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Background/aim To compare the GeneXpert® O6-methylguanine DNA methyltransferase (MGMT) methylation prototype (GX MGMT) assay with pyrosequencing in glioblastomas. Materials and methods The MGMT methylation status was retrospectively assessed in formalin-fixed paraffin embedded (FFPE) tumor blocks from 262 glioblastoma patients obtained from three independent cohorts using either a standard of care pyrosequencing laboratory developed test or the GX MGMT assay. Results The concordance rate was 92.1% (58/63) for Oregon Health and Science University (OSHU) samples, 91.7% (88/96) for Medical University of Vienna (MUV) samples, and 82.5% (85/103) for Kepler University Hospital (KUH) samples. Patients with MGMT promoter hypermethylation assessed by pyrosequencing or the GX MGMT test had a significantly longer overall survival compared to patients without hypermethylation (HR=0.43, 95%CI=0.26-0.72, p=0.001 and HR=0.51, 95%CI=0.31-0.84, p=0.008, respectively). Conclusion Standardized, simplified, and on-demand testing of MGMT promoter methylation by the GX MGMT assay is feasible.

Cells require coordinated control over gene expression when responding to environmental stimuli. Here, we apply scATAC-seq and scRNA-seq in resting and stimulated human blood cells. Collectively, we generate ∼91,000 single-cell profiles, allowing us to probe the cis-regulatory landscape of immunological response across cell types, stimuli and time. Advancing tools to integrate multi-omic data, we develop FigR - a framework to computationally pair scATAC-seq with scRNA-seq cells, connect distal cis-regulatory elements to genes, and infer gene regulatory networks (GRNs) to identify candidate TF regulators. Utilizing these paired multi-omic data, we define Domains of Regulatory Chromatin (DORCs) of immune stimulation and find that cells alter chromatin accessibility prior to production of gene expression at time scales of minutes. Further, the construction of the stimulation GRN elucidates TF activity at disease-associated DORCs. Overall, FigR enables the elucidation of regulatory interactions across single-cell data, providing new opportunities to understand the function of cells within tissues.

Recent technical advancements have facilitated the mapping of epigenomes at single-cell resolution; however, the throughput and quality of these methods have limited their widespread adoption. Here we describe a high-quality (10(5) nuclear fragments per cell) droplet-microfluidics-based method for single-cell profiling of chromatin accessibility. We use this approach, named ‘droplet single-cell assay for transposase-accessible chromatin using sequencing’ (dscATAC-seq), to assay 46,653 cells for the unbiased discovery of cell types and regulatory elements in adult mouse brain. We further increase the throughput of this platform by combining it with combinatorial indexing (dsciATAC-seq), enabling single-cell studies at a massive scale. We demonstrate the utility of this approach by measuring chromatin accessibility across 136,463 resting and stimulated human bone marrow-derived cells to reveal changes in the cis- and trans-regulatory landscape across cell types and under stimulatory conditions at single-cell resolution. Altogether, we describe a total of 510,123 single-cell profiles, demonstrating the scalability and flexibility of this droplet-based platform.

While recent technical advancements have facilitated the mapping of epigenomes at single-cell resolution, the throughput and quality of these methods have limited the widespread adoption of these technologies. Here, we describe a droplet microfluidics platform for single-cell assay for transposase accessible chromatin (scATAC-seq) for high-throughput single-cell profiling of chromatin accessibility. We use this approach for the unbiased discovery of cell types and regulatory elements within the mouse brain. Further, we extend the throughput of this approach by pairing combinatorial indexing with droplet microfluidics, enabling single-cell studies at a massive scale. With this approach, we measure chromatin accessibility across resting and stimulated human bone marrow derived cells to reveal changes in the cis- and trans- regulatory landscape across cell types and upon stimulation conditions at single-cell resolution. Altogether, we describe a total of 502,207 single-cell profiles, demonstrating the scalability and flexibility of this droplet-based platform.

RNA is a versatile macromolecule that accommodates functional information in primary sequence and secondary and tertiary structure. We use a combination of chemical probing, RNA structure modeling, comparative sequence analysis, and functional assays to examine the role of RNA structure in the hepatitis C virus (HCV) genome. We describe a set of conserved but functionally diverse structural RNA motifs that occur in multiple coding regions of the HCV genome, and we demonstrate that conformational changes in these motifs influence specific stages in the virus' life cycle. Our study shows that these types of structures can pervade a genome, where they play specific mechanistic and regulatory roles, constituting a "code within the code" for controlling biological processes.

Hepatitis C virus (HCV) NS3-4A is required for viral replication and assembly. We establish that virus assembly is sensitive to mutations in the linker region between the helicase and protease domains of NS3-4A. However, we find that the protease cleavage, RNA binding, and unwinding rates of NS3 are minimally affected in vitro . Thus, we conclude that the NS3 linker is critical for mediating protein-protein interactions and dynamic control rather than for modulating the enzymatic functions of NS3-4A.

Hepatitis C virus (HCV) NS4A is a single-pass transmembrane (TM) protein essential for viral replication and particle assembly. The sequence of the NS4A TM domain is highly conserved, suggesting that it may be important for protein-protein interactions. To test this hypothesis, we measured the potential dimerization of the NS4A TM domain in a well-characterized two-hybrid TM protein interaction system. The NS4A TM domain exhibited a strong homotypic interaction that was comparable in affinity to glycophorin A, a well-studied human blood group antigen that forms TM homodimers. Several mutations predicted to cluster on a common surface of the NS4A TM helix caused significant reductions in dimerization, suggesting that these residues form an interface for NS4A dimerization. Mutations in the NS4A TM domain were further examined in the JFH-1 genotype 2a replicon system; importantly, all mutations that destabilized NS4A dimers also caused defects in RNA replication and/or virus assembly. Computational modeling of NS4A TM interactions suggests a right-handed dimeric interaction of helices with an interface that is consistent with the mutational effects. Furthermore, defects in NS4A oligomerization and virus particle assembly of two mutants were rescued by NS4A A15S, a TM mutation recently identified through forward genetics as a cell culture-adaptive mutation. Together, these data provide the first example of a functionally important TM dimer interface within an HCV nonstructural protein and reveal a fundamental role of the NS4A TM domain in coordinating HCV RNA replication and virus particle assembly.

Double-stranded RNAs are an important class of functional macromolecules in living systems. They are usually found as part of highly specialized intracellular machines that control diverse cellular events, ranging from virus replication, antiviral defense, RNA interference, to regulation of gene activities and genomic integrity. Within different intracellular machines, the RNA duplex is often found in association with specific RNA-dependent ATPases, including Dicer, RIG-I and DRH-3 proteins. These duplex RNA-activated ATPases represent an emerging group of motor proteins within the large and diverse super family 2 nucleic acid-dependent ATPases (which are historically defined as SF2 helicases). The duplex RNA-activated ATPases share characteristic molecular features for duplex RNA recognition, including motifs (e.g., motifs IIa and Vc) and an insertion domain (HEL2i), and they require double-strand RNA binding for their enzymatic activities. Proteins in this family undergo large conformational changes concomitant with RNA binding, ATP binding and ATP hydrolysis in order to achieve their functions, which include the release of signaling domains and the recruitment of partner proteins. The duplex RNA-activated ATPases represent a distinct and fascinating group of nanomechanical molecular motors that are essential for duplex RNA sensing and processing in diverse cellular pathways.

Summary Retinoic acid inducible gene-I (RIG-I) is a key intracellular immune receptor for pathogenic RNAs, particularly from RNA viruses. Here, we report the crystal structure of human RIG-I bound to a 5′ triphosphorylated RNA hairpin and ADP nucleotide at 2.8 A resolution. The RNA ligand contains all structural features that are essential for optimal recognition by RIG-I, as it mimics the panhandle-like signatures within the genome of negative-stranded RNA viruses. RIG-I adopts an intermediate, semiclosed conformation in this product state of ATP hydrolysis. The structure of this complex allows us to visualize the first steps in RIG-I recognition and activation upon viral infection.

Hepatitis C virus NS3-4A is a membrane-bound enzyme complex that exhibits serine protease, RNA helicase, and RNA-stimulated ATPase activities. This enzyme complex is essential for viral genome replication and has been recently implicated in virus particle assembly. To help clarify the role of NS4A in these processes, we conducted alanine scanning mutagenesis on the C-terminal acidic domain of NS4A in the context of a chimeric genotype 2a reporter virus. Of 13 mutants tested, two (Y45A and F48A) had severe defects in replication, while seven (K41A, L44A, D49A, E50A, M51A, E52A, and E53A) efficiently replicated but had severe defects in virus particle assembly. Multiple strategies were used to identify second-site mutations that suppressed these NS4A defects. The replication defect of NS4A F48A was partially suppressed by mutation of NS4B I7F, indicating that a genetic interaction between NS4A and NS4B contributes to RNA replication. Furthermore, the virus assembly defect of NS4A K41A was suppressed by NS3 Q221L, a mutation previously implicated in overcoming other virus assembly defects. We therefore examined the known enzymatic activities of wild-type or mutant forms of NS3-4A but did not detect specific defects in the mutants. Taken together, our data reveal interactions between NS4A and NS4B that control genome replication and between NS3 and NS4A that control virus assembly.

BACKGROUND: Underdeveloped countries reported 882,900 new cases of breast cancer and 324,000 deaths in 2012, likely to be a gross underestimation according to recent reports. Often, mammography screening is not available, primary care services are limited, and pathology and treatment services are available only in the regional hospitals. Because of the lack of access to diagnostic and treatment services, it is estimated that more than 90% of patients with breast cancer never present for medical treatment. To address this situation, an accurate, easy-to-perform diagnostic test appropriate for use in remote clinics is desperately needed. Johns Hopkins (JH) and Cepheid partnered to translate a robust Quantitative Multiplex Methylation-Specific PCR (QM-MSP) assay to an automated, cartridge-based system that provides quantitative measures of DNA methylation within hours of fine needle aspiration or core biopsy of image-detected suspicious lesions. METHODS: With a goal of discriminating malignant from benign breast disease with high sensitivity and specificity, we evaluated 24 breast cancer-specific DNA methylation markers (selected through comprehensive methylome analysis) in 119 invasive ductal carcinomas and 186 benign breast tissues. QM-MSP was performed on sections of formalin-fixed paraffin-embedded (FFPE) tissues to quantify DNA methylation. The dynamic range and performance of quantitative methylation detection was tested using a subset of 9 genes in the cartridge-based system. RESULTS: QM-MSP was performed in a Training set consisting of 93 tissues [n=43 IDC, n=50 benign lesions (25 usual ductal hyperplasia, UDH, and 25 papilloma)] from the US. We selected 9 DNA markers significantly (p CONCLUSIONS: We identified a panel of methylated DNA markers that discriminate malignant from benign breast lesions and built a prototype automated cartridge-based assay with promising sensitivity and specificity for breast cancer. Such an assay has the potential to aid in specimen triage in the pathology lab and provide fast, low cost and accurate diagnosis of breast cancer in LMIC settings. Citation Format: Fackler MJ, Downs BM, Mercado-Rodriguez C, Cimino-Mathews A, Chen C, Yuan J, Cope LM, Kohlway A, Kocmond K, Lai E, Weidler J, Visvanathan K, Umbricht CB, Harvey S, Wolff AC, Bates M, Sukumar S. An automated DNA methylation assay (QM-MSP) for rapid breast cancer diagnosis in underdeveloped countries [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-03-07.

The nature of specific RNA-RNA and protein-RNA interactions involved in the process of genome dimerization and isomerization in HIV-1, which is mediated in vitro by the stemloop 1 (SL1) of the packaging signal and by the nucleocapsid (NC) domain of the viral Gag polyprotein, was investigated by using archetypical nucleic acid ligands as non-covalent probes. Small-molecule ligands make contact with their target substrates through complex combinations of H-bonds, salt bridges, and hydrophobic interactions. Therefore, their binding patterns assessed by electrospray ionization (ESI) mass spectrometry can provide valuable insights into the factors determining specific recognition between species involved in biopolymer assemblies. In the case of SL1, dimerization and isomerization create unique structural features capable of sustaining stable interactions with classic nucleic acid ligands. The binding modes exhibited by intercalators and minor groove binders were adversely affected by the significant distortion of the duplex formed by palindrome annealing in the kissing-loop (KL) dimer, whereas the modes observed for the corresponding extended duplex (ED) confirmed a more regular helical structure. Consistent with the ability to establish electrostatic interactions with highly negative pockets typical of helix anomalies, polycationic aminoglycosides bound to the stem-bulge motif conserved in all SL1 conformers, to the unpaired nucleotides located at the hinge between kissing hairpins in KL, and to the exposed bases flanking the palindrome duplex in ED. The patterns afforded by intercalators and minor groove binders did not display detectable variations when the corresponding NC-SL1 complexes were submitted to probing. In contrast, aminoglycosides displayed the ability to compete with the protein for overlapping sites, producing opposite effects on the isomerization process. Indeed, displacing NC from the stem-bulges of the KL dimer induced inhibition of stem melting and decreased the efficiency of isomerization. Competition for the hinge region, instead, eliminated the NC stabilization of a grip motif formed by nucleobases of opposite strands, thus facilitating the strand-exchange required for isomerization. These non-covalent probes provided further evidence that the structural context of the actual binding sites has significant influence on the chaperone activities of NC, which should be taken in account when developing potential drug candidates aimed at disrupting genome dimerization and isomerization in HIV-1.

The binding modes and structural determinants of the noncovalent complexes formed by aminoglycoside antibiotics with conserved domains of the HIV-1 packaging signal (Psi-RNA) were investigated using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The location of the aminoglycoside binding sites on the different stemloop structures was revealed by characteristic coverage gaps in the ion series obtained by sustained off-resonance irradiation collision induced dissociation (SORI-CID) of the antibiotic-RNA assemblies. The site positions were confirmed using mutants that eliminated salient structural features of the Psi-RNA domains. The effects of the mutations on the binding properties of the different substrates served to validate the position of the aminoglycoside site on the wild-type structures. Additional information was provided by docking experiments performed on the different aminoglycoside-stemloop complexes. The results have shown that, in the absence of features disrupting the regular A-helix of the double-stranded stem, aminoglycosides tend to bind in an area situated between the upper stem and the loop regions, as demonstrated for stemloop SL3. The presence of a tandem wobbles motif in SL4 modifies the regular geometry of the upper stem, which does not affect the general site location, but greatly increases its solution binding affinity compared with SL3. The platform motif in SL2 locates the binding site in the stem midsection and confers upon this stemloop an intermediate affinity toward aminoglycosides. In SL3 and SL4, the extensive overlap of the antibiotic site with the region used to bind the nucleocapsid (NC) protein provides the basis for a competition mechanism that could explain the aminoglycoside inhibition of the NC.SL3 and NC.SL4 assemblies. In contrast, the minimal overlap between the aminoglycoside and the NC sites in SL2 accounts for the absence of inhibition of the NC.SL2 complex.

Background: Epigenetic silencing of the O6-methylguanine DNA methyltransferase (MGMT) gene by promoter methylation is observed in 40 to 50% of glioblastomas and is associated with improved overall survival on radiation and/or temozolamide therapy. Currently, pyrosequencing is commonly used to assess the methylation status of the MGMT gene in formalin-fixed, paraffin-embedded (FFPE) samples. We examined the concordance of the recently developed GeneXpert (GX) MGMT Methylation RUO Assay with pyrosequencing in three independent glioblastoma cohorts. Patients and methods: The GX MGMT Methylation RUO Assay is a research use only, cartridge-based methylation specific PCR assay performed on the GeneXpert® Instrument (Cepheid) that automates DNA purification, bisulfite conversion, and methylation-specific PCR after sample preparation. Cartridge results are reported in less than 4 hours as delta cycle threshold (dCt) measurements (dCt = beta-actin/ACTB Ct - MGMT Ct). The MGMT methylation status was assessed on FFPE tumor blocks from 262 glioblastoma patients obtained from OHSU (n=63), MUV (n=96), and KUL (n=103). Two adjacent sections from each block were prepared, one 4 µm section for H&E staining to determine the % tumor cell content/tumor area and one 4 µm section for lysate preparation and GX testing. Both GX MGMT Methylation testing and pyrosequencing results were correlated with overall survival of the patients in the MUV cohort. Results: All of the 262 (100%) glioblastoma samples tested yielded valid results for both GX MGMT Methylation testing and pyrosequencing. The cut-offs were set at dCt -6.2 for the GX MGMT Methylation RUO Assay and 8% methylation for pyrosequencing. The concordance rate between GX MGMT Methylation testing and pyrosequencing was 92% (58/63 OHSU samples, 92% (88/96 MUV samples) and 83% (85/103 KUL samples). The overall concordance rate was 88% (231/262 samples). Sensitivity and specificity was 84% (27/32) and 100% (31/31) for OHSU samples, 89% (33/37) and 93% (55/59) for MUV samples, and 70% (31/44/) and 92% (54/59) for KUL samples, respectively. Overall sensitivity was 81% (91/113) and overall specificity was 94% (140/149). Overall survival data were available for 95 MUV patients. Patients with MGMT promoter hypermethylation based on pyrosequencing had a significantly longer overall survival compared to patients without hypermethylation (median 16 vs. 11 months, p = 0.001). Similar results were observed with the GX MGMT Methylation RUO Assay (median 15 vs. 11 months, p = 0.005). Conclusion: GX MGMT Methylation testing is highly reproducible and shows good concordance with pyrosequencing in three independent cohorts of glioblastoma patients. Our results suggest that standardized, simplified, and on-demand testing of MGMT promoter methylation by the GX MGMT Methylation RUO Assay is feasible. Citation Format: Martin Filipits, Anita Brandstetter, Matthias Preusser, Johannes Hainfellner, Sabine Spiegl-Kreinecker, Edwin W. Lai, Kriszten Kocmond, Andrew Kohlway, Jodi Weidler, Michael Bates, Christopher Corless. Evaluation of an assay for on-demand and easy assessment of MGMT gene promoter methylation in glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-046. doi:10.1158/1538-7445.AM2017-LB-046

Retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor expressed in metazoan cells that is responsible for eliciting the production of type I interferons and pro-inflammatory cytokines upon detection of intracellular, non-self RNA. Structural studies of RIG-I have identified a novel Pincer domain composed of two alpha helices that physically tethers the C-terminal domain to the SF2 helicase core. We find that the Pincer plays an important role in mediating the enzymatic and signaling activities of RIG-I. We identify a series of mutations that additively decouple the Pincer motif from the ATPase core and show that this decoupling results in impaired signaling. Through enzymological and biophysical analysis, we further show that the Pincer domain controls coupled enzymatic activity of the protein through allosteric control of the ATPase core. Further, we show that select regions of the HEL1 domain have evolved to potentiate interactions with the Pincer domain, resulting in an adapted ATPase cleft that is now responsive to adjacent domains that selectively bind viral RNA. Published version

Edited Information Flow RNA is generally a faithful mirror of the information encoded in DNA. However, posttranscriptional RNA editing is a widespread phenomenon that can result in a translated protein differing from its gene code. A class of deaminase enzymes related to human APOBEC (apolipoprotein B editing complex protein) edit adenosines in eukaryotes. Randau et al. (p. 657 ) have discovered an addition to this enzyme family in prokaryotes that edits cytosine to uridine at the highly conserved and structurally critical position 8 in the majority of transfer RNAs of the hyperthermophilic archaebacterium Methanopyrus kandleri .

RIG-I is an innate immune receptor that detects and responds to infection by deadly RNA viruses such as influenza, and Hepatitis C. In the cytoplasm, RIG-I is faced with a difficult challenge: it must sensitively detect viral RNA while ignoring the abundance of host RNA. It has been suggested that RIG-I has a ‘proof-reading’ mechanism for rejecting host RNA targets, and that disruptions of this selectivity filter give rise to autoimmune diseases. Here, we directly monitor RNA proof-reading by RIG-I and we show that it is controlled by a set of conserved amino acids that couple RNA and ATP binding to the protein (Motif III). Mutations of this motif directly modulate proof-reading by eliminating or enhancing selectivity for viral RNA, with major implications for autoimmune disease and cancer. More broadly, the results provide a physical explanation for the ATP-gated behavior of SF2 RNA helicases and receptor proteins.

Many positive-strand RNA viruses encode genes that can function in trans, whereas other genes are required in cis for genome replication. The mechanisms underlying trans- and cis-preferences are not fully understood. Here, we evaluate this concept for hepatitis C virus (HCV), an important cause of chronic liver disease and member of the Flaviviridae family. HCV encodes five nonstructural (NS) genes that are required for RNA replication. To date, only two of these genes, NS4B and NS5A, have been trans-complemented, leading to suggestions that other replicase genes work only in cis. We describe a new quantitative system to measure the cis- and trans-requirements for HCV NS gene function in RNA replication and identify several lethal mutations in the NS3, NS4A, NS4B, NS5A, and NS5B genes that can be complemented in trans, alone or in combination, by expressing the NS3–5B polyprotein from a synthetic mRNA. Although NS5B RNA binding and polymerase activities can be supplied in trans, NS5B protein expression was required in cis, indicating that NS5B has a cis-acting role in replicase assembly distinct from its known enzymatic activity. Furthermore, the RNA binding and NTPase activities of the NS3 helicase domain were required in cis, suggesting that these activities play an essential role in RNA template selection. A comprehensive complementation group analysis revealed functional linkages between NS3-4A and NS4B and between NS5B and the upstream NS3–5A genes. Finally, NS5B polymerase activity segregated with a daclatasvir-sensitive NS5A activity, which could explain the synergy of this antiviral compound with nucleoside analogs in patients. Together, these studies define several new aspects of HCV replicase structure-function, help to explain the potency of HCV-specific combination therapies, and provide an experimental framework for the study of cis- and trans-acting activities in positive-strand RNA virus replication more generally., Author Summary RNA viruses encode one or more proteins that form an RNA replicase, which serves to propagate the viral RNA genome. Some replication genes can function in trans, meaning that proteins expressed by one viral genome can complement and assist in the replication of a defective mutant virus within the same cell. Other viral genes work in cis, meaning that they are unable to complement defective mutants; these cis-acting genes therefore link the replication of a viral RNA genome to its translation. Because genetic complementation reflects the formation of molecular interactions that were previously absent or nonfunctional, understanding the cis- and trans-activities of viral replication proteins can provide insights into the structure and function of viral replicases. Here we develop a novel methodology to study interactions between components of the viral replicase to define essential cis- and trans-activities for all five hepatitis C virus (HCV) replication gene products. Our studies show that representative defects in each HCV replication protein can be complemented in trans. However, specific features of NS3 and NS5B were required in cis, indicating that these proteins specifically function to replicate the genome that encodes them. These data define important activities of the HCV replication proteins and provide new insight into how these proteins function together as an enzyme complex.

SummaryIntracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP2) recognize viral RNAs as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral immune response. To understand the molecular basis of this process, we determined the crystal structure of RIG-I in complex with double-stranded RNA (dsRNA). The dsRNA is sheathed within a network of protein domains that include a conserved “helicase” domain (regions HEL1 and HEL2), a specialized insertion domain (HEL2i), and a C-terminal regulatory domain (CTD). A V-shaped pincer connects HEL2 and the CTD by gripping an α-helical shaft that extends from HEL1. In this way, the pincer coordinates functions of all the domains and couples RNA binding with ATP hydrolysis. RIG-I falls within the Dicer-RIG-I clade of the superfamily 2 helicases, and this structure reveals complex interplay between motor domains, accessory mechanical domains, and RNA that has implications for understanding the nanomechanical function of this protein family and other ATPases more broadly.