Exploiting activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications.

Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here, we use four cryoelectron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9 that strongly inhibit Neisseria lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through allosteric inhibition, whereas AcrIC9 achieves so through direct competition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for type I CRISPR eukaryotic genome engineering.

Experimental Development of an Injection Molding Process Window.

Injection molding is one of the most common and effective manufacturing processes used to produce plastic products and impacts industries around the world. However, injection molding is a complex process that requires careful consideration of several key control variables. These variables and how they are utilized greatly affect the resulting polymer parts of any molding operation. The bounds of the acceptable values of each Control Process Variable (CPV) must be analyzed and delimited to ensure manufacturing success and produce injected molded parts efficiently and effectively. One such method by which the key CPVs of an injection molding operation can be delimited is through the development of a process window. Once developed, operating CPVs at values inside the boundaries of the window or region will allow for the consistent production of parts that comply with the desired Performance Measures (PM), promoting a stable manufacturing process. This work proposes a novel approach to experimentally developing process windows and illustrates the methodology with a specific molding operation. A semicrystalline material was selected as it is more sensitive to process conditions than amorphous materials.

Exploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications.

Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets, and the nuclease-helicase Cas3 to degrade them. Among seven subtypes, Type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here we use four cryo-electron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling efficient NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9, that strongly inhibit N. lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through direct competition, whereas AcrIC9 achieves so through allosteric inhibition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for controllable Type I CRISPR genome engineering.

4-Aminoquinolines modulate RNA structure and function: Pharmacophore implications of a conformationally restricted polyamine.

RNA structure plays an important role in regulating cellular function and there is a significant emerging interest in targeting RNA for drug discovery. Here we report the identification of 4-aminoquinolines as modulators of RNA structure and function. Aminoquinolines have a broad range of pharmacological activities, but their specific mechanism of action is often not fully understood. Using electrophoretic mobility shift assays and enzymatic probing we identified 4-aminoquinolines that bind the stem-loop II motif (s2m) of SARS-CoV-2 RNA site-specifically and induce dimerization. Using fluorescence-based RNA binding and T-box riboswitch functional assays we identified that hydroxychloroquine binds the T-box riboswitch antiterminator RNA element and inhibits riboswitch function. Based on its structure and riboswitch dose-response activity we identified that the antagonist activity of hydroxychloroquine is consistent with it being a conformationally restricted analog of the polyamine spermidine. Given the known role that polyamines play in RNA function, the identification of an RNA binding ligand with the pharmacophore of a conformationally restricted polyamine has significant implications for further elucidation of RNA structure-function relationships and RNA-targeted drug discovery.

RNA sequence and ligand binding alter conformational profile of SARS-CoV-2 stem loop II motif.

Antiviral drug discovery continues to be an essential complement to vaccine development for overcoming the global pandemic caused by SARS-CoV-2. The genomic RNA of SARS-CoV-2 contains structural elements important for viral replication and/or pathogenesis making them potential therapeutic targets. Here we report on the stem-loop II motif, a highly conserved noncoding RNA element. Based on our homology model we determined that the G to U transversion in the SARS-CoV-2 stem-loop II motif (S2MG35U) forms a C-U base-pair isosteric to the C-G base-pair in the early 2000's SARS-CoV (S2M). In addition, chemo-enzymatic probing and molecular dynamics simulations indicate the S2MG35U conformational profile is altered compared to S2M in the apical loop region. We explored S2MG35U as a potential drug target by docking a library of FDA approved drugs. Enzymatic probing of the best docking ligands (aminoglycosides and polymyxins) indicated that polymyxin binding alters the conformational profile and/or secondary structure of the RNA. The SARS-CoV-2 stem-loop II motif conformational differences due to nucleotide transversion and ligand binding are highly significant and provide insight for future drug discovery efforts since the conformation of noncoding RNA elements affects their function.

Identification of a unique glomerular factor X activator in murine lupus nephritis.

The role of glomerular procoagulant activity (PCA) was studied in mice (MRL/lpr, NZBxWF,, and BXSB) that are known to develop lupus nephritis. In young mice (6 to 8 wk) without renal disease, there was no increase in spontaneous glomerular PCA. In contrast, older (5 to 8 mo) autoimmune mice had significant augmentation in glomerular PCA, coinciding with the histologic appearance of severe glomerulonephritis and renal fibrin deposition. The PCA was characterized as a serine protease that directly activated factor X. This factor X activator is not tissue factor because (1) expression of PCA was not dependent on factor VII; (2) a monoclonal antibody against the factor X activator inhibited glomerular PCA, but not tissue factor; (3) the molecular weight (66 kD) of the activator was different from that of tissue factor; and (4) concanavalin A inhibited tissue factor but not glomerular PCA. Immunohistochemical studies localized the factor X activator to the glomerular mesangium and capillary wall of 4- to 6-mo-old diseased MRL/lpr mice. Immunogold-labeled antibody bound to the dense deposits, macrophages, and endothelial cells of diseased glomeruli. These studies define the role of a unique glomerular factor X activator in murine lupus nephritis.

Association of mouse fibrinogen-like protein with murine hepatitis virus-induced prothrombinase activity.

Previously, we demonstrated induction of a unique macrophage prothrombinase during infection of BALB/cJ mice by mouse hepatitis virus strain 3 (MHV-3). By immunologic screening, a clone representing this prothrombinase was isolated from a cDNA library and sequenced. The sequence identified this clone as representing part of a gene, musfiblp, that encodes a fibrinogen-like protein. Six additional clones were isolated, and one clone, p11-3-1, encompassed the entire coding region of musfiblp. Murine macrophages did not constitutively express musfiblp but, when infected with MHV-3, synthesized musfiblp-specific mRNA. musfiblp mRNA induction was earlier and significantly greater in BALB/cJ than A/J macrophages. Prothrombinase activity was demonstrated when musfiblp was expressed from p11-3-1 in RAW 264.7 cells. These data suggest that musfiblp encodes the MHV-induced prothrombinase.

Characterization of the procoagulant-inducing factor derived from the plasma of BXSB mice.

Plasma procoagulant activity inducing factor (PIF) is a spontaneously occurring, potent inducer of macrophage procoagulant activity (PCA) in the male BXSB murine model of systemic lupus erythematosus. The physical characteristics of PCA induction by PIF, aggregated mouse IgG, and lipopolysaccharide (LPS) were compared. Both aggregated IgG and PIF-induced PCA were heat, acid and alkali sensitive. In contrast, LPS-induced PCA was heat resistant and only partially acid and alkali sensitive. Plasma containing PIF was fractionated on Sephacryl S-300. The PIF activity localized to the first protein peak, molecular weight 400,000 to 900,000 daltons. Analysis of peak 1 by an enzyme-linked immunosorbent assay showed the presence of IgM, IgA and IgG. This was confirmed by Western blot analysis using 125I-labelled goat anti-mouse IgM, IgA and IgG probes. The concentration of PIF increased with Sephacryl S-300 chromatography and was reduced by removal of IgG, but not IgA or IgM by affinity chromatography. Peak 1 did not contain DNA as revealed by ethidium bromide staining. Thus, IgG from the plasma of BXSB mice, a strain which develops lupus nephritis, stimulates macrophages to express PCA, accounting for PCA induction in the BXSB model of murine lupus.

MHV-3 induced prothrombinase is encoded by musfiblp.

Previously, we demonstrated induction of a unique macrophage prothrombinase, PCA, in MHV-3 infected BALB/cJ mice. By immunologic screening, a clone representing PCA was isolated from a cDNA library and sequenced. The sequence identified this clone as representing part of a gene, musfiblp, that encodes a fibrinogen-like protein. Six additional clones were isolated, and one clone, p11-3-1, encompassed the entire coding region of musfiblp. Murine macrophages did not constitutively express musfiblp, but when infected with MHV-3, synthesized musfiblp-specific mRNA. Musfiblp mRNA induction was earlier and significantly greater in BALB/cJ than A/J macrophages. Prothrombinase activity was demonstrate when musfiblp was expressed from p11-3-1 in RAW 264.7 cells. These data suggest that musfiblp encodes the MHV-induced prothrombinase.

Monoclonal antiprothrombinase (3D4.3) prevents mortality from murine hepatitis virus (MHV-3) infection.

The induction of monocyte/macrophage procoagulant activity (PCA) has been implicated in the pathogenesis of murine hepatitis virus strain 3 (MHV-3) infection and disease. Previously, we have shown that induction of PCA by MHV-3 correlated with resistance/susceptibility to infection in different mouse strains. In this study, all BALB/cJ mice that were infected with 10(3) plaque-forming units of MHV-3 developed severe liver disease and died within 96-120 h. Examination of the livers of these animals showed marked hepatic necrosis, deposition of fibrin, and cellular expression of PCA by direct immunofluorescence staining in areas of necrosis as well as in hepatic sinusoids. Splenic mononuclear cells recovered from these mice expressed high concentrations of PCA with time after infection. Infusion into mice of a high-titered monoclonal antibody that neutralized PCA (3D4.3) attenuated the development of hepatic necrosis and enhanced survival in a dose-dependent manner. All of the animals receiving 100 micrograms, and 44% and 22% of the animals that received 50 and 25 micrograms per day, respectively, survived for 10 d and made a full recovery. Administration of the antibody resulted in a dose-dependent reduction in fibrin deposition, PCA expression as detected by direct immunofluorescence staining and by a functional assay. In animals treated with high concentrations of antibody, titers of antibody to PCA fell from 87 +/- 15 micrograms/ml to 100 +/- 7 ng/ml during the active phase of the disease, consistent with sequestration due to binding of the immunoglobulin to cells expressing PCA. Surviving animals, when rechallenged with MHV-3, had a 40% mortality, consistent with the known rates of metabolism of immunoglobulin. This further suggested that protection was by a passive mechanism. The results reported here demonstrate that a neutralizing antibody to PCA protects animals from fulminant hepatitis and death associated with MHV-3 infection, and supports the notion that PCA is a potent inflammatory mediator that plays a pivotal role in the pathogenesis of liver injury resulting from MHV-3 infection.