A Multi-tissue Transcriptome Analysis of Human Metabolites Guides Interpretability of Associations Based on Multi-SNP Models for Gene Expression.

There is particular interest in transcriptome-wide association studies (TWAS) gene-level tests based on multi-SNP predictive models of gene expression-for identifying causal genes at loci associated with complex traits. However, interpretation of TWAS associations may be complicated by divergent effects of model SNPs on phenotype and gene expression. We developed an iterative modeling scheme for obtaining multi-SNP models of gene expression and applied this framework to generate expression models for 43 human tissues from the Genotype-Tissue Expression (GTEx) Project. We characterized the performance of single- and multi-SNP models for identifying causal genes in GWAS data for 46 circulating metabolites. We show that: (A) multi-SNP models captured more variation in expression than did the top cis-eQTL (median 2-fold improvement); (B) predicted expression based on multi-SNP models was associated (false discovery rate < 0.01) with metabolite levels for 826 unique gene-metabolite pairs, but, after stepwise conditional analyses, 90% were dominated by a single eQTL SNP; (C) among the 35% of associations where a SNP in the expression model was a significant cis-eQTL and metabolomic-QTL (met-QTL), 92% demonstrated colocalization between these signals, but interpretation was often complicated by incomplete overlap of QTLs in multi-SNP models; and (D) using a "truth" set of causal genes at 61 met-QTLs, the sensitivity was high (67%), but the positive predictive value was low, as only 8% of TWAS associations (19% when restricted to colocalized associations at met-QTLs) involved true causal genes. These results guide the interpretation of TWAS and highlight the need for corroborative data to provide confident assignment of causality.

A Multi-omic Integrative Scheme Characterizes Tissues of Action at Loci Associated with Type 2 Diabetes.

Resolving the molecular processes that mediate genetic risk remains a challenge because most disease-associated variants are non-coding and functional characterization of these signals requires knowledge of the specific tissues and cell-types in which they operate. To address this challenge, we developed a framework for integrating tissue-specific gene expression and epigenomic maps to obtain "tissue-of-action" (TOA) scores for each association signal by systematically partitioning posterior probabilities from Bayesian fine-mapping. We applied this scheme to credible set variants for 380 association signals from a recent GWAS meta-analysis of type 2 diabetes (T2D) in Europeans. The resulting tissue profiles underscored a predominant role for pancreatic islets and, to a lesser extent, adipose and liver, particularly among signals with greater fine-mapping resolution. We incorporated resulting TOA scores into a rule-based classifier and validated the tissue assignments through comparison with data from cis-eQTL enrichment, functional fine-mapping, RNA co-expression, and patterns of physiological association. In addition to implicating signals with a single TOA, we found evidence for signals with shared effects in multiple tissues as well as distinct tissue profiles between independent signals within heterogeneous loci. Lastly, we demonstrated that TOA scores can be directly coupled with eQTL colocalization to further resolve effector transcripts at T2D signals. This framework guides mechanistic inference by directing functional validation studies to the most relevant tissues and can gain power as fine-mapping resolution and cell-specific annotations become richer. This method is generalizable to all complex traits with relevant annotation data and is made available as an R package.

Dehydrogenation of ammonia on free-standing and epitaxial hexagonal boron nitride.

We report a thermodynamically feasible mechanism for producing H2 from NH3 using hBN as a catalyst. 2D catalysts have exceptional surface areas with unique thermal and electronic properties suited for catalysis. Metal-free, 2D catalysts, are highly desirable materials that can be more sustainable than the ubiquitously employed precious and transition metal-based catalysts. Here, using density functional theory (DFT) calculations, we demonstrate that metal-free hexagonal boron nitride (hBN) is a valid alternative to precious metal catalysts for producing H2via reaction of ammonia with a boron and nitrogen divacancy (VBN). Our results show that the decomposition of ammonia proceeds on monolayer hBN with an activation energy barrier of 0.52 eV. Furthermore, the reaction of ammonia with epitaxially grown hBN on a Ru(0001) substrate was investigated, and we observed similar NH3 decomposition energy barriers (0.61 eV), but a much more facile H2 associative desorption barrier (0.69 eV vs 5.89 eV). H2 generation from the free-standing monolayer would instead occur through a diffusion process with an energy barrier of 3.36 eV. A detailed analysis of the electron density and charge distribution along the reaction pathways was carried out to rationalise the substrate effects on the catalytic reaction.

Twelve tips for developing and implementing curriculum in dedicated 'collaborative classrooms'.

Many health professional schools may be investing time and resources on dedicated educational spaces intended to promote collaborative learning. Alone, innovative physical space or technologies are not sufficient to ensure success in this. Lesson plans informed by collaborative praxis, individual motivation, faculty development, learner feedback, and team interactions also play a necessary and substantial role. We have used faculty observations, quantitative and qualitative student evaluation data, and the existing educational literature to provide twelve tips on leveraging curricular content, activity setup, physical space, learner behavior, and faculty facilitation to make the most of collaborative learning spaces.

The postauricular fascia: classification, anatomy, and potential surgical applications.

In recent times, there has been evolving interest in the fascial structure of the ear, especially in relation to otoplasty techniques. Although the fascial tissues used in these procedures are referred to as "postauricular/retroauricular fascia," the sparse anatomical studies that exist use this terminology to describe what is the adjacent thicker and more fibrous structure of the superficial temporal area continuous with the mastoid region, rather than the tissue actually used in these procedures which is adherent to the posterior surface of the ear. There are clear clinical differences in the properties of these two structures, and this study set out to identify the anatomical nature of these differences, looking in detail at the anatomy and vascularity of the fascia directly posterior and adherent to the ear itself, highlighting its unique properties, and how it interfaces with the rest of the fascia. We provide a nomenclature to differentiate the fascia adherent to the posterior of the ear (the intrinsic postauricular fascia) from the more fibrous tissues continuous with the scalp fascia (the extrinsic postauricular fascia). Clinical applications for the fascia are suggested based on the vascularity and anatomy described, and our clinical experience.

Theoretical insights into the methane catalytic decomposition on graphene nanoribbons edges.

Catalytic methane decomposition (CMD) is receiving much attention as a promising application for hydrogen production. Due to the high energy required for breaking the C-H bonds of methane, the choice of catalyst is crucial to the viability of this process. However, atomistic insights for the CMD mechanism on carbon-based materials are still limited. Here, we investigate the viability of CMD under reaction conditions on the zigzag (12-ZGNR) and armchair (AGRN) edges of graphene nanoribbons employing dispersion-corrected density functional theory (DFT). First, we investigated the desorption of H and H2 at 1200 K on the passivated 12-ZGNR and 12-AGNR edges. The diffusion of hydrogen atom on the passivated edges is the rate determinant step for the most favourable H2 desorption pathway, with a activation free energy of 4.17 eV and 3.45 eV on 12-ZGNR and 12-AGNR, respectively. The most favourable H2 desorption occurs on the 12-AGNR edges with a free energy barrier of 1.56 eV, reflecting the availability of bare carbon active sites on the catalytic application. The direct dissociative chemisorption of CH4 is the preferred pathway on the non-passivated 12-ZGNR edges, with an activation free energy of 0.56 eV. We also present the reaction steps for the complete catalytic dehydrogenation of methane on 12-ZGNR and 12-AGNR edges, proposing a mechanism in which the solid carbon formed on the edges act as new active sites. The active sites on the 12-AGNR edges show more propensity to be regenerated due lower free energy barrier of 2.71 eV for the H2 desorption from the newly grown active site. Comparison is made between the results obtained here and experimental and computational data available in the literature. We provide fundamental insights for the engineering of carbon-based catalysts for the CMD, showing that the bare carbon edges of graphene nanoribbons have performance comparable to commonly used metallic and bi-metallic catalysts for methane decomposition.

Evolution of ordered nanoporous phases during h-BN growth: controlling the route from gas-phase precursor to 2D material by in situ monitoring.

Large-area single-crystal monolayers of two-dimensional (2D) materials such as graphene and hexagonal boron nitride (h-BN) can be grown by chemical vapour deposition (CVD). However, the high temperatures and fast timescales at which the conversion from a gas-phase precursor to the 2D material appears, make it extremely challenging to simultaneously follow the atomic arrangements. We utilise helium atom scattering to discover and control the growth of novel 2D h-BN nanoporous phases during the CVD process. We find that prior to the formation of h-BN from the gas-phase precursor, a metastable (3 × 3) structure is formed, and that excess deposition on the resulting 2D h-BN leads to the emergence of a (3 × 4) structure. We illustrate that these nanoporous structures are produced by partial dehydrogenation and polymerisation of the borazine precursor upon adsorption. These steps are largely unexplored during the synthesis of 2D materials and we unveil the rich phases during CVD growth. Our results provide significant foundations for 2D materials engineering in CVD, by adjusting or carefully controlling the growth conditions and thus exploiting these intermediate structures for the synthesis of covalent self-assembled 2D networks.

Loss of skeletal muscle strength by ablation of the sarcoplasmic reticulum protein JP45.

Skeletal muscle constitutes approximately 40% of the human body mass, and alterations in muscle mass and strength may result in physical disability. Therefore, the elucidation of the factors responsible for muscle force development is of paramount importance. Excitation-contraction coupling (ECC) is a process during which the skeletal muscle surface membrane is depolarized, causing a transient release of calcium from the sarcoplasmic reticulum that activates the contractile proteins. The ECC machinery is complex, and the functional role of many of its protein components remains elusive. This study demonstrates that deletion of the gene encoding the sarcoplasmic reticulum protein JP45 results in decreased muscle strength in young mice. Specifically, this loss of muscle strength in JP45 knockout mice is caused by decreased functional expression of the voltage-dependent Ca(2+) channel Ca(v)1.1, which is the molecule that couples membrane depolarization and calcium release from the sarcoplasmic reticulum. These results point to JP45 as one of the molecules involved in the development or maintenance of skeletal muscle strength.

The vascular anatomy of the rat superficial epigastric flap by vascular corrosion casting and technical refinement for the study of choke vessels in cadaveric flap models.

Accurate depiction of cutaneous vascular microanatomy is of relevance to plastic surgical flap research, and to descriptive anatomy. Yet current techniques have not permitted full visualization of the subdermal plexus, or potential angiosomal connections. Nor has endothelial visualization been facilitated. Vascular corrosion casting techniques are promising in that regard, and were applied in an extended lateral thoracoabdominal suprafascial adipocutaneous flap in the rat (based on the superficial epigastric bundle). Technical refinements for application to further study of human cadaveric flap models are presented. The intraflap vascular branching pattern of the superficial epigastric artery is described, with filling of the lateral thoracic, intercostals, and iliolumbar angiosomes found when coagulation of vessels at the periphery was delayed until after clearance. The vascular casting protocol presented is an effective and promising tool for the study of macro- and microvascular anatomy.

The anatomic and radiologic basis of the circumflex scapular artery perforator flap.

Microsurgical development has recently focused upon the perforator paradigm and primary thinning. Existing perforator flaps may require intramuscular dissection or lack reliable surface markings, whereas traditional scapular/parascapular flaps have low donor morbidity and reliable anatomy, but can be excessively bulky. Clinical application of a new flap based on a perforator from the circumflex scapular axis (CSA) has recently been published, but the vessel's anatomy has not been adequately characterized. The CSA was dissected in 115 sites in 69 cadavers. The number, external vessel diameter, and site of origin of perforators were measured relative to the CSA bifurcation. Color Doppler ultrasound was used to delineate the CSA and its perforators bilaterally in 40 volunteers. The number, origin relative to CSA bifurcation, diameter, length, and flow velocity of cutaneous perforators were determined. A CSA perforator was always present, running into the subdermal plexus, arising within 2.4 cm of the bifurcation. Cadaver studies: mean perforator diameter, 1.3 mm (SD, 0.66); 13% arose at bifurcation, 36% arose proximal (mean, 1.1 mm; SD, 0.63), and 52% distal to bifurcation (mean, 1.5 mm; SD, 0.88). Ultrasound: mean perforator diameter, 1.18 mm (SD, 0.41); mean flow velocity, 16.3 cm/s (SD, 3.65); perforator arose in 36% proximal, in 40% distal to bifurcation, and in 24% from the bifurcation. We definitively describe the anatomy of the perforator from the circumflex scapular artery upon which a new flap has been based. Its origin and dimensions are anatomically and radiologically reliable. The flap has certain potential benefits over existing perforator flaps.

Genetic variant effects on gene expression in human pancreatic islets and their implications for T2D.

Most signals detected by genome-wide association studies map to non-coding sequence and their tissue-specific effects influence transcriptional regulation. However, key tissues and cell-types required for functional inference are absent from large-scale resources. Here we explore the relationship between genetic variants influencing predisposition to type 2 diabetes (T2D) and related glycemic traits, and human pancreatic islet transcription using data from 420 donors. We find: (a) 7741 cis-eQTLs in islets with a replication rate across 44 GTEx tissues between 40% and 73%; (b) marked overlap between islet cis-eQTL signals and active regulatory sequences in islets, with reduced eQTL effect size observed in the stretch enhancers most strongly implicated in GWAS signal location; (c) enrichment of islet cis-eQTL signals with T2D risk variants identified in genome-wide association studies; and (d) colocalization between 47 islet cis-eQTLs and variants influencing T2D or glycemic traits, including DGKB and TCF7L2. Our findings illustrate the advantages of performing functional and regulatory studies in disease relevant tissues.

The impact of sex on gene expression across human tissues.

Many complex human phenotypes exhibit sex-differentiated characteristics. However, the molecular mechanisms underlying these differences remain largely unknown. We generated a catalog of sex differences in gene expression and in the genetic regulation of gene expression across 44 human tissue sources surveyed by the Genotype-Tissue Expression project (GTEx, v8 release). We demonstrate that sex influences gene expression levels and cellular composition of tissue samples across the human body. A total of 37% of all genes exhibit sex-biased expression in at least one tissue. We identify cis expression quantitative trait loci (eQTLs) with sex-differentiated effects and characterize their cellular origin. By integrating sex-biased eQTLs with genome-wide association study data, we identify 58 gene-trait associations that are driven by genetic regulation of gene expression in a single sex. These findings provide an extensive characterization of sex differences in the human transcriptome and its genetic regulation.

In vitro and in vivo glucuronidation of midazolam in humans.

AIMS: Midazolam (MDZ) is a benzodiazepine used as a CYP3A4 probe in clinical and in vitro studies. A glucuronide metabolite of MDZ has been identified in vitro in human liver microsome (HLM) incubations. The primary aim of this study was to understand the in vivo relevance of this pathway. METHODS: An authentic standard of N-glucuronide was generated from microsomal incubations and isolated using solid-phase extraction. The structure was confirmed using proton nuclear magnetic resonance (NMR) and (1)H-(13)C long range correlation experiments. The metabolite was quantified in vivo in human urine samples. Enzyme kinetic behaviour of the pathway was investigated in HLM and recombinant UGT (rUGT) enzymes. Additionally, preliminary experiments were performed with 1'-OH midazolam (1'-OH MDZ) and 4-OH-midazolam (4-OH MDZ) to investigate N-glucuronidation. RESULTS: NMR data confirmed conjugation of midazolam N-glucuronide (MDZG) standard to be on the alpha-nitrogen of the imidazole ring. In vivo, MDZG in the urine accounted for 1-2% of the administered dose. In vitro incubations confirmed UGT1A4 as the enzyme of interest. The pathway exhibited atypical kinetics and a substrate inhibitory cooperative binding model was applied to determine K(m) (46 microM, 64 microM), V(max) (445 pmol min(-1) mg(-1), 427 pmol min(-1) mg(-1)) and K(i) (58 microM, 79 microM) in HLM and rUGT1A4, respectively. From incubations with HLM and rUGT enzymes, N-glucuronidation of 1'-OH MDZ and 4-OH MDZ is also inferred. CONCLUSIONS: A more complete picture of MDZ metabolism and the enzymes involved has been elucidated. Direct N-glucuronidation of MDZ occurs in vivo. Pharmacokinetic modelling using Simcyp illustrates an increased role for UGT1A4 under CYP3A inhibited conditions.

Loss of ZnT8 function protects against diabetes by enhanced insulin secretion.

A rare loss-of-function allele p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8), which is enriched in Western Finland, protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, particularly when compared with individuals matched for the genotype of a common T2D-risk allele in SLC30A8, p.Arg325. In genome-edited human induced pluripotent stem cell (iPSC)-derived ß-like cells, we establish that the p.Arg138* allele results in reduced SLC30A8 expression due to haploinsufficiency. In human ß cells, loss of SLC30A8 leads to increased glucose responsiveness and reduced KATP channel function similar to isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aimed at maintaining insulin secretion capacity in T2D.

Type 2 diabetes risk alleles in PAM impact insulin release from human pancreatic ß-cells.

The molecular mechanisms underpinning susceptibility loci for type 2 diabetes (T2D) remain poorly understood. Coding variants in peptidylglycine α-amidating monooxygenase (PAM) are associated with both T2D risk and insulinogenic index. Here, we demonstrate that the T2D risk alleles impact negatively on overall PAM activity via defects in expression and catalytic function. PAM deficiency results in reduced insulin content and altered dynamics of insulin secretion in a human ß-cell model and primary islets from cadaveric donors. Thus, our results demonstrate a role for PAM in ß-cell function, and establish molecular mechanisms for T2D risk alleles at this locus.

Fine-mapping type 2 diabetes loci to single-variant resolution using high-density imputation and islet-specific epigenome maps.

We expanded GWAS discovery for type 2 diabetes (T2D) by combining data from 898,130 European-descent individuals (9% cases), after imputation to high-density reference panels. With these data, we (i) extend the inventory of T2D-risk variants (243 loci, 135 newly implicated in T2D predisposition, comprising 403 distinct association signals); (ii) enrich discovery of lower-frequency risk alleles (80 index variants with minor allele frequency <5%, 14 with estimated allelic odds ratio >2); (iii) substantially improve fine-mapping of causal variants (at 51 signals, one variant accounted for >80% posterior probability of association (PPA)); (iv) extend fine-mapping through integration of tissue-specific epigenomic information (islet regulatory annotations extend the number of variants with PPA >80% to 73); (v) highlight validated therapeutic targets (18 genes with associations attributable to coding variants); and (vi) demonstrate enhanced potential for clinical translation (genome-wide chip heritability explains 18% of T2D risk; individuals in the extremes of a T2D polygenic risk score differ more than ninefold in prevalence).

Motor neuron targeting of IGF-1 attenuates age-related external Ca2+-dependent skeletal muscle contraction in senescent mice.

A population of fast muscle fibers from aging mice is dependent on external Ca(2+) to maintain tetanic force during repeated contractions. We hypothesized that age-related denervation in muscle fibers plays a role in initiating this contractile deficit, and that prevention of denervation by IGF-1 overexpression would prevent external Ca(2+)-dependent contraction in aging mice. IGF-1 overexpression in skeletal muscle prevents age-related denervation, and prevented external Ca(2+)-dependent contraction in this work. To determine if the effects of IGF-1 overexpression are on muscle or nerve, aging mice were injected with a tetanus toxin fragment-C (TTC) fusion protein that targets IGF-1 to spinal cord motor neurons. This treatment prevented external Ca(2+)-dependent contraction. We also show evidence that injections of the IGF-1-TTC fusion protein prevent age-related alterations to the nerve terminals at the neuromuscular junctions. We conclude that the slow age-related denervation of fast muscle fibers underlies dependence on external Ca(2+) to maintain tetanic force in a population of muscle fibers from senescent mice.

Exploiting hypoxia in solid tumors to achieve oncolysis.

Chemo- and radio-resistant cancer cells within solid tumors undermine the effectiveness of these approaches to achieving oncolysis. These resistant cells and clusters of cells typically thrive at low oxygen tensions and are reliant on anaerobic metabolic pathways that churn out lactate. This hypoxic state is one that can be exploited and in this paper a novel method is advanced involving tumor cell infiltration by bifidobacterium species which should bring about prodigious lactate synthesis; concomitant blocking of its enzymatic degradation by urea as well as export (from the cell) by use of quercetin; depletion of ATP using exogenous thyroid; and compromised oxidative catabolism of free fatty acids and amino acids via oral intake of l-hydroxycitrate, melatonin and nontoxic NDGA. This "anaerobic pathway cocktail", it is hypothesized, will bring about a profound reduction in intracellular pH and a compromised state of cellular energetics sufficient to effect oncolysis.

Regional transport, source apportionment and health impact of PM10 bound polycyclic aromatic hydrocarbons in Singapore's atmosphere.

A study of 16 United States Environmental Protection Agency (USEPA) priority listed PAHs associated with particulate matter ≤ 10 µm (PM10) was conducted in Singapore during the period 29th May 2015 to 28th May 2016. The sampling period coincided with an extensive, regional smoke haze episode (5th September to 25th October) that occurred as a result of forest and peat fires in neighboring Indonesia. Throughout this study, 54 atmospheric PM10 samples were collected in 24 h periods using a high volume sampler (HVS) and quarts fiber filters (QFF) as the collection medium. Hysplit software for computing 3-D backward air mass trajectories, diagnostic ratio analysis and ring number distribution calculations were used to examine the sources of PAHs in the atmosphere in Singapore. Under normal conditions the total PAH concentrations were in a range from 0.68 ng m-3 to 3.07 ng m-3, while for the high haze period the results showed approximately double the concentrations with a maximum value of 5.97 ng m-3. Diagnostic ratio (DR) and principal component analysis (PCA) were conducted and indicated the contribution of the traffic as a dominant pyrogenic source of PAHs during normal periods, while results from the haze dataset showed relatively strong influence of smoke from peat and forest fires in Indonesia. Environmental and health risk from PAHs were assessed for both regular and hazy days.

Segmental Aging Underlies the Development of a Parkinson Phenotype in the AS/AGU Rat.

There is a paucity of information on the molecular biology of aging processes in the brain. We have used biomarkers of aging (SA ß-Gal, p16Ink4a, Sirt5, Sirt6, and Sirt7) to demonstrate the presence of an accelerated aging phenotype across different brain regions in the AS/AGU rat, a spontaneous Parkinsonian mutant of PKCγ derived from a parental AS strain. P16INK4a expression was significantly higher in AS/AGU animals compared to age-matched AS controls (p < 0.001) and displayed segmental expression across various brain regions. The age-related expression of sirtuins similarly showed differences between strains and between brain regions. Our data clearly show segmental aging processes within the rat brain, and that these are accelerated in the AS/AGU mutant. The accelerated aging, Parkinsonian phenotype, and disruption to dopamine signalling in the basal ganglia in AS/AGU rats, suggests that this rat strain represents a useful model for studies of development and progression of Parkinson's disease in the context of biological aging and may offer unique mechanistic insights into the biology of aging.