Perspective: Quality Versus Quantity; Is It Important to Assess the Role of Enhancers in Complex Disease from an In Vivo Perspective?

AMcE was funded by BBSRC project grant (BB/N017544/1).Peer reviewedPublisher PD

Context-dependant enhancers as a reservoir of functional polymorphisms and epigenetic markers linked to alcohol use disorders and comorbidities

© 2022 The Authors. Acknowledgements AMcE was funded by BBSRC project grant (BB/N017544/1) and EH was funded by Medical Research Scotland (PhD-719–2013).Peer reviewedPublisher PD

Disease associated polymorphisms within the conserved ECR1 enhancer differentially regulate the tissue specific activity of the cannabinoid‐1 receptor gene promoter; implications for cannabinoid pharmacogenetics

EH was funded by Medical Research Scotland (PhD-719-2013) and GW Pharmaceuticals. AMcE was funded by BBSRC project grant (BB/N017544/1). PB and DW are funded by the Scottish Government Rural and Environment Science and Analytical Services Division to the Rowett Institute. The authors declare no conflicts of interest.Peer reviewedPublisher PD

Zinc is essential for high-affinity DNA binding and recombinase activity of φC31 integrase

The mechanism through which the large serine recombinases bind DNA is poorly understood. Alignments of ϕC31 integrase (Int) and its relatives indicate the presence of a conserved motif containing four cysteines resembling a zinc finger. Inductively coupled plasma–mass spectrometry (ICP–MS) confirmed that an Int monomer contains one atom of zinc. Pre-incubation of Int with ethylenediaminetetraacetic acid (EDTA) was detrimental for both recombination activity and DNA binding affinities but full activity could be restored by adding back Zn2+. Mutations in the cysteines and other highly conserved residues yielded proteins that were hypersensitive to proteases, suggesting that without zinc the domain is unfolded. Substitutions in the highly charged region between the conserved cysteines led to lowered DNA binding affinities while circular dichroism revealed that these variant Ints were not greatly affected in overall folding. Int was protected from inhibition by EDTA when DNA containing an attachment site was present suggesting that the zinc finger and the DNA are in close proximity. A truncated mutant of Int, hInt V371SUGA, lacking the putative zinc finger could bind DNA with low affinity. The data are consistent with there being at least two DNA binding motifs in Int one of which is the zinc finger-like motif

CRISPR disruption and UK Biobank analysis of a highly conserved polymorphic enhancer suggests a role in male anxiety and alcohol intake.

Excessive alcohol intake is associated with 5.9% of global deaths. However, this figure is especially acute in men such that 7.6% of deaths can be attributed to alcohol intake. Previous studies identified a significant interaction between genotypes of the galanin (GAL) gene with anxiety and alcohol abuse in different male populations but were unable to define a mechanism. To address these issues the current study analysed the human UK Biobank cohort and identified a significant interaction (n = 115,865; p = 0.0007) between allelic variation (GG or CA genotypes) in the highly conserved human GAL5.1 enhancer, alcohol intake (AUDIT questionnaire scores) and anxiety in men. Critically, disruption of GAL5.1 in mice using CRISPR genome editing significantly reduced GAL expression in the amygdala and hypothalamus whilst producing a corresponding reduction in ethanol intake in KO mice. Intriguingly, we also found the evidence of reduced anxiety-like behaviour in male GAL5.1KO animals mirroring that seen in humans from our UK Biobank studies. Using bioinformatic analysis and co-transfection studies we further identified the EGR1 transcription factor, that is co-expressed with GAL in amygdala and hypothalamus, as being important in the protein kinase C (PKC) supported activity of the GG genotype of GAL5.1 but less so in the CA genotype. Our unique study uses a novel combination of human association analysis, CRISPR genome editing in mice, animal behavioural analysis and cell culture studies to identify a highly conserved regulatory mechanism linking anxiety and alcohol intake that might contribute to increased susceptibility to anxiety and alcohol abuse in men

VANG-1 and PRKL-1 Cooperate to Negatively Regulate Neurite Formation in Caenorhabditis elegans

Neuritogenesis is a critical early step in the development and maturation of neurons and neuronal circuits. While extracellular directional cues are known to specify the site and orientation of nascent neurite formation in vivo, little is known about the genetic pathways that block inappropriate neurite emergence in order to maintain proper neuronal polarity. Here we report that the Caenorhabditis elegans orthologues of Van Gogh (vang-1), Prickle (prkl-1), and Dishevelled (dsh-1), core components of planar cell polarity (PCP) signaling, are required in a subset of peripheral motor neurons to restrict neurite emergence to a specific organ axis. In loss-of-function mutants, neurons display supernumerary neurites that extend inappropriately along the orthogonal anteroposterior (A/P) body axis. We show that autonomous and non-autonomous gene activities are required early and persistently to inhibit the formation or consolidation of growth cone protrusions directed away from organ precursor cells. Furthermore, prkl-1 overexpression is sufficient to suppress neurite formation and reorient neuronal polarity in a vang-1– and dsh-1–dependent manner. Our findings suggest a novel role for a PCP–like pathway in maintaining polarized neuronal morphology by inhibiting neuronal responses to extrinsic or intrinsic cues that would otherwise promote extraneous neurite formation

Cold-adapted arsenite oxidase from a psychrotolerant Polaromonas species.

Polaromonas sp. str. GM1 is an aerobic, psychrotolerant, heterotrophic member of the Betaproteobacteria and is the only isolate capable of oxidising arsenite at temperatures below 10 °C. Sequencing of the aio gene cluster in GM1 revealed the presence of the aioB and aioA genes, which encode the arsenite oxidase but the regulatory genes typically found upstream of aioB in other members of the Proteobacteria were absent. The GM1 Aio was purified to homogeneity and was found to be a heterodimer. The enzyme contained Mo and Fe as cofactors and had, using the artificial electron acceptor 2,6-dichlorophenolindophenol, a Km for arsenite of 111.70 ± 0.88 μM and a Vmax of 12.16 ± 0.30 U mg(-1), which is the highest reported specific activity for any known Aio. The temperature-activity profiles of the arsenite oxidases from GM1 and the mesophilic betaproteobacterium Alcaligenes faecalis were compared and showed that the GM1 Aio was more active at low temperatures than that of A. faecalis. A homology model of the GM1 Aio was made using the X-ray crystal structure of the Aio from A. faecalis as the template. Structural changes that account for cold adaptation were identified and it was found that these resulted in increased enzyme flexibility and a reduction in the hydrophobicity of the core

Plasma gut hormone levels in 37 patients with pheochromocytomas

Pheochromocytomas are usually recognized by the effects of overproduction of catecholamines, but there are clinical features that cannot be ascribed to catecholamine excess that may be due to vasoactive peptides. We, therefore, measured blood levels of vasoactive intestinal peptides (VIP), substance P, somatostatin (SS), and motilin in 50 instances in 37 patients with pheochromocytomas-21 malignant, 10 benign intra-adrenal, and 6 ectopic (5 paracardial and 1 perirenal). Hormone levels were considered raised if the level was more than 3 S.D. above the mean value found in 52 healthy subjects. Of the 37 patients, 20 (54%) had an abnormality in 1 or more gut hormone levels. The most common abnormality was a raised SS in 9/37 (24%). In addition to these, however, 3 (8%) others had raised VIP, 5 (13.5%) raised motilin, and 3 (8%) raised substance P. Patients with benign adrenal adenomas had raised levels of SS and substance P. Ectopic pheochromocytomas produced only SS in addition to catecholamines, but malignant pheochromocytomas could secrete all 4 peptides, and more than 1 in the same patient. We conclude that pheochromocytomas may secrete multiple vasoactive peptides, and they are more likely to do so if malignant. Somatostatin is the most commonly secreted peptide and is found with benign adrenal and ectopic (paracardiac) tumors. If the level of more than 1 peptide is elevated, the likelihood of malignancy is significantly increased . Les phéochromocytomes sont généralement déceléspar les effets dûs à la surproduction de catécholamines, mais certains troubles ne peuvent être attribués à ce phénomène et relèvent peut être de l'action de peptides vasoactifs. Les auteurs se sont donc attachés à doser dans le sang le VIP, la substance P, la somatostatine (SS), et la motiline. Ces dosages furent pratiqués 50 fois chez 37 malades porteurs de phéochromocytomes: 21 malins, 10 bénins et 6 ectopiques (5 paracardiaque et 1 péri-rénal). Les taux des hormones furent considérés comme élevés lorsque leur niveau fut supérieur à plus de 3 fois le taux de 52 sujets sains. Sur les 37 malades 20 (54%) présentaient un excès d'une ou de plusieurs hormones digestives. L'anomalie constatée la plus fréquente fut l'élévation de la SS (9 fois sur 37 soit 24%). Ajoutée à ce fait fut l'élévation de la VIP chez 3 sujets (8%), de la motiline chez 5 (13.5%) et de la substance P chez 3 (8%). Les phéochromocytomes bénins surrénaliens présentaient à la fois une élévation du taux de la SS et de la substance P. Les phéochromocytomes ectopiques en revanche présentaient seulement une élévation de la SS. Les phéochromocytomes malins pouvaient sécréter les 4 peptides ou plus d'un chez le même malade. En conclusion les phéochromocytomes peuvent secréter de multiples peptides vasoactifs et plus particulièrement lorsqu'ils sont malins. La SS est la substance qui est la plus souvent secrétée et elle est trouvée dans les tumeurs bénignes surrénaliennes ou ectopiques. Si plus d'une de ces substances est produite en excès les risques de malignité de la tumeur sont significativement plus importants. Los feocromocitomas generalmente son diagnosticados por los efectos del exceso de producción de catecolaminas pero hay características clínicas que no pueden ser atribuidas al exceso de catecolaminas y que pueden ser más bien manifestación de péptidos vasoactivos. Hemos establecido los niveles sanguíneos del péptido intestinal vasoactivo (VIP), de la sustancia P, de la somatostatina (SS), y de la motilina en 50 determinaciones en 37 pacientes con feocromocitomas; 21 malignos, 10 benignos intra-adrenales, y 6 ectópicos (5 paracardiales y 1 perirrenal). Se consideró que los niveles hormonales estaban elevados cuando el nivel era de más de 3 de desviación estandar sobre el valor promedio en 52 individuos normales. De 37 pacientes, 20 (54%) presentaron un valor anormal en 1 o más determinaciones del nivel de hormonas intestinales. La anormalidad más común fue la elevación de la SS en 9/37 (24%). Además de esto, sinembargo, otros 3 (8%) presentaban elevación de VIP, 5 (13.5%) elevación de sustancia P. Los adenomas suprarrenales benignos exhibieron niveles elevados de SS y de sustancia P. Los feocromocitomas ectópicos demostraron producción sólo de SS además de catecolaminas, pero los feocromocitomas malignos demostraron ser capaces de secretar todos los 4 péptidos, y más de 1 en el mismo paciente. Hemos llegado a la conclusión de que los feocromocitomas pueden secretar múltiples peptidos vasoactivos y que ésto tiende a ocurrir cuando son malignos. La SS es el péptido más frecuentemente secretado y se lo encuentra en los tumores suprarrenales benigno y ectópico (paracardiacos). Si se encuentran niveles elevados de más de 1 péptido, la posibilidad de malignidad aparece significativamente aumentada.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41274/1/268_2005_Article_BF01655534.pd