Massively parallel reporter assays and variant scoring identified functional variants and target genes for melanoma loci and highlighted cell-type specificity.

The most recent genome-wide association study (GWAS) of cutaneous melanoma identified 54 risk-associated loci, but functional variants and their target genes for most have not been established. Here, we performed massively parallel reporter assays (MPRAs) by using malignant melanoma and normal melanocyte cells and further integrated multi-layer annotation to systematically prioritize functional variants and susceptibility genes from these GWAS loci. Of 1,992 risk-associated variants tested in MPRAs, we identified 285 from 42 loci (78% of the known loci) displaying significant allelic transcriptional activities in either cell type (FDR < 1%). We further characterized MPRA-significant variants by motif prediction, epigenomic annotation, and statistical/functional fine-mapping to create integrative variant scores, which prioritized one to six plausible candidate variants per locus for the 42 loci and nominated a single variant for 43% of these loci. Overlaying the MPRA-significant variants with genome-wide significant expression or methylation quantitative trait loci (eQTLs or meQTLs, respectively) from melanocytes or melanomas identified candidate susceptibility genes for 60% of variants (172 of 285 variants). CRISPRi of top-scoring variants validated their cis-regulatory effect on the eQTL target genes, MAFF (22q13.1) and GPRC5A (12p13.1). Finally, we identified 36 melanoma-specific and 45 melanocyte-specific MPRA-significant variants, a subset of which are linked to cell-type-specific target genes. Analyses of transcription factor availability in MPRA datasets and variant-transcription-factor interaction in eQTL datasets highlighted the roles of transcription factors in cell-type-specific variant functionality. In conclusion, MPRAs along with variant scoring effectively prioritized plausible candidates for most melanoma GWAS loci and highlighted cellular contexts where the susceptibility variants are functional.

An Application Exploiting Aurophilic Bonding and iClick to Produce White Light Emitting Materials.

The paper focuses on exploiting aurophilic bonding to produce white light emitting materials. Inorganic Click (iClick) is employed to link two or four Au(I) metal ions through a triazolate bridge. Depending on the choice of phosphine ligand (PEt3 or PPh3), dinuclear Au2-FO or tetranuclear Au4-FO complexes can be controllably synthesized (FO = 2-(9,9-dioctylfluoreneyl-)). The iClick products Au2-FO and Au4-FO are characterized by combustion analysis and multinuclear NMR, TOCSY 1D, 1H-13C gHMBC, and 1H-13C gHSQC. In addition, the photophysical properties of Au2-FO and Au4-FO were examined in THF solution. Transient absorption spectroscopy was employed to elucidate the excited state features of the gold compounds. Solution processed OLEDs were fabricated and characterized, which gave white light electroluminescence with CIE coordinates (0.34, 0.36), as seen referenced to CIE standard illuminant D65 (0.31, 0.32). TDDFT computational analysis of Au2-FO and Au4-FO reveals the origin of light emission. In the case of Au4-FO, direct excitation leads to increased aurophilic bonding in the excited state, and as a result the emission profile is broadened to cover a larger region of the visible spectrum, thus giving white light emission. Designing molecules that can access or increase aurophilic bonding in the excited state provides another tool for fine-tuning the emission profiles of gold complexes.

Architected Poly(ionic liquid) Composites with Spatially Programmable Mechanical Properties and Mixed Conductivity.

Structural electrolytes present advantages over liquid varieties, which are critical to myriad applications. In particular, structural electrolytes based on polymerized ionic liquids or poly(ionic liquids) (pILs) provide wide electrochemical windows, high thermal stability, nonvolatility, and modular chemistry. However, current methods of fabricating structural electrolytes from pILs and their composites present limitations. Recent advances have been made in 3D printing pIL electrolytes, but current printing techniques limit the complexity of forms that can be achieved, as well as the ability to control mechanical properties or conductivity. We introduce a method for fabricating architected pIL composites as structural electrolytes via embedded 3D (EMB3D) printing. We present a modular design for formulating ionic liquid (IL) monomer composite inks that can be printed into sparse, lightweight, free-standing lattices with different functionalities. In addition to characterizing the rheological and mechanical behaviors of IL monomer inks and pIL lattices, we demonstrate the self-sensing capabilities of our printed structural electrolytes during cyclic compression. Finally, we use our inks and printing method to spatially program self-sensing capabilities in pIL lattices through heterogeneous architectures as well as ink compositions that provide mixed ionic-electronic conductivity. Our free-form approach to fabricating structural electrolytes in complex, 3D forms with programmable, anisotropic properties has broad potential use in next-generation sensors, soft robotics, bioelectronics, energy storage devices, and more.

Physical removal of metallic carbon nanotubes from nanotube network devices using a thermal and fluidic process.

Electronic and optoelectronic devices based on thin films of carbon nanotubes are currently limited by the presence of metallic nanotubes. Here we present a novel approach based on nanotube alkyl functionalization to physically remove the metallic nanotubes from such network devices. The process relies on preferential thermal desorption of the alkyls from the semiconducting nanotubes and the subsequent dissolution and selective removal of the metallic nanotubes in chloroform. The approach is versatile and is applied to devices post-fabrication.

Identification and sequencing of temperature sensitive alleles of the Anaphase Promoting Complex component mat-3 in C. elegans.

The Anaphase Promoting Complex (APC) regulates the transition from metaphase to anaphase during cell division and is important to prevent defects in chromosome segregation. In a recent temperature sensitive genetic screen looking for further genes involved in fertilization, we isolated a new temperature sensitive allele of mat-3 (as49) . We also sequenced a previously identified mat-3 ( or344 ) allele that did not previously have an annotated sequence. We determined that the as49 allele was an Alanine to Threonine (A451T) mutation in the sixth exon and the or344 mutation was a Leucine to Phenylalanine (L474F) mutation in the seventh exon. These locations of the mutant alleles are consistent with other previously annotated alleles that displayed the same metaphase to anaphase transition defect phenotype and further reinforce the importance of the tetratricopeptide repeats to mediate protein interactions.

Context-aware single-cell multiome approach identified cell-type specific lung cancer susceptibility genes.

Genome-wide association studies (GWAS) identified over fifty loci associated with lung cancer risk. However, the genetic mechanisms and target genes underlying these loci are largely unknown, as most risk-associated-variants might regulate gene expression in a context-specific manner. Here, we generated a barcode-shared transcriptome and chromatin accessibility map of 117,911 human lung cells from age/sex-matched ever- and never-smokers to profile context-specific gene regulation. Accessible chromatin peak detection identified cell-type-specific candidate cis-regulatory elements (cCREs) from each lung cell type. Colocalization of lung cancer candidate causal variants (CCVs) with these cCREs prioritized the variants for 68% of the GWAS loci, a subset of which was also supported by transcription factor abundance and footprinting. cCRE colocalization and single-cell based trait relevance score nominated epithelial and immune cells as the main cell groups contributing to lung cancer susceptibility. Notably, cCREs of rare proliferating epithelial cell types, such as AT2-proliferating (0.13%) and basal cells (1.8%), overlapped with CCVs, including those in TERT. A multi-level cCRE-gene linking system identified candidate susceptibility genes from 57% of lung cancer loci, including those not detected in tissue- or cell-line-based approaches. cCRE-gene linkage uncovered that adjacent genes expressed in different cell types are correlated with distinct subsets of coinherited CCVs, including JAML and MPZL3 at the 11q23.3 locus. Our data revealed the cell types and contexts where the lung cancer susceptibility genes are functional.

The promise of graphene-based transistors for democratizing multiomics studies.

As biological research has synthesized genomics, proteomics, metabolomics, and transcriptomics into systems biology, a new multiomics approach to biological research has emerged. Today, multiomics studies are challenging and expensive. An experimental platform that could unify the multiple omics approaches to measurement could increase access to multiomics data by enabling more individual labs to successfully attempt multiomics studies. Field effect biosensing based on graphene transistors have gained significant attention as a potential unifying technology for such multiomics studies. This review article highlights the outstanding performance characteristics that makes graphene field effect transistor an attractive sensing platform for a wide variety of analytes important to system biology. In addition to many studies demonstrating the biosensing capabilities of graphene field effect transistors, they are uniquely suited to address the challenges of multiomics studies by providing an integrative multiplex platform for large scale manufacturing using the well-established processes of semiconductor industry. Furthermore, the resulting digital data is readily analyzable by machine learning to derive actionable biological insight to address the challenge of data compatibility for multiomics studies. A critical stage of systems biology will be democratizing multiomics study, and the graphene field effect transistor is uniquely positioned to serve as an accessible multiomics platform.

Hydrogen sensing and sensitivity of palladium-decorated single-walled carbon nanotubes with defects.

Individual single-walled carbon nanotubes (SWCNTs) become sensitive to H(2) gas when their surfaces are decorated with Pd metal, and previous reports measure typical chemoresistive increases to be approximately 2-fold. Here, thousand-fold increases in resistance are demonstrated in the specific case where a Pd cluster decorates a SWCNT sidewall defect site. Measurements on single SWCNTs, performed both before and after defect incorporation, prove that defects have extraordinary consequences on the chemoresistive response, especially in the case of SWCNTs with metallic band structure. Undecorated defects do not contribute to H(2) chemosensitivity, indicating that this amplification is due to a specific but complex interdependence between a defect site's electronic transmission and the chemistry of the defect-Pd-H(2) system. Dosage experiments suggest a primary role is played by spillover of atomic H onto the defect site.

Discovery of novel allosteric modulators of metabotropic glutamate receptor subtype 5 reveals chemical and functional diversity and in vivo activity in rat behavioral models of anxiolytic and antipsychotic activity.

Modulators of metabotropic glutamate receptor subtype 5 (mGluR5) may provide novel treatments for multiple central nervous system (CNS) disorders, including anxiety and schizophrenia. Although compounds have been developed to better understand the physiological roles of mGluR5 and potential usefulness for the treatment of these disorders, there are limitations in the tools available, including poor selectivity, low potency, and limited solubility. To address these issues, we developed an innovative assay that allows simultaneous screening for mGluR5 agonists, antagonists, and potentiators. We identified multiple scaffolds that possess diverse modes of activity at mGluR5, including both positive and negative allosteric modulators (PAMs and NAMs, respectively). 3-Fluoro-5-(3-(pyridine-2-yl)-1,2,4-oxadiazol-5-yl)benzonitrile (VU0285683) was developed as a novel selective mGluR5 NAM with high affinity for the 2-methyl-6-(phenylethynyl)-pyridine (MPEP) binding site. VU0285683 had anxiolytic-like activity in two rodent models for anxiety but did not potentiate phencyclidine-induced hyperlocomotor activity. (4-Hydroxypiperidin-1-yl)(4-phenylethynyl)phenyl)methanone (VU0092273) was identified as a novel mGluR5 PAM that also binds to the MPEP site. VU0092273 was chemically optimized to an orally active analog, N-cyclobutyl-6-((3-fluorophenyl)ethynyl)nicotinamide hydrochloride (VU0360172), which is selective for mGluR5. This novel mGluR5 PAM produced a dose-dependent reversal of amphetamine-induced hyperlocomotion, a rodent model predictive of antipsychotic activity. Discovery of structurally and functionally diverse allosteric modulators of mGluR5 that demonstrate in vivo efficacy in rodent models of anxiety and antipsychotic activity provide further support for the tremendous diversity of chemical scaffolds and modes of efficacy of mGluR5 ligands. In addition, these studies provide strong support for the hypothesis that multiple structurally distinct mGluR5 modulators have robust activity in animal models that predict efficacy in the treatment of CNS disorders.

Graphitic electrical contacts to metallic single-walled carbon nanotubes using Pt electrodes.

We investigate electronic devices consisting of individual, metallic, single-walled carbon nanotubes contacted by Pt electrodes in a field effect transistor configuration, focusing on improvements to the metal-nanotube contact resistance as the devices are annealed in inert environments including ultrahigh vacuum. At moderate temperatures (T < 880 K), thermal processing results in high resistance contacts with thermally activated barriers. Higher temperatures (T > 880 K) achieve nearly transparent contacts. In the latter case, analytical surface measurements reveal the catalytic decomposition of hydrocarbons into graphene layers on the Pt surface, suggesting that improved electronic behavior is primarily due to the formation of an all-carbon nanotube-graphite interface rather than to the improvement of the nanotube-Pt one.

Controlling Magnetization Vector Depth Profiles of La0.7Sr0.3CoO3/La0.7Sr0.3MnO3 Exchange Spring Bilayers via Interface Reconstruction.

The La0.7Sr0.3CoO3-δ/La0.7Sr0.3MnO3-δ (LSCO/LSMO) bilayer system is an ideal perovskite oxide platform for investigating interface reconstruction and its effect on their magnetic properties. Previous studies have shown that LSCO can separate into magnetic sublayers, which possess distinct trends as the total LSCO thickness increases. In this study, we used polarized neutron reflectometry to quantify changes in the magnetic and chemical depth profiles, and it confirms the formation of ∼12 Å-thick interfacial LSCO and LSMO layers, characterized by a decreased nuclear scattering length density compared to the bulk of the layers. This decrease is attributed to the combined effects of oxygen vacancy formation and interfacial charge transfer, which lead to magnetically active Co2+ ions with ionic radii larger than the Co3+/Co4+ ions typically found in bulk LSCO or single-layer films. The interfacial magnetization values, as well as Co2+ ion and oxygen vacancy concentrations, depend strongly on the LSCO layer thickness. These results highlight the sensitive interplay of the cation valence states, oxygen vacancy concentration, and magnetization at interfaces in perovskite oxide multilayers, demonstrating the potential to tune their functional properties via careful design of their structure.

Novel selective allosteric activator of the M1 muscarinic acetylcholine receptor regulates amyloid processing and produces antipsychotic-like activity in rats.

Recent studies suggest that subtype-selective activators of M(1)/M(4) muscarinic acetylcholine receptors (mAChRs) may offer a novel approach for the treatment of psychotic symptoms associated with schizophrenia and Alzheimer's disease. Previously developed muscarinic agonists have provided clinical data in support of this hypothesis, but failed in clinical development because of a lack of true subtype specificity and adverse effects associated with activation of other mAChR subtypes. We now report characterization of a novel highly selective agonist for the M(1) receptor with no agonist activity at any of the other mAChR subtypes, termed TBPB [1-(1'-2-methylbenzyl)-1,4'-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one]. Mutagenesis and molecular pharmacology studies revealed that TBPB activates M(1) through an allosteric site rather than the orthosteric acetylcholine binding site, which is likely critical for its unprecedented selectivity. Whole-cell patch-clamp recordings demonstrated that activation of M(1) by TBPB potentiates NMDA receptor currents in hippocampal pyramidal cells but does not alter excitatory or inhibitory synaptic transmission, responses thought to be mediated by M(2) and M(4). TBPB was efficacious in models predictive of antipsychotic-like activity in rats at doses that did not produce catalepsy or peripheral adverse effects of other mAChR agonists. Finally, TBPB had effects on the processing of the amyloid precursor protein toward the non-amyloidogenic pathway and decreased Abeta production in vitro. Together, these data suggest that selective activation of M(1) may provide a novel approach for the treatment of symptoms associated with schizophrenia and Alzheimer's disease.

A novel selective muscarinic acetylcholine receptor subtype 1 antagonist reduces seizures without impairing hippocampus-dependent learning.

Previous studies suggest that selective antagonists of specific subtypes of muscarinic acetylcholine receptors (mAChRs) may provide a novel approach for the treatment of certain central nervous system (CNS) disorders, including epileptic disorders, Parkinson's disease, and dystonia. Unfortunately, previously reported antagonists are not highly selective for specific mAChR subtypes, making it difficult to definitively establish the functional roles and therapeutic potential for individual subtypes of this receptor subfamily. The M(1) mAChR is of particular interest as a potential target for treatment of CNS disorders. We now report the discovery of a novel selective antagonist of M(1) mAChRs, termed VU0255035 [N-(3-oxo-3-(4-(pyridine-4-yl)piperazin-1-yl)propyl)-benzo[c][1,2,5]thiadiazole-4 sulfonamide]. Equilibrium radioligand binding and functional studies demonstrate a greater than 75-fold selectivity of VU0255035 for M(1) mAChRs relative to M(2)-M(5). Molecular pharmacology and mutagenesis studies indicate that VU0255035 is a competitive orthosteric antagonist of M(1) mAChRs, a surprising finding given the high level of M(1) mAChR selectivity relative to other orthosteric antagonists. Whole-cell patch-clamp recordings demonstrate that VU0255035 inhibits potentiation of N-methyl-D-aspartate receptor currents by the muscarinic agonist carbachol in hippocampal pyramidal cells. VU0255035 has excellent brain penetration in vivo and is efficacious in reducing pilocarpine-induced seizures in mice. We were surprised to find that doses of VU0255035 that reduce pilocarpine-induced seizures do not induce deficits in contextual freezing, a measure of hippocampus-dependent learning that is disrupted by nonselective mAChR antagonists. Taken together, these data suggest that selective antagonists of M(1) mAChRs do not induce the severe cognitive deficits seen with nonselective mAChR antagonists and could provide a novel approach for the treatment certain of CNS disorders.

Mechanism-guided improvements to the single molecule oxidation of carbon nanotube sidewalls.

Real-time monitoring of carbon nanotube conductance during electrochemical and chemical etching reveals the electronic signatures of individual bond alteration events on the nanotube sidewall. Tracking the conductance of multiple single-molecule experiments through different synthetic protocols supports putative mechanisms for sidewall derivatization. Insights gained from these mechanistic observations imply the formation of sidewall carboxylates, which are useful as handles for bioconjugation. We describe an electronic state required for efficacious chemical treatment. Such real-time monitoring can improve carboxylate yields to 45 % or more. The experiments illustrate the power of molecular nanocircuits to uncover and direct the mechanisms of chemical reactions.

Engineered superlattices with crossover from decoupled to synthetic ferromagnetic behavior.

The extent of interfacial charge transfer and the resulting impact on magnetic interactions were investigated as a function of sublayer thickness in La0.7Sr0.3MnO3/La0.7Sr0.3CoO3 ferromagnetic superlattices. Element-specific soft x-ray magnetic spectroscopy reveals that the electronic structure is altered within 5-6 unit cells of the chemical interface, and can lead to a synthetic ferromagnet with strong magnetic coupling between the sublayers. The saturation magnetization and coercivity depends sensitively on the sublayer thickness due to the length scale of this interfacial effect. For larger sublayer thicknesses, the La0.7Sr0.3MnO3 and La0.7Sr0.3CoO3 sublayers are magnetically decoupled, displaying two independent magnetic transitions with little sublayer thickness dependence. These results demonstrate how interfacial phenomena at perovskite oxide interfaces can be used to tailor their functional properties at the atomic scale.

Polarity-directed analog electro-optic switching in a low-polarization chiral smectic liquid crystal with positive dielectric anisotropy.

We describe an analog electro-optic (EO) switching mechanism occurring in thin cells filled with a low-polarization ferroelectric liquid crystal mixture with positive dielectric anisotropy. The mixture is composed of an achiral nonpolar smectic-C (Sm-C) host doped with a small amount of a commercially available unichiral compound. The switching mechanism provides analog EO behavior, and thus could be attractive for information display applications. The process is polarization-driven for weak fields, while for higher field strength the dielectric coupling dominates the process.

Etching of surfactant from solution-processed, type-separated carbon nanotubes and impact on device behavior.

Semiconducting single-walled carbon nanotubes (SWCNTs) have great potential for use in electronic and optoelectronic devices. However, methods for synthesizing SWCNTs produce a mixture of metallic and semiconducting materials, which require additional processing to separate by electronic type. Purification and enrichment of the semiconducting fraction is readily achieved by using the centrifugation of aqueous suspensions of SWCNTs with the help of surfactants, but this leaves residual surfactant on the SWCNT surface that can impact their electronic and optical properties. Here, we present a detailed study of the sodium taurodeoxycholate (STDC) surfactant removal process during vacuum annealing, showing that it occurs through fragmentation of the surfactant, and that complete removal requires exceedingly high temperatures, which indicates strong binding to the SWCNTs. We then present an approach based on air oxidation and mild annealing to completely remove the surfactant while maintaining the SWCNT properties. Using this approach, we compare single SWCNT electronic devices with and without STDC and show that, despite the very strong surfactant binding, it does not affect device performance substantially.

Photothermoelectric p-n junction photodetector with intrinsic broadband polarimetry based on macroscopic carbon nanotube films.

Light polarization is used in the animal kingdom for communication, navigation, and enhanced scene interpretation and also plays an important role in astronomy, remote sensing, and military applications. To date, there have been few photodetector materials demonstrated to have direct polarization sensitivity, as is usually the case in nature. Here, we report the realization of a carbon-based broadband photodetector, where the polarimetry is intrinsic to the active photodetector material. The detector is based on p-n junctions formed between two macroscopic films of single-wall carbon nanotubes. A responsivity up to ~1 V/W was observed in these devices, with a broadband spectral response spanning the visible to the mid-infrared. This responsivity is about 35 times larger than previous devices without p-n junctions. A combination of experiment and theory is used to demonstrate the photothermoelectric origin of the responsivity and to discuss the performance attributes of such devices.

(3-Cyano-5-fluorophenyl)biaryl negative allosteric modulators of mGlu(5): Discovery of a new tool compound with activity in the OSS mouse model of addiction.

Glutamate is the major excitatory transmitter in the mammalian CNS, exerting its effects through both ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors (mGlus) belong to family C of the G-protein-coupled receptors (GPCRs). The eight mGlus identified to date are classified into three groups based on their structure, preferred signal transduction mechanisms, and pharmacology (Group I: mGlu(1) and mGlu(5); Group II: mGlu(2) and mGlu(3); Group III: mGlu(4), mGlu(6), mGlu(7), and mGlu(8)). Non-competitive antagonists, also known as negative allosteric modulators (NAMs), of mGlu(5) offer potential therapeutic applications in diseases such as pain, anxiety, gastroesophageal reflux disease (GERD), Parkinson's disease (PD), fragile X syndrome, and addiction. The development of SAR in a (3-cyano-5-fluorophenyl)biaryl series using our functional cell-based assay is described in this communication. Further characterization of a selected compound, 3-fluoro-5-(2-methylbenzo[d]thiazol-5-yl)benzonitrile, in additional cell based assays as well as in vitro assays designed to measure its metabolic stability and protein binding indicated its potential utility as an in vivo tool. Subsequent evaluation of the same compound in a pharmacokinetic study using intraperitoneal dosing in mice showed good exposure in both plasma and brain samples. The compound was efficacious in a mouse marble burying model of anxiety, an assay known to be sensitive to mGlu(5) antagonists. A new operant model of addiction termed operant sensation seeking (OSS) was chosen as a second behavioral assay. The compound also proved efficacious in the OSS model and constitutes the first reported example of efficacy with a small molecule mGlu(5) NAM in this novel assay.

Discovery and characterization of novel subtype-selective allosteric agonists for the investigation of M(1) receptor function in the central nervous system.

Cholinergic transmission in the forebrain is mediated primarily by five subtypes of muscarinic acetylcholine receptors (mAChRs), termed M(1)-M(5). Of the mAChR subtypes, M(1) is among the most heavily expressed in regions that are critical for learning and memory, and has been viewed as the most critical mAChR subtype for memory and attention mechanisms. Unfortunately, it has been difficult to develop selective activators of M(1) and other individual mAChR subtypes, which has prevented detailed studies of the functional roles of selective activation of M(1). Using a functional HTS screen and subsequent diversity-oriented synthesis approach we have discovered a novel series of highly selective M(1) allosteric agonists. These compounds activate M(1) with EC(50) values in the 150 nM to 500 nM range and have unprecedented, clean ancillary pharmacology (no substantial activity at 10µM across a large panel of targets). Targeted mutagenesis revealed a potentially novel allosteric binding site in the third extracellular loop of the M(1) receptor for these allosteric agonists. Optimized compounds, such as VU0357017, provide excellent brain exposure after systemic dosing and have robust in vivo efficacy in reversing scopolamine-induced deficits in a rodent model of contextual fear conditioning. This series of selective M(1) allosteric agonists provides critical research tools to allow dissection of M(1)-mediated effects in the CNS and potential leads for novel treatments for Alzheimer's disease and schizophrenia.