FEAR antiviral response pathway is independent of interferons and countered by poxvirus proteins.

The human facilitates chromatin transcription (FACT) complex is a chromatin remodeller composed of human suppressor of Ty 16 homologue (hSpt16) and structure-specific recognition protein-1 subunits that regulates cellular gene expression. Whether FACT regulates host responses to infection remained unclear. We identify a FACT-mediated, interferon-independent, antiviral pathway that restricts poxvirus replication.

Poxvirus A51R proteins regulate microtubule stability and antagonize a cell-intrinsic antiviral response.

Numerous viruses alter host microtubule (MT) networks during infection, but how and why they induce these changes is unclear in many cases. We show that the vaccinia virus (VV)-encoded A51R protein is a MT-associated protein (MAP) that directly binds MTs and stabilizes them by both promoting their growth and preventing their depolymerization. Furthermore, we demonstrate that A51R-MT interactions are conserved across A51R proteins from multiple poxvirus genera, and highly conserved, positively charged residues in A51R proteins mediate these interactions.

Influenza virus mRNAs encode determinants for nuclear export via the cellular TREX-2 complex.

Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)-2 to the NPC for exporting a subset of cellular mRNAs.

A FACT-ETS-1 Antiviral Response Pathway Restricts Viral Replication and is Countered by Poxvirus A51R Proteins.

The FACT complex is an ancient chromatin remodeling factor comprised of Spt16 and SSRP1 subunits that regulates specific eukaryotic gene expression programs. However, whether FACT regulates host immune responses to infection was unclear. Here, we identify an antiviral pathway mediated by FACT, distinct from the interferon response, that restricts poxvirus replication.

Discovery of JNJ-64264681: A Potent and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK) is a Tec family kinase that plays an essential role in B-cell receptor (BCR) signaling as well as Fcγ receptor signaling in leukocytes. Pharmacological inhibition of BTK has been shown to be effective in treating hematological malignancies and is hypothesized to provide an effective strategy for the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. We report the discovery and preclinical properties of JNJ-64264681 (), a covalent, irreversible BTK inhibitor with potent whole blood activity and exceptional kinome selectivity.

How to Inhibit Nuclear Factor-Kappa B Signaling: Lessons from Poxviruses.

The Nuclear Factor-kappa B (NF-κB) family of transcription factors regulates key host inflammatory and antiviral gene expression programs, and thus, is often activated during viral infection through the action of pattern-recognition receptors and cytokine-receptor interactions. In turn, many viral pathogens encode strategies to manipulate and/or inhibit NF-κB signaling. This is particularly exemplified by vaccinia virus (VV), the prototypic poxvirus, which encodes at least 18 different inhibitors of NF-κB signaling.

Discovery of a Potent and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase with Oral Anti-Inflammatory Activity.

Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase that plays a critical role in the activation of B cells, macrophages, and osteoclasts. Given the key role of these cell types in the pathology of autoimmune disorders, BTK inhibitors have the potential to improve treatment outcomes in multiple diseases. Herein, we report the discovery and characterization of a novel potent and selective covalent 4-oxo-4,5-dihydro-3-1-thia-3,5,8-triazaacenaphthylene-2-carboxamide BTK inhibitor chemotype.

Development of phenotypic assays for identifying novel blockers of L-type calcium channels in neurons.

L-type calcium channels (LTCCs) are highly expressed in the heart and brain and are critical for cardiac and neuronal functions. LTCC-blocking drugs have a long and successful record in the clinic for treating cardiovascular disorders. In contrast, establishment of their efficacy for indications of the central nervous system remains challenging given the tendency of existing LTCC drugs being functionally and mechanistically more selective for peripheral tissues.

Functional characterization of ABCC8 variants of unknown significance based on bioinformatics predictions, splicing assays, and protein analyses: Benefits for the accurate diagnosis of congenital hyperinsulinism.

ABCC8 encodes the SUR1 subunit of the β-cell ATP-sensitive potassium channel whose loss of function causes congenital hyperinsulinism (CHI). Molecular diagnosis is critical for optimal management of CHI patients. Unfortunately, assessing the impact of ABCC8 variants on RNA splicing remains very challenging as this gene is poorly expressed in leukocytes.

α7 nicotinic acetylcholine receptor upregulation by anti-apoptotic Bcl-2 proteins.

Nicotinic acetylcholine receptors (nAChRs) mediate and modulate synaptic transmission throughout the brain, and contribute to learning, memory, and behavior. Dysregulation of α7-type nAChRs in neuropsychiatric as well as immunological and oncological diseases makes them attractive targets for pharmaceutical development. Recently, we identified NACHO as an essential chaperone for α7 nAChRs.

Gypsy moth genome provides insights into flight capability and virus-host interactions.

Since its accidental introduction to Massachusetts in the late 1800s, the European gypsy moth (EGM; ) has become a major defoliator in North American forests. However, in part because females are flightless, the spread of the EGM across the United States and Canada has been relatively slow over the past 150 years. In contrast, females of the Asian gypsy moth (AGM; ) subspecies have fully developed wings and can fly, thereby posing a serious economic threat if populations are established in North America.

Shape Analysis of the Patellar Bone Surface and Cutting Plane for Knee Replacement Surgery.

Geometry of the patella (kneecap) remains poorly understood yet is highly relevant to performing the correct patellar cut to reduce pain and to improve function and satisfaction after knee replacement surgery. Although studies routinely refer to "parallel to the anterior surface" and "the patellar horizon," a quantitative definition of these is lacking and significant variability exists between observers for this irregularly-shaped bone. A 2D-3D shape analysis technique was developed to determine the optimal device configuration for contacting the patellar surface.

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens.

Guiding Device for the Patellar Cut in Total Knee Arthroplasty: Design and Validation.

An incorrect cut of the patella (kneecap) during total knee arthroplasty, affects the thickness in different quadrants of the patella, leading to pain and poor function. Because of the disadvantages of existing devices, many surgeons choose to perform the cut freehand. Given this mistrust of existing devices, a quick, but accurate, method is needed that guides the cut, without constraining the surgeon.

Infection of with Vesicular Stomatitis Virus via Microinjection.

Over the past 15 years, the free-living nematode, has become an important model system for exploring eukaryotic innate immunity to bacterial and fungal pathogens. More recently, infection models using either natural or non-natural nematode viruses have also been established in . These models offer new opportunities to use the nematode to understand eukaryotic antiviral defense mechanisms.

Probing Subunits Interactions in K Channels Using Photo-Crosslinking via Genetically Encoded p-Azido-L-phenylalanine.

Potassium channels are multimeric protein complexes regulated by diverse physiological and pharmacological ligands. The key to understanding mechanisms of channel regulation is the ability to detect structural changes associated with ligand binding. While high-resolution structural methods such as X-ray crystallography and single-particle cryo-electron microscopy offer direct visualization of channel structures, these methods do have limitations and may not be suitable for the question of interest.

Cryo-EM structure of the ATP-sensitive potassium channel illuminates mechanisms of assembly and gating.

K channels are metabolic sensors that couple cell energetics to membrane excitability. In pancreatic β-cells, channels formed by SUR1 and Kir6.2 regulate insulin secretion and are the targets of antidiabetic sulfonylureas.

A Genome-Wide Arrayed cDNA Screen to Identify Functional Modulators of α7 Nicotinic Acetylcholine Receptors.

Cellular signaling is in part regulated by the composition and subcellular localization of a series of protein interactions that collectively form a signaling complex. Using the α7 nicotinic acetylcholine receptor (α7nAChR) as a proof-of-concept target, we developed a platform to identify functional modulators (or auxiliary proteins) of α7nAChR signaling. The Broad cDNA library was transiently cotransfected with α7nAChR cDNA in HEK293T cells in a high-throughput fashion.

Pharmacological Correction of Trafficking Defects in ATP-sensitive Potassium Channels Caused by Sulfonylurea Receptor 1 Mutations.

ATP-sensitive potassium (K) channels play a key role in mediating glucose-stimulated insulin secretion by coupling metabolic signals to β-cell membrane potential. Loss of K channel function due to mutations in ABCC8 or KCNJ11, genes encoding the sulfonylurea receptor 1 (SUR1) or the inwardly rectifying potassium channel Kir6.2, respectively, results in congenital hyperinsulinism.

Pharmacological Modulation of GluK1 and GluK2 by NETO1, NETO2, and PSD95.

The association between the kainate receptors (KARs) GluK1 and GluK2 and the modifying proteins neuropilin- and tolloid-like 1 (NETO1), neuropilin- and tolloid-like 2 (NETO2), and postsynaptic density protein 95 (PSD95) is likely to produce distinct GluK1 and GluK2 pharmacology in postsynaptic neurons. However, little is known about their corresponding modulatory effects on GluK1 and GluK2 activity in high-throughput assays for cell-based drug discovery. Using heterologous cells that potentially mimic the response in native cells in a fluorescence imaging plate reader (FLIPR) assay, we have investigated assays that incorporate (1) coexpression of GluK1 or GluK2 with their modulatory proteins (NETO1, NETO2, PSD95) and/or (2) enablement of assays with physiological concentration of native GluK1 and GluK2 agonist (glutamate) in the absence of an artificial potentiator (e.

Phenotypic Approaches to Identify Inhibitors of B Cell Activation.

An EPIC label-free phenotypic platform was developed to explore B cell receptor (BCR) and CD40R-mediated B cell activation. The phenotypic assay measured the association of RL non-Hodgkin's lymphoma B cells expressing lymphocyte function-associated antigen 1 (LFA-1) to intercellular adhesion molecule 1 (ICAM-1)-coated EPIC plates. Anti-IgM (immunoglobulin M) mediated BCR activation elicited a response that was blocked by LFA-1/ICAM-1 specific inhibitors and a panel of Bruton's tyrosine kinase (BTK) inhibitors.

Computed tomography analysis of knee pose and geometry before and after total knee arthroplasty.

Using a three-dimensional (3D) modality to image patients' knees before and after total knee arthroplasty (TKA) allows researchers and clinicians to evaluate causes of pain after TKA, differences in implant design, and changes in the articular geometry as a result of surgery. Computed tomography (CT) has not been fully utilized to date for evaluating the knee after TKA due to metal artifacts obscuring part of the image. We describe an accurate, validated protocol, which has been implemented in vivo, that improves visibility of the patellofemoral joint, matches implant models automatically in 3D, segments preoperative bone semi-automatically, detects and sets coordinate systems automatically, determines the six degrees of freedom of knee pose and geometry, and allows for multiple other measurements that are clinically relevant.

Identification of RanBP 9/10 as interacting partners for protein kinase C (PKC) gamma/delta and the D1 dopamine receptor: regulation of PKC-mediated receptor phosphorylation.

We reported previously that ethanol treatment regulates D(1) receptor phosphorylation and signaling in a protein kinase C (PKC) delta- and PKCgamma-dependent fashion by a mechanism that may involve PKC isozyme-specific interacting proteins. Using a PKC isozyme-specific coimmunoprecipitation approach coupled to mass spectrometry, we report the identification of RanBP9 and RanBP10 as novel interacting proteins for both PKCgamma and PKCdelta. Both RanBP9 and RanBP10 were found to specifically coimmunoprecipitate with both PKCgamma and PKCdelta; however, this association did not seem to mediate the ethanol regulation of the PKCs.

Ethanol regulation of D(1) dopamine receptor signaling is mediated by protein kinase C in an isozyme-specific manner.

Ethanol consumption potentiates dopaminergic signaling that is partially mediated by the D(1) dopamine receptor; however, the mechanism(s) underlying ethanol-dependent modulation of D(1) signaling is unclear. We now show that ethanol treatment of D(1) receptor-expressing cells decreases D(1) receptor phosphorylation and concurrently potentiates dopamine-stimulated cAMP accumulation. Protein kinase C (PKC) inhibitors mimic the effects of ethanol on D(1) receptor phosphorylation and dopamine-stimulated cAMP levels in a manner that is non-additive with ethanol treatment.