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An unbiased genome-scale screen for unmutated genes that drive cancer growth when overexpressed identified methyl cytosine-guanine dinucleotide (CpG) binding protein 2 (MECP2) as a novel oncogene. MECP2 resides in a region of the X-chromosome that is significantly amplified across 18% of cancers, and many cancer cell lines have amplified, overexpressed MECP2 and are dependent on MECP2 expression for growth. MECP2 copy-number gain and RAS family member alterations are mutually exclusive in several cancer types. The MECP2 splicing isoforms activate the major growth factor pathways targeted by activated RAS, the MAPK and PI3K pathways. MECP2 rescued the growth of a KRASG12C-addicted cell line after KRAS downregulation, and activated KRAS rescues the growth of an MECP2-addicted cell line after MECP2 downregulation. MECP2 binding to the epigenetic modification 5-hydroxymethylcytosine is required for efficient transformation. These observations suggest that MECP2 is a commonly amplified oncogene with an unusual epigenetic mode of action.Significance:MECP2 is a commonly amplified oncogene in human malignancies with a unique epigenetic mechanism of action. Cancer Discov; 6(1); 45–58. ©2015 AACR.This article is highlighted in the In This Issue feature, p. 1

Analysis of Uterine Cancer Subtypes. This file contains analysis of mutual exclusivity relationship between MECP2 amplification and KRAS mutation in uterine cancer subtypes.

Supplementary Figure S1. The Amplification of the MECP2 Gene Drives Its Expression, and the MECP2 Gene on the Active X Chromosome is Preferentially Amplified, Related to Figure 1. Supplementary Figure S2. MECP2 Overexpression Allows Soft Agar Growth of N minus RAS Cells in Two Different Types of Human Breast Epithelial Cells, and MECP2 Splicing Isoforms Differ In Their Ability to Confer Anchorage Independent Growth, Related to Figure 2A. Supplementary Figure S3: MECP2 Overexpression Allows Two Different Types of N minus RAS Breast Epithelial Cells To Grow As Tumor Xenograft In Nude Mice, Related to Figure 2E, 2F. Supplementary Figure S4. The MECP2 e2 Short Splicing Isoform Allows Sustained Activation of the MAPK Pathway after Prolonged Starvation in Minimal Medium without Growth Factors, Related to Figure 4A-C. Supplementary Figure S5. Both MECP2 Isoforms Activate the PI3K Pathway, Related to Figure 4D. Supplementary Figure S6. Additional human cancer cell lines with high level of MECP2 are growth-inhibited upon inhibition of MECP2 expression, Related to Figure 4E.

Supplementary Table S1. List of validated hits from N minus RAS screen. Supplementary Table S2. Amplicons in Human Cancer (from Broad Institute TCGA Copy Number Portal). Supplementary Table S3. Cancer cell lines used in current study with their MECP2 expression and RAS/MAPK/PI3K pathways mutation status (based on COSMIC, canSAR, ATCC, and CCLE databases).

Supplementary Methods from MECP2 Is a Frequently Amplified Oncogene with a Novel Epigenetic Mechanism That Mimics the Role of Activated RAS in Malignancy

The study and application of transition metal hydrides (TMH), an active area of chemical research since the early 1960’s(1), has been broadly bifurcated into fields focused on energy storage through the reduction of protons to generate hydrogen(2,3) and in organic synthesis for the functionalization of unsaturated C–C, C–O, and C–N bonds.(4,5) In the former instance, electrochemical means for driving such reactivity has been commonplace since the 1950’s(6) but the use of stoichiometric exogenous organic- and metal-based reductants to harness the power of TMHs in synthetic chemistry remains the norm. In particular, Co-based TMHs have found widespread use for the derivatization of olefins and alkynes in complex molecule construction, often via a net hydrogen atom transfer (HAT).(7) Here, we show how an electrocatalytic approach inspired by decades of energy storage precedent can be leveraged in the context of modern organic synthesis. This strategy not only offers benefits in terms of sustainability and efficiency but also enables enhanced chemoselectivity and unique and tunable reactivity. Ten different reaction manifolds across dozens of substrates are thus exemplified, along with detailed mechanistic insight on this scalable electrochemical entry to Co-H generation that uniquely takes place through a low-valent intermediate.

SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratory’s refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics which inhibit coronavirus budding.

Enzymatic pockets such as those of histone deacetylases (HDACs) are among the most favored targets for drug development. However, enzymatic inhibitors often exhibit low selectivity and high toxicity due to targeting multiple enzyme paralogs, which are often involved in distinct multisubunit complexes. Here, we report the discovery and characterization of a non-enzymatic small molecule inhibitor of HDAC transcriptional repression functions with comparable anti-tumor activity to the enzymatic HDAC inhibitor Vorinostat, and anti-psychedelic activity of anHDAC2knockoutin vivo. We highlight that these phenotypes are achieved while modulating the expression of 20- and 80-fold fewer genes than enzymatic and genetic inhibition in the respective models. Thus, by achieving the same biological outcomes as established therapeutics while impacting a dramatically smaller number of genes, inhibitors of protein-protein interactions can offer important advantages in improving the selectivity of epigenetic modulators.GRAPHICAL ABSTRACT

Generating reference maps of the interactome networks underlying most cellular functions can greatly illuminate genetic studies by providing a protein-centric approach to finding new components of existing pathways, complexes, and processes. Here, we applied state-of-the-art experimental and bioinformatics methods to identify high-confidence binary protein-protein interactions (PPIs) for Drosophila melanogaster. We performed four all-by-all yeast two-hybrid (Y2H) screens of >10,000 Drosophila proteins, resulting in the ‘FlyBi’ dataset of 8,723 PPIs among 2,939 proteins. As part of this effort, we tested subsets of our data and data from previous PPI datasets using an orthogonal assay, which allowed us to normalize data quality across datasets. Next, we integrated our FlyBi data with previous PPI data, resulting in an expanded, high-confidence binary Drosophila reference interaction network, DroRI, comprising 17,232 interactions among 6,511 proteins. These data are accessible through the Molecular Interaction Search Tool (MIST) and other databases. To assess the utility of the PPI resource, we used novel interactions from the FlyBi dataset to generate an autophagy interaction network that we validated in vivo using two different autophagy-related assays. We found that deformed wings (dwg) encodes a protein that is both a regulator and a target of autophagy. Altogether, the resources generated in this project provide a strong foundation for building high-confidence new hypotheses regarding protein networks and function.

Technological and computational advances in genomics and interactomics have made it possible to identify how disease mutations perturb protein–protein interaction (PPI) networks within human cells. Here, we show that disease-associated germline variants are significantly enriched in sequences encoding PPI interfaces compared to variants identified in healthy participants from the projects 1000 Genomes and ExAC. Somatic missense mutations are also significantly enriched in PPI interfaces compared to noninterfaces in 10,861 tumor exomes. We computationally identified 470 putative oncoPPIs in a pan-cancer analysis and demonstrate that oncoPPIs are highly correlated with patient survival and drug resistance/sensitivity. We experimentally validate the network effects of 13 oncoPPIs using a systematic binary interaction assay, and also demonstrate the functional consequences of two of these on tumor cell growth. In summary, this human interactome network framework provides a powerful tool for prioritization of alleles with PPI-perturbing mutations to inform pathobiological mechanism- and genotype-based therapeutic discovery. Human disease mutations affect protein–protein interfaces in a three-dimensional structurally resolved interaction network. Predicted oncoPPIs in cancer correlate with survival and drug sensitivity, and affect growth in vitro, supporting their relevance to disease pathogenesis.

Human pluripotent stem cells (hPSCs) offer an unprecedented opportunity to model diverse cell types and tissues. To enable systematic exploration of the programming landscape mediated by transcription factors (TFs), we present the Human TFome, a comprehensive library containing 1,564 TF genes and 1,732 TF splice isoforms. By screening the library in three hPSC lines, we discovered 290 TFs, including 241 that were previously unreported, that induce differentiation in 4 days without alteration of external soluble or biomechanical cues. We used four of the hits to program hPSCs into neurons, fibroblasts, oligodendrocytes and vascular endothelial-like cells that have molecular and functional similarity to primary cells. Our cell-autonomous approach enabled parallel programming of hPSCs into multiple cell types simultaneously. We also demonstrated orthogonal programming by including oligodendrocyte-inducible hPSCs with unmodified hPSCs to generate cerebral organoids, which expedited in situ myelination. Large-scale combinatorial screening of the Human TFome will complement other strategies for cell engineering based on developmental biology and computational systems biology.

Most human protein-coding genes are expressed as multiple isoforms, which greatly expands the functional repertoire of the encoded proteome. While at least one reliable open reading frame (ORF) model has been assigned for every coding gene, the majority of alternative isoforms remains uncharacterized due to (i) vast differences of overall levels between different isoforms expressed from common genes, and (ii) the difficulty of obtaining full-length transcript sequences. Here, we present ORF Capture-Seq (OCS), a flexible method that addresses both challenges for targeted full-length isoform sequencing applications using collections of cloned ORFs as probes. As a proof-of-concept, we show that an OCS pipeline focused on genes coding for transcription factors increases isoform detection by an order of magnitude when compared to unenriched samples. In short, OCS enables rapid discovery of isoforms from custom-selected genes and will accelerate mapping of the human transcriptome., Most human protein-coding genes are expressed as multiple isoforms. Here the authors present ORF Capture-seq that uses cloned ORFs as probes to capture and sequence full length transcript sequences. This enables highly sensitive characterization of eukaryotic transcriptomes.

Protein-protein interactions (PPIs) are essential in understanding numerous aspects of protein function. Here, we significantly scaled and modified analyses of the recently developed all-vs-all sequencing (AVA-Seq) approach using a gold-standard human protein interaction set (hsPRS-v2) containing 98 proteins. Binary interaction analyses recovered 20 of 47 (43%) binary PPIs from this reference set, comparing favorably with other methods. However, the increase of 20X in the interaction search space for all-vs-all sequencing analysis in this manuscript resulted in numerous changes to the method required for future use in genome-wide interaction studies. We show that standard sequencing analysis methods must be modified to consider the possible recovery of thousands of positives among millions of tested interactions in a single sequencing run. The PRS data was used to optimize data scaling, auto-activator removal, rank interaction features (such as orientation and unique fragment pairs), and statistical cutoffs. Using these modifications to the method, AVA-Seq recovered >500 known and novel PPIs, including interactions between wild-type fragments of tumor protein p53 and minichromosomal maintenance complex proteins 2 and 5 (MCM2 and MCM5) that could be of interest in human disease. This article is protected by copyright. All rights reserved.

SummaryHundreds of different protein complexes that perform important functions across all cellular processes, collectively comprising the “complexome” of an organism, have been identified1. However, less is known about the fraction of the interactome that exists outside the complexome, in the “outer-complexome”. To investigate features of “inner”- versus outer-complexome organisation in yeast, we generated a high-quality atlas of binary protein-protein interactions (PPIs), combining three previous maps2–4 and a new reference all-by-all binary interactome map. A greater proportion of interactions in our map are in the outer-complexome, in comparison to those found by affinity purification followed by mass spectrometry5–7 or in literature curated datasets8–11. In addition, recent advances in deep learning predictions of PPI structures12 mirror the existing experimentally resolved structures in being largely focused on the inner complexome and missing most interactions in the outer-complexome. Our new PPI network suggests that the outer-complexome contains considerably more PPIs than the inner-complexome, and integration with functional similarity networks13–15 reveals that interactions in the inner-complexome are highly detectable and correspond to pairs of proteins with high functional similarity, while proteins connected by more transient, harder-to-detect interactions in the outer-complexome, exhibit higher functional heterogeneity.

The study and application of transition metal hydrides (TMH) has been an active area of chemical research since the early 1960’s. The use of TMHs has been broadly bifurcated into fields focused on energy storage through the reduction of protons to generate hydrogen and in organic synthesis for the functionalization of unsaturated C–C, C–O, and C–N bonds. In the former instance, electrochemical means for driving such reactivity has been commonplace since the 1950’s. In contrast, the use of stoichiometric exogenous organic and metal-based reductants to harness the power of TMHs in synthetic chemistry remains the norm. In particular, Co-based TMHs have found widespread use for the derivatization of olefins and alkynes in complex molecule construction, often via a net hydrogen atom transfer (HAT). Here, we show how an electrocatalytic approach inspired by decades of energy storage precedent can be leveraged in the context of modern organic synthesis. Such an approach not only offers benefits in terms of sustainability and efficiency but also enables enhanced chemoselectivity and unique and tunable reactivity. Ten different reaction manifolds across dozens of substrates are thus exemplified, along with a detailed mechanistic and computational analysis of this scalable electrochemical entry into Co-H chemistry.

Protein-protein interactions (PPIs) are important in understanding numerous aspects of protein function. Here, the recently developed all-vs-all sequencing (AVA-Seq) approach to determine protein-protein interactions was tested on a gold-standard human protein interaction set (hsPRS-v2). Initially, these data were interpreted strictly from a binary PPI perspective to compare AVA-Seq to other binary PPI methods tested on the same hsPRS-v2. AVA-Seq recovered 20 of 47 (43%) binary PPIs from this reference set comparing favorably with other methods. The same experimental data allowed for the determination of >500 known and novel PPIs including interactions between wildtype fragments of tumor protein p53 and minichromosomal maintenance complex proteins 2, and 5 (MCM2 and MCM5) that could be of interest in human disease. Additional results gave a better understanding of why interactions might be missed using AVA-Seq and aide future PPI experimental design for maximum recovery of information.

Integrative analysis that combines genome-wide association data with expression quantitative trait analysis and network representation may illuminate causal relationships between genes and diseases. To identify causal lipid genes, we utilized genotype, gene expression, protein-protein interaction networks, and phenotype data from 5,257 Framingham Heart Study participants and performed Mendelian randomization to investigate possible mechanistic explanations for observed associations. We selected three putatively causal candidate genes (ABCA6, ALDH2, and SIDT2) for lipid traits (LDL cholesterol, HDL cholesterol and triglycerides) in humans and conducted mouse knockout studies for each gene to confirm its causal effect on the corresponding lipid trait. We conducted the RNA-seq from mouse livers to explore transcriptome-wide alterations after knocking out the target genes. Our work builds upon a lipid-related gene network and expands upon it by including protein-protein interactions. These resources, along with the innovative combination of emerging analytical techniques, provide a groundwork upon which future studies can be designed to more fully understand genetic contributions to cardiovascular diseases.

One of the most oft-employed methods for C-C bond formation involving the coupling of vinyl-halides with aldehydes catalyzed by Ni and Cr (Nozaki-Hiyama-Kishi, NHK) has been rendered more practical using an electroreductive manifold. Although early studies pointed to the feasibility of such a process, those precedents were never applied by others due to cumbersome setups and limited scope. Here we show that a carefully optimized electroreductive procedure can enable a more sustainable approach to NHK, even in an asymmetric fashion on highly complex medicinally relevant systems. The e-NHK can even enable non-canonical substrate classes, such as redox-active esters, to participate with low loadings of Cr when conventional chemical techniques fail. A combination of detailed kinetics, cyclic voltammetry, and in situ UV-vis spectroelectrochemistry of these processes illuminates the subtle features of this mechanistically intricate process.

Recent advances in DNA/RNA sequencing have made it possible to identify new targets rapidly and to repurpose approved drugs for treating heterogeneous diseases by the ‘precise’ targeting of individualized disease modules. In this study, we develop a Genome-wide Positioning Systems network (GPSnet) algorithm for drug repurposing by specifically targeting disease modules derived from individual patient’s DNA and RNA sequencing profiles mapped to the human protein-protein interactome network. We investigate whole-exome sequencing and transcriptome profiles from ~5,000 patients across 15 cancer types from The Cancer Genome Atlas. We show that GPSnet-predicted disease modules can predict drug responses and prioritize new indications for 140 approved drugs. Importantly, we experimentally validate that an approved cardiac arrhythmia and heart failure drug, ouabain, shows potential antitumor activities in lung adenocarcinoma by uniquely targeting a HIF1α/LEO1-mediated cell metabolism pathway. In summary, GPSnet offers a network-based, in silico drug repurposing framework for more efficacious therapeutic selections., Identification of disease modules in the human interactome can guide more efficacious therapeutic selections. Here, the authors introduce a network-based methodology to identify individualized disease modules by mapping patients’ DNA and RNA sequencing profiles to the interactome, enabling prediction of cancer type-specific drug responses.

Site-selective chemical bioconjugation reactions are enabling tools for the chemical biologist. Guided by a careful study of the selenomethionine (SeM) benzylation, we have refined the reaction to meet the requirements of practical protein bioconjugation. SeM is readily introduced through auxotrophic expression and exhibits unique nucleophilic properties that allow it to be selectively modified even in the presence of cysteine. The resulting benzylselenonium adduct is stable at physiological pH, is selectively labile to glutathione, and embodies a broadly tunable cleavage profile. Specifically, a 4-bromomethylphenylacetyl (BrMePAA) linker has been applied for efficient conjugation of complex organic molecules to SeM-containing proteins. This expansion of the bioconjugation toolkit has broad potential in the development of chemically enhanced proteins.

SUMMARYWhile viral infections are known to hijack the transcription and translation of the host cell, the extent to which encoded viral proteins coordinate these perturbations remains unclear. Here we demonstrate that the oncoviral proteins Tax and HBZ interact with specific components of the spliceosome machinery, including the U2 auxiliary factor large subunit (U2AF2), and the complementary factor for APOBEC-1 (A1CF), respectively. Tax and HBZ perturb the splicing landscape in T-cells by altering cassette exons in opposing manners, with Tax inducing exon inclusion while HBZ induces exon exclusion. Among Tax- and HBZ-dependent splicing changes, we identify events that are also altered in Adult T cell leukemia (ATL) patients, and in well-known cancer census genes. Our interactome mapping approach, applicable to other viral oncogenes, has identified spliceosome perturbation as a novel mechanism coordinately used by Tax and HBZ to reprogram the transcriptome.HighlightsTax and HBZ interact with RNA-binding proteins as well as transcription factorsHTLV-1 encoded proteins Tax and HBZ alter the splicing landscape in T-cellsTax and HBZ expression affect alternative splicing of 33 and 63 cancer genes, respectivelyOpposing roles for Tax and HBZ in deregulation of gene expressionGraphical abstract

Mechanistic investigations uncover a novel role for 2-pyridone ligands and interrogate the origin of enantioselectivity in the (+)-norbornene-mediated Pd-catalyzed meta-C(aryl)-H functionalization of diarylmethylamines. Observations from kinetic analysis in concert with in situ 19F NMR monitoring allow us to propose that the pyridone ligand plays a role in enhancing the rate- and enantio-determining insertion of an arylpalladium species into a chiral norbornene derivative. The unprecedented features of 2-pyridone ligands in asymmetric 1,2 migratory insertion, and norbornene as a transient chiral mediator in relay chemistry, provide new insights into this ligand scaffold for future developments in stereoselective transition-metal-catalyzed C-H functionalization.

The molecular functions of the majority of RNA-binding proteins (RBPs) remain unclear, highlighting a major bottleneck to a full understanding of gene expression regulation. Here, we develop a plasmid resource of 690 human RBPs that we subject to luciferase-based 3ʹ-untranslated-region tethered function assays to pinpoint RBPs that regulate RNA stability or translation. Enhanced UV-cross-linking and immunoprecipitation of these RBPs identifies thousands of endogenous mRNA targets that respond to changes in RBP level, recapitulating effects observed in tethered function assays. Among these RBPs, the ubiquitin-associated protein 2-like (UBAP2L) protein interacts with RNA via its RGG domain and cross-links to mRNA and rRNA. Fusion of UBAP2L to RNA-targeting CRISPR–Cas9 demonstrates programmable translational enhancement. Polysome profiling indicates that UBAP2L promotes translation of target mRNAs, particularly global regulators of translation. Our tethering survey allows rapid assignment of the molecular activity of proteins, such as UBAP2L, to specific steps of mRNA metabolism. A survey of human RNA-binding proteins based on luciferase-based 3ʹ-untranslated-region tethered function assays and identification of their target mRNAs provides insights into their role in RNA metabolism.

Technological and computational advances in genomics and interactomics have made it possible to identify rapidly how disease mutations perturb interaction networks within human cells. In this study, we investigate at large-scale the effects of network perturbations caused by disease mutations within the human three-dimensional (3D), structurally-resolved macromolecular interactome. We show that disease-associated germline mutations are significantly enriched in sequences encoding protein-protein interfaces compared to mutations identified in healthy subjects from the 1000 Genomes and ExAC projects; these interface mutations correspond to protein-protein interaction (PPI)-perturbing alleles including p.Ser127Arg in PCSK9 at the PCSK9-LDLR interface. In addition, somatic missense mutations are significantly enriched in PPI interfaces compared to non-interfaces in 10,861 human exomes across 33 cancer subtypes/types from The Cancer Genome Atlas. Using a binomial statistical model, we computationally identified 470 PPIs harboring a statistically significant excess number of missense mutations at protein-protein interfaces (termed putative oncoPPIs) in pan-cancer analysis. We demonstrate that the oncoPPIs, including histone H4 complex in individual cancer types, are highly correlated with patient survival and drug resistance/sensitivity in human cancer cell lines and patient-derived xenografts. We experimentally validate the network effects of 13 oncoPPIs using a systematic binary interaction assay. We further showed that ALOX5 p.Met146Lys at the ALOX5-MAD1L1 interface and RXRA p.Ser427Phe at the RXRA-PPARG interface promote significant tumor cell growth using cell line-based functional assays, providing a functional proof-of-concept. In summary, if broadly applied, this human 3D interactome network analysis offers a powerful tool for prioritizing alleles with mutations altering PPIs that may contribute to the pathobiology of human diseases, and may offer disease-specific targets for genotype-informed therapeutic discovery.

et al., Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype–phenotype relationships1,2. Here we present a human ‘all-by-all’ reference interactome map of human binary protein interactions, or ‘HuRI’. With approximately 53,000 protein–protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome3, transcriptome4 and proteome5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein–protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes., This work was primarily supported by the National Institutes of Health (NIH) National Human Genome Research Institute (NHGRI) grant U41HG001715 (M.V., F.P.R., D.E.H., M.A.C., G.D.B. and J.T.) with additional support from NIH grants P50HG004233 (M.V. and F.P.R.), U01HL098166 (M.V.), U01HG007690 (M.V.), R01GM109199 (M.A.C.), Canadian Institute for Health Research (CIHR) Foundation Grants (F.P.R. and J. Rak), the Canada Excellence Research Chairs Program (F.P.R.) and an American Heart Association grant 15CVGPS23430000 (M.V.). D.-K.K. was supported by a Banting Postdoctoral Fellowship through the Natural Sciences and Engineering Research Council (NSERC) of Canada and by the Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education (2017R1A6A3A03004385). C. Pons was supported by a Ramon Cajal fellowship (RYC-2017-22959). G.M.S. was supported by NIH Training Grant T32CA009361. M.V. is a Chercheurv Qualifié Honoraire from the Fonds de la Recherche Scientifique (FRS-FNRS, Wallonia-Brussels Federation, Belgium).

The worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SARS-CoV-2 proteins orchestrate complex pathogenic mechanisms that underlie COVID-19 disease. Thus, understanding how viral polypeptides rewire host protein networks enables better-founded therapeutic research. In complement to existing proteomic studies, in this study we define the first proximal interaction network of SARS-CoV-2 proteins, at the whole proteome level in human cells. Applying a proximity-dependent biotinylation (BioID)-based approach greatly expanded the current knowledge by detecting interactions within poorly soluble compartments, transient, and/or of weak affinity in living cells. Our BioID study was complemented by a stringent filtering and uncovered 2,128 unique cellular targets (1,717 not previously associated with SARS-CoV-1 or 2 proteins) connected to the N- and C-ter BioID-tagged 28 SARS-CoV-2 proteins by a total of 5,415 (5,236 new) proximal interactions. In order to facilitate data exploitation, an innovative interactive 3D web interface was developed to allow customized analysis and exploration of the landscape of interactions (accessible at http://www.sars-cov-2-interactome.org/). Interestingly, 342 membrane proteins including interferon and interleukin pathways factors, were associated with specific viral proteins. We uncovered ORF7a and ORF7b protein proximal partners that could be related to anosmia and ageusia symptoms. Moreover, comparing proximal interactomes in basal and infection-mimicking conditions (poly(I:C) treatment) allowed us to detect novel links with major antiviral response pathway components, such as ORF9b with MAVS and ISG20; N with PKR and TARB2; NSP2 with RIG-I and STAT1; NSP16 with PARP9-DTX3L. Altogether, our study provides an unprecedented comprehensive resource for understanding how SARS-CoV-2 proteins orchestrate host proteome remodeling and innate immune response evasion, which can inform development of targeted therapeutic strategies.

Proton irradiation of a thorium (Th) target can produce Actinium-225 (Ac-225), but the irradiated thorium fissions results in the production of 100’s of other isotopes. SynPhase Lanterns containing sulfonic acid groups were evaluated for the purification of Ac-225 from Th and other fission metals. The SynPhase Lanterns were able to quantitatively and selectively capture Ac-225 and lanthanum (La) over Th. A second purification step would be needed to purify Ac-225 from trace amounts of Th and other fission products. The results from these studies were compared to similar studies performed with cation exchange resins, and the resins were superior at removing impurities.

The potential role of dimeric catalyst species on or off the catalytic cycle is considered for a case of Pd-catalyzed C–H functionalization, leading to the development of a general experimental protocol that uses the reaction itself to report on the presence and role of dimeric species in asymmetric catalytic reactions.

A catalytic γ-selective syn-hydroarylation of alkenyl carbonyl compounds using arylboronic acids has been developed using a substrate directivity approach with a palladium(II) catalyst. This method tolerates a wide range of functionalized (hetero)arylboronic acids and a variety of substitution patterns on the alkene. Preliminary mechanistic studies suggest that transmetalation is rate-limiting.

A new tool for probing enantioselective reaction mechanisms is introduced. Monitoring the temporal change in product enantiomeric excess after addition of the opposite enantiomer of the ligand during the reaction provides a means of probing dynamic ligand exchange in enantioselective C-H iodination catalyzed by Pd with monoprotected amino acid ligands (MPAAs). This work has general potential to provide insights about the dynamics of catalyst and ligand molecularity and exchange.

Kinetic and mechanistic studies of the desymmetrization of benzhydrylamine using Pd/monoprotected amino acid ligands (Pd/MPAA) via C–H functionalization with molecular iodine provide mechanistic insight into the rate-determining step and the oxidation state of Pd in the C–H functionalization step. Enantiomeric excess is strikingly insensitive to temperature from ambient temperature up to over 70 °C, and reaction rate is insensitive to the electronic characteristics of the ligand’s benzoyl protecting group. The reaction is highly robust with no evidence of catalyst deactivation. Intriguingly, C–H bond breaking does not occur prior to the addition of I2 to the reaction mixture. Electrochemical experiments demonstrate the viability of oxidative addition of I2 to Pd(II). Together with 19F NMR studies, these observations suggest that iodine oxidizes Pd prior to addition of the amine substrate. This work may lead to a better general understanding of the subtle variations in the reaction mechanisms for C–H funct...

Identifying causal genetic variants and understanding their mechanisms of effect on traits remains a challenge in genome-wide association studies (GWASs). In particular, how genetic variants (i.e., trans -eQTLs) affect expression of remote genes (i.e., trans -eGenes) remains unknown. We hypothesized that some trans -eQTLs regulate expression of distant genes by altering the expression of nearby genes ( cis -eGenes). Using published GWAS datasets with 39,165 single-nucleotide polymorphisms (SNPs) associated with 1,960 traits, we explored whole blood gene expression associations of trait-associated SNPs in 5,257 individuals from the Framingham Heart Study. We identified 2,350 trans -eQTLs (at p −7 ); more than 80% of them were found to have cis -associated eGenes. Mediation testing suggested that for 35% of trans- eQTL -trans- eGene pairs in different chromosomes and 90% pairs in the same chromosome, the disease-associated SNP may alter expression of the trans -eGene via cis -eGene expression. In addition, we identified 13 trans -eQTL hotspots, affecting from ten to hundreds of genes, suggesting the existence of master genetic regulators. Using causal inference testing, we searched causal variants across eight cardiometabolic traits (BMI, systolic and diastolic blood pressure, LDL cholesterol, HDL cholesterol, total cholesterol, triglycerides, and fasting blood glucose) and identified several cis -eGenes ( ALDH2 for systolic and diastolic blood pressure, MCM6 and DARS for total cholesterol, and TRIB1 for triglycerides) that were causal mediators for the corresponding traits, as well as examples of trans- mediators ( TAGAP for LDL cholesterol). The finding of extensive evidence of genome-wide mediation effects suggests a critical role of cryptic gene regulation underlying many disease traits.

The kinetics of phosphine substitution in CpRu(PPh3)2X (X = Br, 1b, X = I, 1c, X = N3, 1d, and X = NCO, 1e) have been measured under pseudo-first order conditions in THF solution and compared with data for CpRu(PPh3)2X (1a). The relative rate of substitution is found to be 1a > 1d > 1b > 1e > 1c. Substitution rates decrease in the presence of added PPh3 and are independent of added X consistent with a dissociative process. Activation parameters for 1a–1c (ΔH† = 113–135 kJ mol−1, ΔS† = 21–102 J mol−1 K−1) and DFT calculations support a dissociative or dissociative interchange pathway even though negative activation entropies (ΔS† = −48 ± 16 to −105 ± 5 J mol−1 K−1) are observed for 1d–e. Differences in Ru–ligand bond angles in 1d–e point to different π-acceptor properties of the pseudohalide ligands, contributing to the faster rate of substitution for the azide complexes, 1d relative to the cyanate derivative 1e. Substitution is not observed when X = F, 1f, X = H, 1g, X = SnF3, 1h, or X = SnCl3, 1i. Compounds 1b–1e also react with chloroform to yield 1a. The rates of halide exchange are comparable to phosphine substitution for 1c and 1d. The latter reaction is inhibited by excess triphenylphosphine and is unaffected by both radical inhibitors and radical traps suggesting that a radical mechanism is unlikely.

Maratus mungaich Waldock 1995 This was the first described species in this group (Waldock 1995), now known only from a relatively small area to the east of Perth (Waldock 2013; Figure 12:2). J��rgen Otto collected most of the M. mungaich illustrated here at Mt. Dale east of Perth (S 32�� 07' 39.2'', E 116�� 17' 25.0'', elevation ~414 m; Figure 11: locality 5), and additional photographs of this species from the Talbot Road Nature Reserve (Bokhari 2012, Hort 2012; Figure 11: locality 6) have also been examined. Field marks for identification of the male (Figures 16-18). Important features of the male M. mungaich are enumerated here and compared with the male M. sarahae in the next section. Among the many differences between the two species, the anterior lateral field of iridescent scales that are extensive and usually appear dull green in M. mungaich (Figures 16-17: feature 10) are perhaps the most obvious distinction. The carapace of both species has a thick marginal band comprised of white setae. The eye region of M. mungaich is covered with relatively bright red-brown setae. Tibia III is dark red-brown, fringed below with uniform black setae. Metatarsus III is dark red-brown to black, and the entire tarsus III is covered with long white, and no black, setae. We have tentatively identified a form that has been consistently found near Mt. Talbot (Figure 17:2) as this species. Since this is not far from Mt. Dale, one suspects that dispersal may be limited in this species, and additional variations associated with local populations may be found in the future. Female features (Figures 19-20). Female M. mungaich are, like those of most other Maratus, relatively uniform in colouration and difficult to identify when not associated with the males. Waldock's (1995) figure of a paratype female specimen suggests a uniform black eye region in this species, although she describes a covering of setae as we have observed (Figure 19). With respect to the structure of the epigynum, our specimens varied but generally differed from this paratype description with repect to the shape of the spermathecae (oval but circular in outline in the paratype), and the relative separation of the fossae (greater in the paratype). This suggests that, in the absence of a study of individual variation, one must not depend too much on the details of epigynal structure for identification. Male courtship display (Figures 21-22). Male M. mungaich often side-step as they hold legs III in the extended position shown here, and rotate the fan from side to side. The observed movement of M. mungaich was relatively simple and patterned. The male would side step in front of the female, assume this characteristic pose as it moved the pedipalps up and down in that new position, then remained in place as it rotated ('twitched') the fan from side to side several times. It would then side step to a position on the other side of the female, as shown here, and repeat this sequence. Notable differences between this display and that of M. avibus include the wide separation of legs III (brought together to frame the fan in M. avibus) and the use of the pedipalps by M. mungaich. Habitat. These spiders were found on or near the ground in a wooded area at Mt. Dale (Figure 23). Many specimens have been collected in pitfall traps in old growth jarrah forest areas (Waldock 2013)., Published as part of J��rgen C. Otto & David E. Hill, 2014, Spiders of the mungaich group from Western Australia (Araneae: Salticidae: Euophryinae: Maratus), with one new species from Cape Arid, pp. 1-35 in Peckhamia 112 (1) on pages 13-17, DOI: 10.5281/zenodo.1292576, {"references":["Waldock, J. M. 1995. A new species of Maratus from southwestern Australia (Araneae: Salticidae). Records of the Western Australian Museum. Supplement No. 52: 165 - 169.","Waldock, J. M. 2013. A review of the peacock spiders of the Maratus mungaich species-group (Araneae: Salticidae), with descriptions of four new species. Records of the Western Australian Museum 28 (1): 66 - 81.","Bokhari, F. 2012. Maratus mungaich. Photo at flickr ®: http: // www. flickr. com / photos / myrmician / 7386460584 /","Hort, J., and F. Hort. 2012. Little charmer. Photo at flickr ®: http: // www. flickr. com / photos / jean _ hort / 7356879882 /"]}

© The Author(s) 2019., Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose a framework for binary protein-protein interaction (PPI) mapping based on optimally combining assays and/or assay versions to maximize detection of true positive interactions, while avoiding detection of random protein pairs. We have engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions, differing by protein expression systems and tagging configurations. The resulting union of N2H versions recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our findings should be applicable to systematic mapping of other biological landscapes., This work was supported by a Claudia Adams Barr Award to S.G.C., a Fonds de la Recherche Scientifique (F.R.S.- FNRS)-Télévie Grant and a Wallonia-Brussels International (WBI)-World Excellence Fellowship to J.O., NHGRI grants U41HG001715 awarded to M.V., D.E.H. and M.A.C, and P50HG004233 awarded to M.V. We acknowledge the support of Labex IBEID (10- LABX-0062). M.V. is a Chercheur Qualifié Honoraire from the Fonds de la Recherche Scientifique (FRS-FNRS, Wallonia-Brussels Federation, Belgium).

Gastric and small intestinal organoids differentiated from human pluripotent stem cells (hPSCs) have revolutionized the study of gastrointestinal development and disease. Distal gut tissues such as cecum and colon, however, have proven considerably more challenging to derive in vitro. Here we report the differentiation of human colonic organoids (HCOs) from hPSCs. We found BMP signaling is required to establish a posterior Satb2+ domain in developing and postnatal intestinal epithelium. Brief activation of BMP signaling is sufficient to activate a posterior HOX code and direct human PSC-derived gut tube cultures into HCOs. In vitro, HCOs express colonic markers and contained colon-specific cell populations. Following transplantation into mice, HCOs undergo morphogenesis and maturation to form tissue that exhibits molecular, cellular, and morphologic properties of human colon. Together these data show BMP-dependent patterning of human hindgut into HCOs, which will be valuable for studying diseases including colitis and colon cancer.

Most human protein-coding genes are expressed as multiple isoforms. This in turn greatly expands the functional repertoire of the encoded proteome. While at least one reliable open reading frame (ORF) model has been assigned for every gene, the majority of alternative isoforms remains uncharacterized experimentally. This is primarily due to: i) vast differences of overall levels between different isoforms expressed from common genes, and ii) the difficulty of obtaining contiguous full-length ORF sequences. Here, we present ORF Capture-Seq (OCS), a flexible and cost-effective method that addresses both challenges for targeted full-length isoform sequencing applications using collections of cloned ORFs as probes. As proof-of-concept, we show that an OCS pipeline focused on genes coding for transcription factors increases isoform detection by an order of magnitude, compared to unenriched sample. In short, OCS enables rapid discovery of isoforms from custom-selected genes and will allow mapping of the full set of human isoforms at reasonable cost.

Global insights into cellular organization and function require comprehensive understanding of interactome networks. Similar to how a reference genome sequence revolutionized human genetics, a reference map of the human interactome network is critical to fully understand genotype-phenotype relationships. Here we present the first human “all-by-all” binary reference interactome map, or “HuRI”. With ~53,000 high-quality protein-protein interactions (PPIs), HuRI is approximately four times larger than the information curated from small-scale studies available in the literature. Integrating HuRI with genome, transcriptome and proteome data enables the study of cellular function within essentially any physiological or pathological cellular context. We demonstrate the use of HuRI in identifying specific subcellular roles of PPIs and protein function modulation via splicing during brain development. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms underlying tissue-specific phenotypes of Mendelian diseases. HuRI thus represents an unprecedented, systematic reference linking genomic variation to phenotypic outcomes.