VEGF-B ablation in pancreatic ß-cells upregulates insulin expression without affecting glucose homeostasis or islet lipid uptake.

Type 2 diabetes mellitus (T2DM) affects millions of people and is linked with obesity and lipid accumulation in peripheral tissues. Increased lipid handling and lipotoxicity in insulin producing ß-cells may contribute to ß-cell dysfunction in T2DM. The vascular endothelial growth factor (VEGF)-B regulates uptake and transcytosis of long-chain fatty acids over the endothelium to tissues such as heart and skeletal muscle. Systemic inhibition of VEGF-B signaling prevents tissue lipid accumulation, improves insulin sensitivity and glucose tolerance, as well as reduces pancreatic islet triglyceride content, under T2DM conditions. To date, the role of local VEGF-B signaling in pancreatic islet physiology and in the regulation of fatty acid trans-endothelial transport in pancreatic islet is unknown. To address these questions, we have generated a mouse strain where VEGF-B is selectively depleted in ß-cells, and assessed glucose homeostasis, ß-cell function and islet lipid content under both normal and high-fat diet feeding conditions. We found that Vegfb was ubiquitously expressed throughout the pancreas, and that ß-cell Vegfb deletion resulted in increased insulin gene expression. However, glucose homeostasis and islet lipid uptake remained unaffected by ß-cell VEGF-B deficiency.

Cross-national invariance of Attention-Deficit/Hyperactivity Disorder factors in Japanese and U.S. university students.

Prior research with children generally supports the two-dimensional structure of Attention-Deficit/Hyperactivity Disorder (ADHD; inattentive and hyperactive/impulsive factors) of the DSM-IV-TR as well as invariance of the two-factor structure across nations and cultures. Research with adults supports either a two-factor or three-factor structure depending on reporting source and breadth of symptoms assessed. However, research with adults is limited and there are few studies addressing cross-national invariance in adults. The purposes of this study were to (1) assess relative fit of two- versus three-factor solutions for self-report of childhood and recent ADHD symptoms in adults; and (2) further establish cross-national invariance of factors. Participants included 271 U.S. and 712 Japanese university students who completed a rating scale assessing the 18 DSM-IV-TR ADHD symptoms. Confirmatory factor analysis using Mplus (Version 6) and the mean and variance-adjusted weighted least squares (WLSMV) procedure showed invariance of two- and three-factor models across U.S. and Japanese samples. The two- and three-factor models showed similar fit indices. Neither a two-factor or three-factor model was clearly superior. The two-factor model was favored, however, because it is more parsimonious and consistent with current theory, and because of high correlations between hyperactive and impulsive factors in the three-factor models. Invariance across nations is consistent with previous studies and supports ADHD as a universally valid syndrome rather than a cultural construct. These results add to the limited knowledge of assessment of ADHD symptoms in Japan.

Passageless comprehension on the Nelson-Denny reading test: well above chance for university students.

The comprehension section of the Nelson-Denny Reading Test (NDRT) is widely used to assess the reading comprehension skills of adolescents and adults in the United States. In this study, the authors explored the content validity of the NDRT Comprehension Test (Forms G and H) by asking university students (with and without at-risk status for learning disorders) to answer the multiple-choice comprehension questions without reading the passages. Overall accuracy rates were well above chance for both NDRT forms and both groups of students. These results raise serious questions about the validity of the NDRT and its use in the identification of reading disabilities.

The diagnostic accuracy of symptom validity tests when used with postsecondary students with learning disabilities: a preliminary investigation.

The current exploratory investigation examined the diagnostic accuracy of the Word Memory Test (WMT), Test of Memory Malingering (TOMM), and Word Reading Test (WRT) with three groups of postsecondary students: controls, learning disability (LD) simulators, and a presumed honest LD group. Each measure achieved high overall diagnostic accuracy, yet each contributed differently to suboptimal effort detection. False-negative classifications varied by measure, yet no simulator went undetected by all three tests. The WMT and WRT identified different members of the presumed honest LD group as demonstrating poor effort, whereas the TOMM identified none. Each measure contributed unique variance in a logistic regression, with effort status best predicted by WMT Consistency. Findings provided preliminary evidence that all three measures may be useful when assessing effort during postsecondary LD evaluations. Implications for future practice and research are discussed.

AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility.

We describe the testing and release of AutoDock4 and the accompanying graphical user interface AutoDockTools. AutoDock4 incorporates limited flexibility in the receptor. Several tests are reported here, including a redocking experiment with 188 diverse ligand-protein complexes and a cross-docking experiment using flexible sidechains in 87 HIV protease complexes. We also report its utility in analysis of covalently bound ligands, using both a grid-based docking method and a modification of the flexible sidechain technique.

Analysis of HIV wild-type and mutant structures via in silico docking against diverse ligand libraries.

The FightAIDS@Home distributed computing project uses AutoDock for an initial virtual screen of HIV protease structures against a broad range of 1771 ligands including both known protease inhibitors and a diverse library of other ligands. The volume of results allows novel large-scale analyses of binding energy "profiles" for HIV structures. Beyond identifying potential lead compounds, these characterizations provide methods for choosing representative wild-type and mutant protein structures from the larger set. From the binding energy profiles of the PDB structures, a principal component analysis based analysis identifies seven "spanning" proteases. A complementary analysis finds that the wild-type protease structure 2BPZ best captures the central tendency of the protease set. Using a comparison of known protease inhibitors against the diverse ligand set yields an AutoDock binding energy "significance" threshold of -7.0 kcal/mol between significant, strongly binding ligands and other weak/nonspecific binding energies. This threshold captures nearly 98% of known inhibitor interactions while rejecting more than 95% of suspected noninhibitor interactions. These methods should be of general use in virtual screening projects and will be used to improve further FightAIDS@Home experiments.

Rapid discovery and structure-activity profiling of novel inhibitors of human immunodeficiency virus type 1 protease enabled by the copper(I)-catalyzed synthesis of 1,2,3-triazoles and their further functionalization.

Building from the results of a computational screen of a range of triazole-containing compounds for binding efficiency to human immunodeficiency virus type 1 protease (HIV-1-Pr), a novel series of potent inhibitors has been developed. The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), which provides ready access to 1,4-disubstituted-1,2,3-triazoles, was used to unite a focused library of azide-containing fragments with a diverse array of functionalized alkyne-containing building blocks. In combination with direct screening of the crude reaction products, this method led to the rapid identification of a lead structure and readily enabled optimization of both azide and alkyne fragments. Replacement of the triazole with a range of alternative linkers led to greatly reduced protease inhibition; however, further functionalization of the triazoles at the 5-position gave a series of compounds with increased activity, exhibiting Ki values as low as 8 nM.

Testing the double-deficit hypothesis in an adult sample.

The double-deficit hypothesis of dyslexia posits that reading deficits are more severe in individuals with weaknesses in phonological awareness and rapid naming than in individuals with deficits in only one of these reading composite skills. In this study, the hypothesis was tested in an adult sample as a model of reading achievement. Participants were parents of children referred for evaluation of reading difficulties. Approximately half of all participants reported difficulty learning to read in childhood and a small subset demonstrated ongoing weaknesses in reading. Structural equation modeling results suggest that the double-deficit hypothesis is an accurate model for understanding adult reading achievement. Better reading achievement was associated with better phonological awareness and faster rapid automatized naming in adults. Posthoc analyses indicated that individuals with double deficits had significantly lower reading achievement than individuals with single deficits or no deficits.

Bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase): evidence for two distinct stages of methylation under single turnover conditions.

We compared the (pre)steady-state and single turnover methylation kinetics of bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase)-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to oligodeoxynucleotide duplexes containing a single recognition site (palindrome 5'-GATC/5'-GATC) or some modified variant. T4Dam-AdoMet functions as a monomer under steady-state conditions (enzyme/DNA << 1), whereas under single turnover conditions (enzyme/DNA > 1), a catalytically active complex containing two Dam-AdoMet molecules is formed initially, and two methyl groups are transferred per duplex (to produce a methylated duplex and S-adenosyl-l-homocysteine (AdoHcy)). We propose that the single turnover reaction proceeds in two stages. First, two preformed T4Dam-AdoMet complexes bind opposite strands of the unmodified target site, and one enzyme molecule catalyzes the rapid transfer of the AdoMet-methyl group (kmeth1 = 0.21 s-1); this is 2.5-fold slower than the rate observed with monomeric T4Dam-AdoMet bound under pre-steady-state conditions for burst determination. In the second stage, methyl transfer to adenine in GATC on the complementary strand occurs at a rate that is 1 order of magnitude slower (kmeth2 = 0.023 s-1). We suggest that under single turnover conditions, methylation of the second strand is rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. The hemimethylated duplex 5'-GATC/5'-GMTC also interacts with T4Dam-AdoMet complexes in two stages under single turnover reaction conditions. The first stage (kmeth1) reflects methylation by dimeric T4Dam-AdoMet productively oriented to the strand with the adenine residue capable of methylation. The slower second stage (kmeth2) reflects methylation by enzyme molecules non-productively oriented to the GMTC chain, which then have to re-orient to the opposite productive chain. Substitutions of bases and deletions in the recognition site affect the kinetic parameters in different fashions. When the GAT portion of GATC was disrupted, the proportion of the initial productive enzyme-substrate complexes was sharply reduced.

DNA (cytosine-N4-)- and -(adenine-N6-)-methyltransferases have different kinetic mechanisms but the same reaction route. A comparison of M.BamHI and T4 Dam.

We studied the kinetics of methyl group transfer by the BamHI DNA-(cytosine-N(4)-)-methyltransferase (MTase) from Bacillus amyloliquefaciens to a 20-mer oligodeoxynucleotide duplex containing the palindromic recognition site GGATCC. Under steady state conditions the BamHI MTase displayed a simple kinetic behavior toward the 20-mer duplex. There was no apparent substrate inhibition at concentrations much higher than the K(m) for either DNA (100-fold higher) or S-adenosyl-l-methionine (AdoMet) (20-fold higher); this indicates that dead-end complexes did not form in the course of the methylation reaction. The DNA methylation rate was analyzed as a function of both substrate and product concentrations. It was found to exhibit product inhibition patterns consistent with a steady state random bi-bi mechanism in which the dominant order of substrate binding and product release (methylated DNA, DNA(Me), and S-adenosyl-l-homocysteine, AdoHcy) was Ado-Met DNA DNA(Me) AdoHcy. The M.BamHI kinetic scheme was compared with that for the T4 Dam (adenine-N(6)-)-MTase. The two differed with respect to an effector action of substrates and in the rate-limiting step of the reaction (product inhibition patterns are the same for the both MTases). From this we conclude that the common chemical step in the methylation reaction, methyl transfer from AdoMet to a free exocyclic amino group, is not sufficient to dictate a common kinetic scheme even though both MTases follow the same reaction route.

Functional analysis of BamHI DNA cytosine-N4 methyltransferase.

We show that the kinetic mechanism of the DNA (cytosine-N(4)-)-methyltransferase M.BamHI, which modifies the underlined cytosine (GGATCC), differs from cytosine C(5) methyltransferases, and is similar to that observed with adenine N(6) methyltransferases. This suggests that the obligate order of ternary complex assembly and disassembly depends on the type of methylation reaction. In contrast, the single-turnover rate of catalysis for M.BamHI (0.10s(-1)) is closer to the DNA (cytosine-C(5)-)-methyltransferases (0.14s(-1)) than the DNA (adenine-N(6)-)-methyltransferases (>200s(-1)). The nucleotide flipping transition dominates the single-turnover constant for adenine N(6) methyltransferases, and, since the disruption of the guanine-cytosine base-pair is essential for both types of cytosine DNA methyltransferases, this transition may be a common, rate-limiting step for methylation for these two enzyme subclasses. The similar overall rate of catalysis by M.BamHI and other DNA methyltransferases is consistent with a common rate-limiting catalytic step of product dissociation. Our analyses of M.BamHI provide functional insights into the relationship between the three different classes of DNA methyltransferases that complement both prior structural and evolutionary insights.