National characteristics and variation in Arabic handwriting

From each of four Arabic countries; Morocco, Tunisia, Jordan and Oman, 150 participants produced handwriting samples which were examined to assess whether national characteristics were discernible. Ten characters, which have different configurations depending upon their position in the word, along with one short word, were classified into distinguishable forms, and these forms recorded for each handwriting sample. Tests of independence showed that character forms used were not independent of country (p < 0.001) for all but one character-position (this was dropped from subsequent analyses). A correspondence analysis ordination plot and analysis of similarity (R = 0.326, p = 0.0002) showed that whole samples were discernibly grouped by country, and a tree analysis produced a classification which was 71% accurate for the original data and 83% accurate for 80 new handwriting samples that underwent ‘blind’ classification. When the countries were combined into two regions, North Africa and Middle East, the grouping was more marked. Thus, there appears to be some scope for narrowing down the nationality, and particularly the wider geographical region of an author based upon the character forms they use in Arabic handwriting

Identification of C-β-d-Glucopyranosyl Azole-Type Inhibitors of Glycogen Phosphorylase That Reduce Glycogenolysis in Hepatocytes: In Silico Design, Synthesis, in Vitro Kinetics, and ex Vivo Studies

Several C-β-d-glucopyranosyl azoles have recently been uncovered as among the most potent glycogen phosphorylase (GP) catalytic site inhibitors discovered to date. Toward further exploring their translational potential, ex vivo experiments have been performed for their effectiveness in reduction of glycogenolysis in hepatocytes. New compounds for these experiments were predicted in silico where, for the first time, effective ranking of GP catalytic site inhibitor potencies using the molecular mechanics-generalized Born surface area (MM-GBSA) method has been demonstrated. For a congeneric training set of 27 ligands, excellent statistics in terms of Pearson (RP) and Spearman (RS) correlations (both 0.98), predictive index (PI = 0.99), and area under the receiver operating characteristic curve (AU-ROC = 0.99) for predicted versus experimental binding affinities were obtained, with ligand tautomeric/ionization states additionally considered using density functional theory (DFT). Seven 2-aryl-4(5)-(β-d-glucopyranosyl)-imidazoles and 2-aryl-4-(β-d-glucopyranosyl)-thiazoles were subsequently synthesized, and kinetics experiments against rabbit muscle GPb revealed new potent inhibitors with best Ki values in the low micromolar range (5c = 1.97 μM; 13b = 4.58 μM). Ten C-β-d-glucopyranosyl azoles were then tested ex vivo in mouse primary hepatocytes. Four of these (5a–c and 9d) demonstrated significant reduction of glucagon stimulated glycogenolysis (IC50 = 30–60 μM). Structural and predicted physicochemical properties associated with their effectiveness were analyzed with permeability related parameters identified as crucial factors. The most effective ligand series 5 contained an imidazole ring, and the calculated pKa (Epik: 6.2; Jaguar 5.5) for protonated imidazole suggests that cellular permeation through the neutral state is favored, while within the cell, there is predicted more favorable binding to GP in the protonated form

A genome-wide association study identifies protein quantitative trait loci (pQTLs)

There is considerable evidence that human genetic variation influences gene expression. Genome-wide studies have revealed that mRNA levels are associated with genetic variation in or close to the gene coding for those mRNA transcripts - cis effects, and elsewhere in the genome - trans effects. The role of genetic variation in determining protein levels has not been systematically assessed. Using a genome-wide association approach we show that common genetic variation influences levels of clinically relevant proteins in human serum and plasma. We evaluated the role of 496,032 polymorphisms on levels of 42 proteins measured in 1200 fasting individuals from the population based InCHIANTI study. Proteins included insulin, several interleukins, adipokines, chemokines, and liver function markers that are implicated in many common diseases including metabolic, inflammatory, and infectious conditions. We identified eight Cis effects, including variants in or near the IL6R (p = 1.8×10 -57), CCL4L1 (p = 3.9×10-21), IL18 (p = 6.8×10-13), LPA (p = 4.4×10-10), GGT1 (p = 1.5×10-7), SHBG (p = 3.1×10-7), CRP (p = 6.4×10-6) and IL1RN (p = 7.3×10-6) genes, all associated with their respective protein products with effect sizes ranging from 0.19 to 0.69 standard deviations per allele. Mechanisms implicated include altered rates of cleavage of bound to unbound soluble receptor (IL6R), altered secretion rates of different sized proteins (LPA), variation in gene copy number (CCL4L1) and altered transcription (GGT1). We identified one novel trans effect that was an association between ABO blood group and tumour necrosis factor alpha (TNF-alpha) levels (p = 6.8×10-40), but this finding was not present when TNF-alpha was measured using a different assay , or in a second study, suggesting an assay-specific association. Our results show that protein levels share some of the features of the genetics of gene expression. These include the presence of strong genetic effects in cis locations. The identification of protein quantitative trait loci (pQTLs) may be a powerful complementary method of improving our understanding of disease pathways. © 2008 Melzer et al

The effects of benzofury (5-APB) on the dopamine transporter and 5-HT2-dependent vasoconstriction in the rat

5-APB, commonly marketed as ‘benzofury’ is a new psychoactive substance and erstwhile ‘legal high’ which has been implicated in 10 recent drug-related deaths in the UK. This drug was available on the internet and in ‘head shops’ and was one of the most commonly sold legal highs up until its recent UK temporary ban (UK Home Office). Despite its prominence, very little is known about its pharmacology. This study was undertaken to examine the pharmacology of 5-APB in vitro. We hypothesized that 5-APB would activate the dopamine and 5-HT systems which may underlie its putative stimulant and hallucinogenic effects. Autoradiographic studies showed that 5-APB displaced both [125I]RTI-121 and [3H]ketanserin from rat brain tissue suggesting affinity at the dopamine transporter and 5-HT2 receptor sites respectively. Voltammetric studies in rat accumbens brain slices revealed that 5-APB slowed dopamine reuptake, and at high concentrations caused reverse transport of dopamine. 5-APB also caused vasoconstriction of rat aorta, an effect antagonized by the 5-HT2A receptor antagonist ketanserin, and caused contraction of rat stomach fundus, which was reversed by the 5-HT2B receptor antagonist RS-127445. These data show that 5-APB interacts with the dopamine transporter and is an agonist at the 5-HT2A and 5-HT2B receptors in the rat. Thus 5-APB’s pharmacology is consistent with it having both stimulant and hallucinogenic properties. In addition, 5-APB’s activity at the 5-HT2B receptor may cause cardiotoxicity

Integration of GWAS SNPs and tissue specific expression profiling reveal discrete eQTLs for human traits in blood and brain

Our knowledge of the transcriptome has become much more complex since the days of the central dogma of molecular biology. We now know that splicing takes place to create potentially thousands of isoforms from a single gene, and we know that RNA does not always faithfully recapitulate DNA if RNA editing occurs. Collectively, these observations show that the transcriptome is amazingly rich with intricate regulatory mechanisms for overall gene expression, splicing, and RNA editing. Genetic variability can play a role in controlling gene expression, which can be identified by examining expression quantitative trait loci (eQTLs). eQTLs are genomic regions where genetic variants, including single nucleotide polymorphisms (SNPs) show a statistical association with expression of mRNA transcripts. In humans, many SNPs are also associated with disease, and have been identified using genome wide association studies (GWAS) but the biological effects of those SNPs are usually not known. If SNPs found in GWAS are also found in eQTLs, then one could hypothesize that expression levels may contribute to disease risk. Performing eQTL analysis with GWAS SNPs in both blood and brain, specifically the frontal cortex and the cerebellum, we found both shared and tissue unique eQTLS. The identification of tissue-unique eQTLs supports the argument that choice of tissue type is important in eQTL studies (Paper I). Aging is a complex process with the mechanisms underlying aging still being poorly defined. There is evidence that the transcriptome changes with age, and hence we used the brain dataset from our first paper as a discovery set, with an additional replication dataset, to investigate any aging-gene expression associations. We found evidence that many genes were associated with aging. We further found that there were more statically significant expression changes in the frontal cortex versus the cerebellum, indicating that brain regions may age at different rates. As the brain is a heterogeneous tissue including both neurons and non-neuronal cells, we used LCM to capture Purkinje cells as a representative neuronal type and repeated the age analysis. Looking at the discovery, replication and Purkinje cell datasets we found five genes with strong, replicated evidence of age-expression associations (Paper II). Being able to capture and quantify the depth of the transcriptome has been a lengthy process starting with methods that could only measure a single gene to genome-wide techniques such as microarray. A recently developed technology, RNA-Seq, shows promise in its ability to capture expression, splicing, and editing and with its broad dynamic range quantification is accurate and reliable. RNA-Seq is, however, data intensive and a great deal of computational expertise is required to fully utilize the strengths of this method. We aimed to create a small, well-controlled, experiment in order to test the performance of this relatively new technology in the brain. We chose embryonic versus adult cerebral cortex, as mice are genetically homogenous and there are many known differences in gene expression related to brain development that we could use as benchmarks for analysis testing. We found a large number of differences in total gene expression between embryonic and adult brain. Rigorous technical and biological validation illustrated the accuracy and dynamic range of RNA-Seq. We were also able to interrogate differences in exon usage in the same dataset. Finally we were able to identify and quantify both well-known and novel A-to-I edit sites. Overall this project helped us develop the tools needed to build usable pipelines for RNA-Seq data processing (Paper III). Our studies in the developing brain (Paper III) illustrated that RNA-Seq was a useful unbiased method for investigating RNA editing. To extend this further, we utilized a genetically modified mouse model to study the transcriptomic role of the RNA editing enzyme ADAR2. We found that ADAR2 was important for editing of the coding region of mRNA as a large proportion of RNA editing sites in coding regions had a statistically significant decrease in editing percentages in Adar2 -/-Gria2 R/R mice versus controls. However, despite indications in the literature that ADAR2 may also be involved in splicing and expression regulatory machinery we found no changes in gene expression or exon utilization in Adar2 -/-Gria2 R/R mice as compared to their littermate controls (Paper IV). In our final study, based on the methods developed in Papers III and IV, we revisited the idea of age related gene expression associations from Paper II. We used a subset of human frontal cortices for RNA sequencing. Interestingly we found more gene expression changes with aging compared to the previous data using microarrays in Paper II. When the significant gene lists were analysed for gene ontology enrichment, we found that there was a large number of downregulated genes involved in synaptic function while those that were upregulated had enrichment in immune function. This dataset illustrates that the aging brain may be predisposed to the processes found in neurodegenerative diseases (Paper V)

An evaluation of indirubin analogues as phosphorylase kinase inhibitors

Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus S. et al., Oncogene 2012, 31, 4333). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170 – 0.360 µM, with indirubin-3’-acetoxime (1c) the most potent. 7-bromoindirubin-3’-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas J. et al., Oncogene, 2006, 25, 6304) is revealed as a specific inhibitor of PhK (IC50 = 1.8 µM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting -hole effects is highlighted. A new statistical metric, the ‘sum of the modified logarithm of ranks’ (SMLR), has been defined which measures performance of a model for both the “early recognition” (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/β), 6’(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work

Genomic attributes of airway commensal bacteria and mucosa

Microbial communities at the airway mucosal barrier are conserved and highly ordered, in likelihood reflecting co-evolution with human host factors. Freed of selection to digest nutrients, the airway microbiome underpins cognate management of mucosal immunity and pathogen resistance. We show here the initial results of systematic culture and whole-genome sequencing of the thoracic airway bacteria, identifying 52 novel species amongst 126 organisms that constitute 75% of commensals typically present in heathy individuals. Clinically relevant genes encode antimicrobial synthesis, adhesion and biofilm formation, immune modulation, iron utilisation, nitrous oxide (NO) metabolism and sphingolipid signalling. Using whole-genome content we identify dysbiotic features that may influence asthma and chronic obstructive pulmonary disease. We match isolate gene content to transcripts and metabolites expressed late in airway epithelial differentiation, identifying pathways to sustain host interactions with microbiota. Our results provide a systematic basis for decrypting interactions between commensals, pathogens, and mucosa in lung diseases of global significance

A multidisciplinary study of 3-(β-d-glucopyranosyl)-5-substituted-1,2,4-triazole derivatives as glycogen phosphorylase inhibitors: Computation, synthesis, crystallography and kinetics reveal new potent inhibitors

3-(β-d-Glucopyranosyl)-5-substituted-1,2,4-triazoles have been revealed as an effective scaffold for the development of potent glycogen phosphorylase (GP) inhibitors but with the potency very sensitive to the nature of the alkyl/aryl 5-substituent (Kun et al., Eur. J. Med. Chem. 2014, 76, 567). For a training set of these ligands, quantum mechanics-polarized ligand docking (QM-PLD) demonstrated good potential to identify larger differences in potencies (predictive index PI = 0.82) and potent inhibitors with K 's < 10 μM (AU-ROC = 0.86). Accordingly, in silico screening of 2335 new analogues exploiting the ZINC docking database was performed and nine predicted candidates selected for synthesis. The compounds were prepared in O-perbenzoylated forms by either ring transformation of 5-β-d-glucopyranosyl tetrazole by N-benzyl-arenecarboximidoyl chlorides, ring closure of C-(β-d-glucopyranosyl)formamidrazone with aroyl chlorides, or that of N-(β-d-glucopyranosylcarbonyl)arenethiocarboxamides by hydrazine, followed by deprotections. Kinetics experiments against rabbit muscle GPb (rmGPb) and human liver GPa (hlGPa) revealed five compounds as potent low μM inhibitors with three of these on the submicromolar range for rmGPa. X-ray crystallographic analysis sourced the potency to a combination of favorable interactions from the 1,2,4-triazole and suitable aryl substituents in the GP catalytic site. The compounds also revealed promising calculated pharmacokinetic profiles. [Abstract copyright: Copyright © 2018 Elsevier Masson SAS. All rights reserved.

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms