1) treatment were also conducted. RESULTS: Proliferation markers were increased in KO GNPs in vivo and in 24-h cultures, suggesting En2 normally serves to promote cell cycle exit. Significantly, IGF1 stimulated greater DNA synthesis in KO than WT cells in culture, a finding associated with markedly increased phospho-S6 kinase activation. Similarly, there was three-fold greater DNA synthesis in the KO cerebellum in response to IGF1 in vivo. On the other hand, KO GNPs exhibited reduced neurite outgrowth and differentiation. Conversely, En2 overexpression increased cell cycle exit and promoted neuronal differentiation. CONCLUSIONS: In aggregate, our observations suggest that the ASD-associated gene En2 promotes GNP cell cycle exit and differentiation, and modulates IGF1 activity during postnatal cerebellar development. Thus, genetic/epigenetic alterations of EN2 expression may impact proliferation, differentiation and IGF1 signaling as possible mechanisms that may contribute to ASD pathogenesis

Functional Analysis of Cdc42 Residues Required for Guanine Nucleotide Exchange

Guanine nucleotide exchange factors (GEFs) directly engage small GTPases to facilitate the exchange of bound GDP for GTP, leading to GTPase activation. Several recent crystal structures of GEFs in complex with Rho family GTPases highlight the conserved interactions and conformational alterations necessary for catalyzing exchange. In the present study, functional roles were defined for specific residues within Cdc42 implicated by the crystal structures as important for physiological exchange of guanine nucleotides within Rho GTPases. In particular, this study highlights the paramount importance of the phosphate-binding loop and interactions with the magnesium co-factor as critical for proper regulation of RhoGEF-catalyzed exchange. Other conformational alterations of the GTPases affecting interactions with the sugar and base of guanine nucleotides are also important but are secondary. Of particular note, substitution of alanine for cysteine at position 18 of Cdc42 leads to a fast cycling phenotype for Cdc42 with heightened affinity for RhoGEFs and produces a dominant negative form of Cdc42 capable of inhibiting RhoGEFs both in vitro and in vivo

Multifunctional Roles for the PH Domain of Dbs in Regulating Rho GTPase Activation

Dbl family members are guanine nucleotide exchange factors specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. Dbs, a Dbl family member specific for Cdc42 and RhoA, exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. In this study, the PH domain of Dbs was mutated to impair selectively either guanine nucleotide exchange or phosphoinositide binding in vitro and resulting physiological alterations were assessed. As anticipated, substitution of residues within the PH domain of Dbs integral to the interface with GTPases reduced nucleotide exchange and eliminated the ability of Dbs to transform NIH 3T3 cells. More interestingly, substitutions within the PH domain that prevent interaction with phosphoinositides yet do not alter in vitro activation of GTPases also do not transform NIH 3T3 cell and fail to activate RhoA in vivo despite proper subcellular localization. Therefore, the PH domain of Dbs serves multiple roles in the activation of GTPases and cannot be viewed as a simple membrane-anchoring device. In particular, the data suggest that binding of phosphoinositides to the PH domain within the context of membrane surfaces may direct orientations or conformations of the linked DH and PH domains to regulate GTPases activation

Changes associated with Ebola virus adaptation to novel species.

Motivation: Ebola viruses are not pathogenic but can be adapted to replicate and cause disease in rodents. Here, we used a structural bioinformatics approach to analyze the mutations associated with Ebola virus adaptation to rodents to elucidate the determinants of host-specific Ebola virus pathogenicity. Results: We identified 33 different mutations associated with Ebola virus adaptation to rodents in the proteins GP, NP, L, VP24, and VP35. Only VP24, GP and NP were consistently found mutated in rodent-adapted Ebola virus strains. Fewer than five mutations in these genes seem to be required for the adaptation of Ebola viruses to a new species. The role of mutations in GP and NP is not clear. However, three VP24 mutations located in the protein interface with karyopherin 5 may enable VP24 to inhibit karyopherins and subsequently the host interferon response. Three further VP24 mutations change hydrogen bonding or cause conformational changes. Hence, there is evidence that few mutations including crucial mutations in VP24 enable Ebola virus adaptation to new hosts. Since Reston virus, the only non-human pathogenic Ebolavirus species circulates in pigs in Asia, this raises concerns that few mutations may result in novel human pathogenic Ebolaviruses

Semantic technologies for data analysis in health care

A fruitful application of Semantic Technologies in the field of healthcare data analysis has emerged from the collaboration between Oxford and Kaiser Permanente a US healthcare provider (HMO). US HMOs have to annually deliver measurement results on their quality of care to US authorities. One of these sets of measurements is defined in a specification called HEDIS which is infamous amongst data analysts for its complexity. Traditional solutions with either SAS-programs or SQL-queries lead to involved solutions whose maintenance and validation is difficult and binds considerable amount of resources. In this paper we present the project in which we have applied Semantic Technologies to compute the most difficult part of the HEDIS measures. We show that we arrive at a clean, structured and legible encoding of HEDIS in the rule language of the RDF-triple store RDFox. We use RDFox's reasoning capabilities and SPARQL queries to compute and extract the results. The results of a whole Kaiser Permanente regional branch could be computed in competitive time by RDFox on readily available commodity hardware. Further development and deployment of the project results are envisaged in Kaiser Permanente

Afmite, Al_3(OH)_4(H_2O)_3(PO_4)(PO_3OH)•H_2O, a new mineral from Fumade, Tarn, France: description and crystal structure

The new mineral afmite, Al_3(OH)_4(H_2O)_3(PO_4)(PO_3OH)·H_2O, is triclinic with space group Pl and cell parameters a = 7.386(3), b = 7.716(3), c = 11.345(4) Å, a = 99.773(5), β = 91.141(6), y = 115.58(5)°, V = 571.6(3) Å^3 and Z = 2. It occurs, sometimes in association with matulaite and variscite, in fractures and solution cavities in shale/siltstone at Fumade, Tarn, France. The formation is probably largely the result of remobilisation and crystallisation during low-temperature hydrothermal activity and/or weathering and ground water activity. Afmite forms in cockscomb aggregates of diamond-shaped tablets on {001}, ubiquitously contact-twinned on {001} and also commonly twinned by rotation on [010] with {010} and {110} composition planes, forming star-like sixlings. The streak of the mineral is white, the luster is pearly, and the Mohs hardness is about 11/2. The mineral is flexible, but not elastic, has an irregular fracture and three cleavage directions: {001} perfect, {010} and {1Formula 0} good. The measured density is 2.39(3) g/cm^3 and the calculated density is 2.391 g/cm^3 based upon the empirical formula. Optical properties (white light): biaxial (+), α = 1.554(1), β = 1.558(1), y = 1.566(1), 2V_(meas). = 70(5)° and 2V_(calc) = 71°. Electron microprobe analyses provided Al_2O_3 40.20 and P_2O_5 38.84 wt% and CHN analyses provided H_2O 25.64 wt%, total 103.68 wt%. Normalized EMP analyses and water based on the structure yield Al_2O_3 36.41, P_2O_5 35.17 and H_2O 28.42, total 100.00 wt%. Infrared and Raman spectra were consistent with the PO_3OH, OH and H_2O as indicated by the crystal-structure determination. The strongest powder X-ray diffraction lines are [d_(obs)(Å),I_(obs),(hkl)]: 11.089,100,(001), 3.540,81,(013,112), 5.484,79,(002,101), 2.918,60(122), 3.089,33(113,201), 4.022,30,(102,112), 6.826,23,(010). The crystal structure, solved from twinned data, (R_1 = 10.4 % for 1262 F_o > 4σF reflections) consists of chains of AlO_6 octahedra parallel to [110] in which edge-sharing octahedral dimers share corners with individual octahedra. Both PO_4 and PO_3OH tetrahedra link the chains into sheets parallel to {001} and the PO_4 tetrahedra further serve to link two sheets together into a thick slab in which tetrahedral (T) and octahedral (O) layers alternate, forming a T-O-T-O-T sandwich. The linkage between these sandwiches in the c direction is via hydrogen bonding with isolated H_2O groups in the interlayer region. Afmite is closely related structurally to the turquoise-group minerals and specifically to planerite. The recently described mineral kobokoboite probably has a closely related sheet structure

How many Erysiphe-like anamorphs are responsible for the recent outbreak of tomato powdery mildew ?

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Devitoite, a new heterophyllosilicate mineral with astrophyllite-like layers from eastern Fresno County, California

Devitoite, [Ba_6(PO_4)_2(CO_3)] [Fe^(2+)_7Fe^(3+)_2(Si_4O_(12))_2O_2(OH)_4], is a new mineral species from the Esquire #8 claim along Big Creek in eastern Fresno County, California, U.S.A. It is also found at the nearby Esquire #7 claim and at Trumbull Peak in Mariposa County. The mineral is named for Alfred (Fred) DeVito (1937–2004). Devitoite crystallized very late in a sequence of minerals resulting from fluids interacting with a quartz–sanbornite vein along its margin with the country rock. The mineral occurs in subparallel intergrowths of very thin brown blades, flattened on {001} and elongate and striated parallel to [100]. The mineral has a cream to pale brown streak, a silky luster, a Mohs hardness of approximately 4, and two cleavages: {001} perfect and {010} good. The calculated density is 4.044 g/cm^3. It is optically biaxial (+), α 1.730(3), β 1.735(6), γ 1.755(3); 2V_(calc) = 53.6°; orientation: X ≈ b, Y ≈ c, Z ≈ α; pleochroism: brown, Y >>> X > Z. Normalized electron-microprobe analyses provided: BaO 38.83, CaO 0.50, MgO 0.75, FeO 22.02, Fe_2O_3 6.08, Al_2O_3 1.50, SiO_2 20.97, TiO_2 0.80, P_2O_5 4.84, H_2O 1.67, CO_2 2.04, total 100.00 wt%, with FeO and Fe_2O_3 assignments and H_2O and CO_2 based on the structure. The empirical formula is [(Ba_(5.45) Ca_(0.19))_(∑5.64)(PO_4)_(1.47)O_(0.30)(CO_3)] [(Fe^(2+)_(6.60)Mg_(0.40))_(∑7)(Fe^(3+)_(1.64)Ti^(4+)_(0.22)Al^(3+)0.14)_(∑2)(Si_(7.51)Al_(0.49))_(∑8)O_(26)(OH)_4]. Devitoite is triclinic, Pl, α 5.3437(7), b 11.6726(15), c 14.680(2) Å, α91.337(4), β 96.757(4), y 103.233(4)°, V 884.0(2) Å^3 and Z = 1. The crystal-structure determination (R_1 = 9.56% for 1370 F_o > 4σF) shows the mineral to be a heterophyllosilicate with astrophyllite-type HOH layers in which five-coordinated Fe^(3+) takes the place of Ti^(4+). The interlayer region contains Ba atoms, PO_4 groups and CO_3 groups. The configuration of the Ba and PO_4 in the interlayer region is similar to that found in the structure of yoshimuraite

RhoGEF Specificity Mutants Implicate RhoA as a Target for Dbs Transforming Activity

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. Like many RhoGEFs, the in vitro catalytic activity of Dbs is not limited to a single substrate. It can catalyze the exchange of GDP for GTP on RhoA and Cdc42, both of which are expressed in most cell types. This lack of substrate specificity, which is relatively common among members of the RhoGEF family, complicates efforts to determine the molecular basis of their transforming activity. We have recently determined crystal structures of several RhoGEFs bound to their cognate GTPases and have used these complexes to predict structural determinants dictating the specificities of coupling between RhoGEFs and GTPases. Guided by this information, we mutated Dbs to alter significantly its relative exchange activity for RhoA versus Cdc42 and show that the transformation potential of Dbs correlates with exchange on RhoA but not Cdc42. Supporting this conclusion, oncogenic Dbs activates endogenous RhoA but not endogenous Cdc42 in NIH 3T3 cells. Similarly, a competitive inhibitor that blocks RhoA activation also blocks Dbs-mediated transformation. In conclusion, this study highlights the usefulness of specificity mutants of RhoGEFs as tools to genetically dissect the multiple signaling pathways potentially activated by overexpressed or oncogenic RhoGEFs. These ideas are exemplified for Dbs, which is strongly implicated in the transformation of NIH 3T3 cells via RhoA and not Cdc42