Variant-to-function analysis of the childhood obesity chr12q13 locus implicates rs7132908 as a causal variant within the 3' UTR of FAIM2.

The ch12q13 locus is among the most significant childhood obesity loci identified in genome-wide association studies. This locus resides in a non-coding region within FAIM2; thus, the underlying causal variant(s) presumably influence disease susceptibility via cis-regulation. We implicated rs7132908 as a putative causal variant by leveraging our in-house 3D genomic data and public domain datasets. Using a luciferase reporter assay, we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. We generated isogenic human embryonic stem cell lines homozygous for either rs7132908 allele to assess changes in gene expression and chromatin accessibility throughout a differentiation to hypothalamic neurons, a key cell type known to regulate feeding behavior. The rs7132908 obesity risk allele influenced expression of FAIM2 and other genes and decreased the proportion of neurons produced by differentiation. We have functionally validated rs7132908 as a causal obesity variant that temporally regulates nearby effector genes and influences neurodevelopment and survival.

Perturbation of the insomnia WDR90 GWAS locus pinpoints rs3752495 as a causal variant influencing distal expression of neighboring gene, PIG-Q.

Although genome wide association studies (GWAS) have identified loci for sleep-related traits, they do not directly uncover the underlying causal variants and corresponding effector genes. The majority of such variants reside in non-coding regions and are therefore presumed to impact cis-regulatory elements. Our previously reported 'variant-to-gene mapping' effort in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs), combined with validation in both Drosophila and zebrafish, implicated PIG-Q as a functionally relevant gene at the insomnia 'WDR90' GWAS locus. However, importantly that effort did not characterize the corresponding underlying causal variant. Specifically, our previous 3D genomic datasets nominated a shortlist of three neighboring single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium within an intronic enhancer region of WDR90 that contacted the open PIG-Q promoter. We sought to investigate the influence of these SNPs collectively and then individually on PIG-Q modulation to pinpoint the causal "regulatory" variant. Starting with gross level perturbation, deletion of the entire region in NPCs via CRISPR-Cas9 editing and subsequent RNA sequencing revealed expression changes in specific PIG-Q transcripts. Results from individual luciferase reporter assays for each SNP in iPSCs revealed that the region with the rs3752495 risk allele induced a ~2.5-fold increase in luciferase expression. Importantly, rs3752495 also exhibited an allele specific effect, with the risk allele increasing the luciferase expression by ~2-fold versus the non-risk allele. In conclusion, our variant-to-function approach and in vitro validation implicates rs3752495 as a causal insomnia variant embedded within WDR90 while modulating the expression of the distally located PIG-Q.

3D genomic features across >50 diverse cell types reveal insights into the genomic architecture of childhood obesity.

The prevalence of childhood obesity is increasing worldwide, along with the associated common comorbidities of type 2 diabetes and cardiovascular disease in later life. Motivated by evidence for a strong genetic component, our prior genome-wide association study (GWAS) efforts for childhood obesity revealed 19 independent signals for the trait; however, the mechanism of action of these loci remains to be elucidated. To molecularly characterize these childhood obesity loci we sought to determine the underlying causal variants and the corresponding effector genes within diverse cellular contexts. Integrating childhood obesity GWAS summary statistics with our existing 3D genomic datasets for 57 human cell types, consisting of high-resolution promoter-focused Capture-C/Hi-C, ATAC-seq, and RNA-seq, we applied stratified LD score regression and calculated the proportion of genome-wide SNP heritability attributable to cell type-specific features, revealing pancreatic alpha cell enrichment as the most statistically significant. Subsequent chromatin contact-based fine-mapping was carried out for genome-wide significant childhood obesity loci and their linkage disequilibrium proxies to implicate effector genes, yielded the most abundant number of candidate variants and target genes at the BDNF, ADCY3, TMEM18 and FTO loci in skeletal muscle myotubes and the pancreatic beta-cell line, EndoC-BH1. One novel implicated effector gene, ALKAL2 - an inflammation-responsive gene in nerve nociceptors - was observed at the key TMEM18 locus across multiple immune cell types. Interestingly, this observation was also supported through colocalization analysis using expression quantitative trait loci (eQTL) derived from the Genotype-Tissue Expression (GTEx) dataset, supporting an inflammatory and neurologic component to the pathogenesis of childhood obesity. Our comprehensive appraisal of 3D genomic datasets generated in a myriad of different cell types provides genomic insights into pediatric obesity pathogenesis.

Variant-to-function analysis of the childhood obesity chr12q13 locus implicates rs7132908 as a causal variant within the 3' UTR of FAIM2.

The ch12q13 obesity locus is among the most significant childhood obesity loci identified in genome-wide association studies. This locus resides in a non-coding region within FAIM2; thus, the underlying causal variant(s) presumably influence disease susceptibility via an influence on cis-regulation within the genomic region. We implicated rs7132908 as a putative causal variant at this locus leveraging a combination of our inhouse 3D genomic data, public domain datasets, and several computational approaches. Using a luciferase reporter assay in human primary astrocytes, we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. Motivated by this finding, we went on to generate isogenic human embryonic stem cell lines homozygous for either rs7132908 allele with CRISPR-Cas9 homology-directed repair to assess changes in gene expression due to genotype and chromatin accessibility throughout a differentiation to hypothalamic neurons, a key cell type known to regulate feeding behavior. We observed that the rs7132908 obesity risk allele influenced the expression of FAIM2 along with other genes, decreased the proportion of neurons produced during differentiation, up-regulated cell death gene sets, and conversely down-regulated neuron differentiation gene sets. We have therefore functionally validated rs7132908 as a causal obesity variant which temporally regulates nearby effector genes at the ch12q13 locus and influences neurodevelopment and survival.

Perturbation of the insomnia WDR90 GWAS locus pinpoints rs3752495 as a causal variant influencing distal expression of neighboring gene, PIG-Q.

Although genome wide association studies (GWAS) have been crucial for the identification of loci associated with sleep traits and disorders, the method itself does not directly uncover the underlying causal variants and corresponding effector genes. The overwhelming majority of such variants reside in non-coding regions and are therefore presumed to impact the activity of cis-regulatory elements, such as enhancers. Our previously reported 'variant-to-gene mapping' effort in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs), combined with validation in both Drosophila and zebrafish, implicated PIG-Q as a functionally relevant gene at the insomnia 'WDR90' locus. However, importantly that effort did not characterize the corresponding underlying causal variant at this GWAS signal. Specifically, our genome-wide ATAC-seq and high-resolution promoter-focused Capture C datasets generated in this cell setting brought our attention to a shortlist of three tightly neighboring single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium in a candidate intronic enhancer region of WDR90 that contacted the open PIG-Q promoter. The objective of this study was to investigate the influence of the proxy SNPs collectively and then individually on PIG-Q modulation and to pinpoint the causal "regulatory" variant among the three SNPs. Starting at a gross level perturbation, deletion of the entire region harboring all three SNPs in human iPSC-derived neural progenitor cells via CRISPR-Cas9 editing and subsequent RNA sequencing revealed expression changes in specific PIG-Q transcripts. Results from more refined individual luciferase reporter assays for each of the three SNPs in iPSCs revealed that the intronic region with the rs3752495 risk allele induced a ~2.5-fold increase in luciferase expression (n=10). Importantly, rs3752495 also exhibited an allele specific effect, with the risk allele increasing the luciferase expression by ~2-fold compared to the non-risk allele. In conclusion, our variant-to-function approach and subsequent in vitro validation implicates rs3752495 as a causal insomnia risk variant embedded at the WDR90-PIG-Q locus.

Benefit of inhalation aromatherapy as a complementary treatment for stress and anxiety in a clinical setting - A systematic review.

OBJECTIVE: The purpose of this systematic review is to ascertain the impact of inhalation aromatherapy on stress and anxiety in clinical settings. METHODS: A search strategy was developed using various databases. Randomised Controlled Trials (RCTs) as well as single and double-blind pilot clinical studies (non-RCT) using inhalation aromatherapy with an essential oil blend or a single essential oil were examined. All studies included a control intervention and use of a validated measurement tool. The time period under review was years 2000-2021. Due to the high level of heterogeneity and element of bias, a narrative synthesis was conducted. RESULTS: The search strategy initially retrieved 628 studies and through application of the selection criteria and the removal of duplicates, 76 studies were selected for review with a total of 6539 patients. In 42% of the RCTs, physiological measures including vital signs and/or salivary cortisol were used in addition to questionnaires. Over 70% of the studies reported a positive effect on anxiety levels in the aromatherapy intervention groups compared with the control. However, in many cases this is limited by the absence of safety data, imprecise reporting of plant species and dosage of essential oil. CONCLUSION: Inhalation aromatherapy has the potential to reduce stress and anxiety with data emerging to further support this result across a wide modality of clinical treatments. However, there is a clear need for the development of standard protocols for research in this area, generating measurable results which will create the opportunity for more rigorous evidence-based outcomes.

Strategies to identify causal common genetic variants and corresponding effector genes for paediatric obesity.

BACKGROUND: Childhood obesity rates are on the rise, but there are currently no effective therapies available to slow or halt their progression. Although environmental and lifestyle factors have been implicated in its pathogenesis, childhood obesity is considered a complex disorder with a clear genetic component. Intense genome-wide association study (GWAS) efforts through large-scale collaborations have enabled the discovery of genetic loci robustly associated with childhood obesity beyond the classic FTO locus. That said, GWAS itself does not pinpoint the actual underlying causal effector genes, but rather just yields association signals in the genome. OBJECTIVE: This review aims to outline what has been elucidated thus far on the genetic aetiology of commong childhood obesity and to describe strategies to identify and validate both causal common genetic variants and their corresponding effector genes. RESULTS: Relevant cell types for molecular studies can be identified by gene set enrichment analysis and considering known biology of obesity-related physiological processes. Putatively causal single nucleotide polymorphisms (SNPs) can be identified by several methods including statistical fine mapping and 'assay for transposase accessible chromatin sequencing' (ATAC-seq). Variant to gene mapping can then nominate effector genes likely regulated by cis-regulatory elements harbouring putatively causal SNPs. A SNP's cis-regulatory activity can be functionally validated by several in vitro methods including luciferase assay and CRISPR approaches. These CRISPR approaches can also be used to investigate how dysregulatn of effector genes may confer obesity risk. CONCLUSION: Uncovering the causative genes related to GWAS signals and elucidating their functional contributions to paediatric obesity with these strategies will deepen our understanding of this disease and serve better treatment outcomes.

Cis-regulatory architecture of human ESC-derived hypothalamic neuron differentiation aids in variant-to-gene mapping of relevant complex traits.

The hypothalamus regulates metabolic homeostasis by influencing behavior and endocrine systems. Given its role governing key traits, such as body weight and reproductive timing, understanding the genetic regulation of hypothalamic development and function could yield insights into disease pathogenesis. However, given its inaccessibility, studying human hypothalamic gene regulation has proven challenging. To address this gap, we generate a high-resolution chromatin architecture atlas of an established embryonic stem cell derived hypothalamic-like neuron model across three stages of in vitro differentiation. We profile accessible chromatin and identify physical contacts between gene promoters and putative cis-regulatory elements to characterize global regulatory landscape changes during hypothalamic differentiation. Next, we integrate these data with GWAS loci for various complex traits, identifying multiple candidate effector genes. Our results reveal common target genes for these traits, potentially affecting core developmental pathways. Our atlas will enable future efforts to determine hypothalamic mechanisms influencing disease susceptibility.

Noise levels during cesarean delivery: a prospective observational study.

BACKGROUND: Excessive noise has negative implications for both clinicians and patients. Emergency cesarean deliveries require rapid co-ordination and communication, possibly increasing noise pollution. We aimed to determine if noise levels in the Labor and Delivery operating room were higher during emergency cesarean deliveries than during non-emergency cesarean deliveries. METHODS: We conducted a prospective observational study measuring noise levels in Labor and Delivery operating rooms at a single academic medical center. Sound meters placed on anesthesia machines and events charted in the electronic medical record were used to correlate noise levels to clinical activity. Noise levels in all cesarean deliveries were recorded for one year. Deliveries were classified into two groups: non-emergency (routine or urgent) and emergency. We compared noise levels of the groups at eight time points of interest: anesthesia provider enters operating room (T1), induction (T2), five minutes before incision (T3), three minutes before incision (T4), one minute before incision (T5), time of incision (T6), delivery (T7), and five minutes before initiating emergence (T8). RESULTS: Noise levels were measured for 440 cesarean deliveries. Forty were classified emergency and 400 non-emergency (304 routine, 96 urgent) procedures. Emergency cesarean deliveries were noisier at all eight time points, although the absolute difference in decibels between the two groups was modest. The difference in noise level reached statistical significance at five time points (T1, T2, T5, T6, and T7). CONCLUSION: Noise levels were higher during emergency than during non-emergency cesarean deliveries.

Genetic Determinants of Childhood Obesity.

Obesity represents a major health burden to both developed and developing countries. Furthermore, the incidence of obesity is increasing in children. Obesity contributes substantially to mortality in the United States by increasing the risk for type 2 diabetes, cardiovascular-related diseases, and other comorbidities. Despite environmental changes over past decades, including increases in high-calorie foods and sedentary lifestyles, there is very clear evidence of a genetic predisposition to obesity risk. Childhood obesity cases can be categorized in one of two ways: syndromic or non-syndromic. Syndromic obesity includes disorders such as Prader-Willi syndrome, Bardet-Biedl syndrome, and Alström syndrome. Non-syndromic cases of obesity can be further separated into rarer instances of monogenic obesity and much more common forms of polygenic obesity. The advent of genome-wide association studies (GWAS) and next-generation sequencing has driven significant advances in our understanding of the genetic contribution to childhood obesity. Many rare and common genetic variants have been shown to contribute to the heritability in obesity, although the molecular mechanisms underlying most of these variants remain unclear. An important caveat of GWAS efforts is that they do not strictly represent gene target discoveries, rather simply the uncovering of robust genetic signals. One clear example of this is with progress in understanding the key obesity signal harbored within an intronic region of the FTO gene. It has been shown that the non-coding region in which the variant actually resides in fact influences the expression of genes distal to FTO instead, specifically IRX3 and IRX5. Such discoveries suggest that associated non-coding variants can be embedded within or next to one gene, but commonly influence the expression of other, more distal effector genes. Advances in genetics and genomics are therefore contributing to a deeper understanding of childhood obesity, allowing for development of clinical tools and therapeutic agents.

Grazing multispecies swards improves ewe and lamb performance.

A two-year (2015 and 2016) grazing study was established to compare ewe and lamb performance when grazed on a perennial ryegrass only sward compared to more diverse sward types. In that study four sward types were investigated: a perennial ryegrass (Lolium perenne) only sward receiving 163 kg nitrogen per hectare per year (N/ha/yr) (PRG); a perennial ryegrass and white clover (Trifolium repens) sward receiving 90 kg N/ha/yr (PRGWC); a six species sward (two grasses (perennial ryegrass and timothy (Phleum pratense)), two legumes (white and red clover (Trifolium pratense)) and two herbs (ribwort plantain (Plantago lanceolata) and chicory (Cichorium intybus)) receiving 90 kg N/ha/yr (6S); and a nine species sward containing cocksfoot (Dactylis glomerata), greater birdsfoot trefoil (Lotus pedunculatus) and yarrow (Achillea millefolium) in addition to the six species listed above, receiving 90 kg N/ha/yr (9S). Each sward type was managed as a separate farmlet and stocked with 30 twin-rearing ewes at a stocking rate of 12.5 ewes/ha under rotational grazing management from turnout post-lambing until housing. Lamb live weight was recorded fortnightly and lambs were drafted for slaughter at 45 kg. Ewe live weight and body condition score (BCS) were recorded on five occasions annually. Lamb faecal egg count (FEC) was recorded fortnightly and lambs were treated with anthelmintics when mean lamb FEC per sward type was above 400 eggs per gram. Ewes grazing the 6S and 9S swards had heavier (P < 0.01) live weights and BCS throughout the study than the ewes grazing the PRG sward. Lambs grazing the 6S sward were heavier than lambs grazing all other sward types of 14 weeks old (P < 0.05). Lambs grazing the PRG sward required more days to reach slaughter weight than lambs grazing all other sward types (P < 0.001). Lambs grazing the 6S and 9S swards required fewer anthelmintic treatments than lambs grazing the PRG or PRGWC swards. In conclusion, grazing multispecies swards improved ewe and lamb performance and reduced the requirement for chemical anthelmintics.

Palmitoylation and Membrane Binding of Arc/Arg3.1: A Potential Role in Synaptic Depression.

Activity-regulated cytoskeletal-associated protein (Arc, also known as activity-regulated gene 3.1 or Arg3.1) is induced in neurons in response to salient experience and neural activity and is necessary for activity-induced forms of synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD), cellular substrates of learning and memory. The best-characterized function of Arc is enhancement of the endocytic internalization of AMPA receptors in dendritic spines, a process associated with LTD. Arc has also been implicated in the proteolytic processing of amyloid precursor protein on the surface of endosomes. To mediate these activities, Arc must associate with cellular membranes, but it is unclear whether Arc binds directly to the lipid bilayer or requires protein-protein interactions for membrane recruitment. In this study, we show that Arc associates with pure phospholipid vesicles in vitro and undergoes palmitoylation in neurons, a modification that allows it to insert directly into the hydrophobic core of the bilayer. The palmitoylated cysteines are clustered in a motif, 94CLCRC98, located in the N-terminal half of the protein, which has not yet been structurally characterized. Expression of Arc with three mutated cysteines in that motif cannot support synaptic depression induced by the activity-dependent transcription factor, MEF2 (myocyte enhancer factor 2), in contrast to wild-type Arc. Thus, it appears that palmitoylation regulates at least a subset of Arc functions in synaptic plasticity.

A series of 1, 2-coupled indane dimers with mast cell stabilisation and smooth muscle relaxation properties.

Asthma is characterised by bronchoconstriction and inflammation, with infiltration and activation of inflammatory cells such as eosinophils and mast cells, and subsequent release of inflammatory mediators. Much of the therapy directed at the treatment of asthma is either to provide symptomatic relief through bronchodilation or to reduce inflammation to prevent or delay airway remodelling. In an attempt to address both of these issues, a novel series of 1,2-indane dimers has been synthesized and evaluated for smooth muscle relaxant and mast cell stabilising activities. We have identified two lead compounds, 5 and 15, which have substantial mast cell stabilisation activity.

Prevalence and distribution of paratuberculosis (Johne's disease) in cattle herds in Ireland.

A simple random survey was conducted in Ireland during 2005 to estimate the ELISA-prevalence of paratuberculosis, commonly called Johne's disease (JD), in the cattle population. Serum samples were collected from all 20,322 females/breeding bulls over 12 months-of-age in 639 herds. All samples were tested using a commercially available absorbed ELISA. The overall prevalence of infected herds, based on the presence of at least one ELISA-positive animal, was 21.4% (95% CI 18.4%-24.9%). Herd prevalence levels amongst dairy herds (mean 31.5%; 95% CI: 24.6%, 39.3%) was higher than among beef herds (mean 17.9%; 95% CI: 14.6%-21.8%). However, the animal level prevalence was similar. The true prevalence among all animals tested, was calculated to be 2.86% (95%CI: 2.76, 2.97) and for animals >= 2 yrs, it was 3.30% (95%CI: 3.17, 3.43). For animals in beef herds, true prevalence was 3.09% (95%CI: 2.93, 3.24), and for those in dairy herds, 2.74% (95%CI: 2.59, 2.90). The majority of herds had only one ELISA-positive infected animal. Only 6.4% (95% CI 4.7%-8.7%) of all herds had more than one ELISA-positive infected animal; 13.3% (CI 8.7%-19.7%) of dairy herds ranging from two to eight ELISA-positive infected animals; and, 3.9% beef herds (CI 2.4%-6.2%) ranging from two to five ELISA-positive infected animals. The true prevalence of herds infected and shedding Mycobacterium avium subspecies paratuberculosis is estimated to be 9.5% for all herd types; 20.6% for dairy herds; and 7.6% for beef herds. If ELISA positive animals <2-years-of-age are excluded, the true herd prevalene reduces to: 9.3% for all herd types; 19.6% for dairy herds; and 6.3% for beef herds based on a test specificity (Sp) of 99.8% and test sensitivity (Se) (i.e., ability to detect culture-positive, infected animals shedding at any level) of 27.8-28.9%.

Synthesis and pharmacological activity of aminoindanone dimers and related compounds.

A series of N-substituted 3-aminoindanones were synthesised and evaluated for smooth muscle relaxant activity and mediator release inhibition effects. A low level of smooth muscle relaxant activity has been identified in all derivatives. Data have revealed that the significant mediator release inhibition effects observed are related to the nature of the amine substituents. A structure activity relationship is proposed.

Structure of C42D Azotobacter vinelandii FdI. A Cys-X-X-Asp-X-X-Cys motif ligates an air-stable [4Fe-4S]2+/+ cluster.

All naturally occurring ferredoxins that have Cys-X-X-Asp-X-X-Cys motifs contain [4Fe-4S](2+/+) clusters that can be easily and reversibly converted to [3Fe-4S](+/0) clusters. In contrast, ferredoxins with unmodified Cys-X-X-Cys-X-X-Cys motifs assemble [4Fe-4S](2+/+) clusters that cannot be easily interconverted with [3Fe-4S](+/0) clusters. In this study we changed the central cysteine of the Cys(39)-X-X-Cys(42)-X-X-Cys(45) of Azotobacter vinelandii FdI, which coordinates its [4Fe-4S](2+/+) cluster, into an aspartate. UV-visible, EPR, and CD spectroscopies, metal analysis, and x-ray crystallography show that, like native FdI, aerobically purified C42D FdI is a seven-iron protein retaining its [4Fe-4S](2+/+) cluster with monodentate aspartate ligation to one iron. Unlike known clusters of this type the reduced [4Fe-4S](+) cluster of C42D FdI exhibits only an S = 1/2 EPR with no higher spin signals detected. The cluster shows only a minor change in reduction potential relative to the native protein. All attempts to convert the cluster to a 3Fe cluster using conventional methods of oxygen or ferricyanide oxidation or thiol exchange were not successful. The cluster conversion was ultimately accomplished using a new electrochemical method. Hydrophobic and electrostatic interaction and the lack of Gly residues adjacent to the Asp ligand explain the remarkable stability of this cluster.

Novel radicinol derivatives from long-term cultures of alternaria chrysanthemi

Cultures of Alternaria chrysanthemi normally produce radicinin (1) and radicinol (2) when cultured on Czapek-Dox medium or on potato dextrose broth. We have observed that long-term cultures of A. chrysanthemi grown on malt-extract broth produce 3-epiradicinol (3), the novel metabolites 3-methoxy-3-epiradicinol (4) and 9, 10-epoxy-3-methoxy-3-epiradicinol (5), and (2).