Journal: ACS infectious diseases

Article Title: A Sensitive In Vitro High-Throughput Screen to Identify Pan-Filoviral Replication Inhibitors Targeting the VP35-NP Interface

doi: 10.1021/acsinfecdis.6b00209

Figure Lengend Snippet: The EBOV VP35/NP interface is a target for HTS assay development. (A) EBOV RNA synthesis requires viral proteins NP, VP35, and polymerase L. The N-terminal domain (NTD) of NP (blue) binds ssRNA and the VP35 NPBP (VP35 NP binding peptide) (red). The NP C-terminal domain (CTD) is important for viral replication, but the precise function is currently unknown. VP35 oligomerization domain (OD) and interferon inhibitory domain (IID) are labeled as shown. Arrows indicate interactions that are validated at present. (B) Ribbon representation of the crystal structure of the EBOV ΔNPNTD (blue) and VP35 NPBP (red) complex (PDB 4YPI). (C) Schematic of the fluorescence polarization assay based on the NP/NPBP interaction. Outcomes in the polarization assays and the corresponding peptides (NPBP or FITC-NPBP), protein receptor (ΔNPNTD) and competitor are labeled.

Article Snippet: All peptides, including VP35 NPBP, FITC-NPBP, Bio-NPBP, Nrf2 ETGE, and FITC-ETGE were purchased from GenScript (Piscataway, New Jersey).

Techniques: HTS Assay, Binding Assay, Labeling, Fluorescence

Journal: ACS infectious diseases

Article Title: A Sensitive In Vitro High-Throughput Screen to Identify Pan-Filoviral Replication Inhibitors Targeting the VP35-NP Interface

doi: 10.1021/acsinfecdis.6b00209

Figure Lengend Snippet: FITC-NPBP binding to ΔNPNTD results in a highly sensitive FPA with a large dynamic range. (A) Titration with the protein receptor, ΔNPNTD, results in an increase in fluorescence polarization of FITC-NPBP peptide. Apparent KD = 32 ± 3 nM was calculated for FITC-NPBP from non-linear fitting (red) using Origin (San Clemente, California). (B) Competition with unlabeled NPBP (black) and NPBP truncation mutants 28–46 (red) and 26–48 (green) with 1 nM FITC-NPBP in the presence of 20 nM ΔNPNTD resulted in apparent dissociation constants of 0.27 ± 0.07, 1.39 ± 0.36, and 6.61 ± 1.47 µM for NPBP and NPBP mutants, respectively.

Article Snippet: All peptides, including VP35 NPBP, FITC-NPBP, Bio-NPBP, Nrf2 ETGE, and FITC-ETGE were purchased from GenScript (Piscataway, New Jersey).

Techniques: Binding Assay, Titration, Fluorescence

Journal: ACS infectious diseases

Article Title: A Sensitive In Vitro High-Throughput Screen to Identify Pan-Filoviral Replication Inhibitors Targeting the VP35-NP Interface

doi: 10.1021/acsinfecdis.6b00209

Figure Lengend Snippet: Results from the initial screens reveals that the assay is highly reproducible. (A) FP signals from 22 samples containing 1 nM free FITC-NPBP (red, positive controls) and with 20 nM ΔNPNTD (blue, negative controls) were used to determine the Z'-factor, signal-to-noise (S/N) and signal-to-background (S/B) ratios, and assay window values using 1 nM probe (FITC-NPBP). See Table S1. (B) FP signals of 22 samples of 1 nM FITC-NPBP + 20 nM ΔNPNTD (blue, negative control) and with 1 µM NPBP (magenta, positive control) plotted versus sample wells. (C) Pilot screening using 640 FDA-approved drugs (green). 64 samples of 1 nM FITC-NPBP + 1 µM NPBP with 20 nM ΔNPNTD (magenta) and 64 of nM FITC-NPBP with 20 nM ΔNPNTD (blue) are used as positive and negative controls, respectively. (D) Results from duplicate screens of the FDA-approved drugs were converted to Z-scores with respect to the negative control. The correlation coefficient (R2) between the duplicate assays is 0.74.

Article Snippet: All peptides, including VP35 NPBP, FITC-NPBP, Bio-NPBP, Nrf2 ETGE, and FITC-ETGE were purchased from GenScript (Piscataway, New Jersey).

Techniques: Negative Control, Positive Control

Journal: ACS infectious diseases

Article Title: A Sensitive In Vitro High-Throughput Screen to Identify Pan-Filoviral Replication Inhibitors Targeting the VP35-NP Interface

doi: 10.1021/acsinfecdis.6b00209

Figure Lengend Snippet: The NPBP/NP FPA assay identified several hits that show dose-dependent responses. Representative data for a non-hit (A) and three hits (B–D) are shown where FP (blue) is plotted as a function of compound concentration for: (A) trequinsin, (B) epinephrine-(+)-L(−), (C) 5-aminosalicylic acid, and (D) tolcapone. Assay conditions contained 1 nM FITC-NPBP + 20 nM ΔNPNTD with 2-fold dilutions of compounds. Fluorescence intensity is plotted in green.

Article Snippet: All peptides, including VP35 NPBP, FITC-NPBP, Bio-NPBP, Nrf2 ETGE, and FITC-ETGE were purchased from GenScript (Piscataway, New Jersey).

Techniques: Concentration Assay, Fluorescence

Journal: ACS infectious diseases

Article Title: A Sensitive In Vitro High-Throughput Screen to Identify Pan-Filoviral Replication Inhibitors Targeting the VP35-NP Interface

doi: 10.1021/acsinfecdis.6b00209

Figure Lengend Snippet: Validation of compounds identified from FPA with biolayer interferometry (BLI) analysis and infectious assay. (A) BLI analysis of VP35 NPBP binding to ΔNPNTD in the presence of compounds. BLI association and dissociation curves for NPBP (black), epinephrine-(+)-L (−) (red), tolcapone (blue), 5-aminosalicylic acid (cyan), and trequinsin (orange). Baseline curve for DMSO only is shown in gray. (B) Normalized data from (A) at 5 seconds prior to the beginning of dissociation indicated by the arrowhead in (A). 250 nM ΔNPNTD and 10 µM competitor were used in the assays. (C and D) Tolcapone inhibits the infection of Vero cells by EBOV-GFP. (C) Bright-field (top) and fluorescence (bottom) images of EBOV-GFP-infected Vero E6 cells treated with DMSO (mock) or Tolcapone at 10 µM 4dpi. (D) Viral titers were determined by focus-forming assay 3 dpi. Results are in triplicate and plotted as averages and standard deviations. * indicates p value <0.030, ** p value <0.011.

Article Snippet: All peptides, including VP35 NPBP, FITC-NPBP, Bio-NPBP, Nrf2 ETGE, and FITC-ETGE were purchased from GenScript (Piscataway, New Jersey).

Techniques: Biomarker Discovery, Binding Assay, Infection, Fluorescence, Focus Forming Assay

Journal: ACS infectious diseases

Article Title: A Sensitive In Vitro High-Throughput Screen to Identify Pan-Filoviral Replication Inhibitors Targeting the VP35-NP Interface

doi: 10.1021/acsinfecdis.6b00209

Figure Lengend Snippet: EBOV NPBP/NP FP assay is a versatile panfiloviral assay that can function with NP proteins from all ebolaviruses and marburgviruses. (A) Multiple sequence alignment of the NPBP regions from Ebolavirus (accession AAQ55046), Restonvirus (accession AB050936.1), Sudanvirus (accession EU338380.1) and Marburgvirus (accession CAA78115) using ClustalW. Identical residues are shown in blue and labeled with '*'; strongly conserved in green and with ':'; and weakly conserved in dark blue and with '.'. FPA dose response curves for FITC-NPBP competition with corresponding NPBP sequences from (B) Ebolavirus (NPBP), (C) Restonvirus (rNPBP), (D) Sudanvirus (sNPBP), and (E) Marburgvirus (mNPBP). (F) Normalized BLI data for the competitors in B–E in Figure 5 using MARV NP as the protein receptor. 250 nM ΔNPNTD mNPNTD and 10 µM competitor were used in the assays.

Article Snippet: All peptides, including VP35 NPBP, FITC-NPBP, Bio-NPBP, Nrf2 ETGE, and FITC-ETGE were purchased from GenScript (Piscataway, New Jersey).

Techniques: FP Assay, Sequencing, Labeling

Journal: Marine Drugs

Article Title: Evaluation of Rapid, Early Warning Approaches to Track Shellfish Toxins Associated with Dinophysis and Alexandrium Blooms

doi: 10.3390/md16010028

Figure Lengend Snippet: Locations of sampling sites in Northport Bay and Cold Spring Harbor (CSH), New York. Black diamonds = wild shellfish sampling sites in Cold Spring Harbor; black circles = time series sampling sites in Northport Harbor (NPB2 and NPB8), Black star = wild shellfish sampling site Scudder Beach (SB) Northport Harbor.

Article Snippet: During 2012, subsurface water (~0.25 m) was collected on a weekly to twice-weekly basis from March–August at two sites, Britannia (NPB2) and Woodbine (NPB8), in Northport Harbor, which is within the southeastern portion of the Northport-Huntington Bay complex, located on the north shore of Long Island, NY, USA ( ).

Techniques: Sampling

Journal: Marine Drugs

Article Title: Evaluation of Rapid, Early Warning Approaches to Track Shellfish Toxins Associated with Dinophysis and Alexandrium Blooms

doi: 10.3390/md16010028

Figure Lengend Snippet: Comparison of ( A , B ) DSP toxins (OA, DTX1, esterified OA and esterified DTX1; ng g −1 of dry resin) and ( C , D ) pectenotoxins (PTX2, PTX2 isomers, secoPTX2 and PTX-11-like compounds) adsorbed by two resins (HP20 and XAD-2) in passive solid-phase adsorption toxin tracking (SPATT) samplers deployed side-by-side at NPB2 (Britannia) in Northport Harbor, New York. Bars represent the value of a single sample.

Article Snippet: During 2012, subsurface water (~0.25 m) was collected on a weekly to twice-weekly basis from March–August at two sites, Britannia (NPB2) and Woodbine (NPB8), in Northport Harbor, which is within the southeastern portion of the Northport-Huntington Bay complex, located on the north shore of Long Island, NY, USA ( ).

Techniques: Comparison, Adsorption

Journal: Marine Drugs

Article Title: Evaluation of Rapid, Early Warning Approaches to Track Shellfish Toxins Associated with Dinophysis and Alexandrium Blooms

doi: 10.3390/md16010028

Figure Lengend Snippet: ( A ) Log Dinophysis densities (cells L −1 ), the DSP toxins, okadaic acid (OA) dinophysistoxin 1 (DTX1) and their esters (pg mL −1 ) and total pectenotoxins (PTX; pg mL −1 × 10) for NPB2 (Britannia) located in Northport Harbor, during 2012. ( B ) Dynamics of the DSP toxins, OA, DTX1 and their esters, as well as total pectenotoxins (ng g −1 ) in blue mussels ( Mytilus edulis ) hung for monitoring purposes. The red line indicates USFDA action level (160 ng g −1 shellfish tissue). ( C ) DSP toxins (OA, DTX1 and esters) and pectenotoxins (ng g −1 of dry resin) adsorbed by SPATT samplers containing HP20 resin that were hung alongside mussels bags in Northport Harbor. Pie chart insets represent the percent of total toxin each DSP congener represents (colors as in bottom panel). For Dinophysis densities, points are means while error bars represent the standard deviation of duplicate samples. For all toxin samples, points represent the value of a single sample. For shellfish, points represent 15–20 individuals pooled prior to toxin analysis.

Article Snippet: During 2012, subsurface water (~0.25 m) was collected on a weekly to twice-weekly basis from March–August at two sites, Britannia (NPB2) and Woodbine (NPB8), in Northport Harbor, which is within the southeastern portion of the Northport-Huntington Bay complex, located on the north shore of Long Island, NY, USA ( ).

Techniques: Standard Deviation

Journal: Marine Drugs

Article Title: Evaluation of Rapid, Early Warning Approaches to Track Shellfish Toxins Associated with Dinophysis and Alexandrium Blooms

doi: 10.3390/md16010028

Figure Lengend Snippet: Dinophysis densities (cells L −1 ) during the spring and summer of ( A ) 2013 and ( B ) 2014 at Northport Bay (NPB2) and two sites in Cold Spring Harbor (CSH 1 and CSH2). Points are means, while error bars represent the standard deviation of duplicate samples.

Article Snippet: During 2012, subsurface water (~0.25 m) was collected on a weekly to twice-weekly basis from March–August at two sites, Britannia (NPB2) and Woodbine (NPB8), in Northport Harbor, which is within the southeastern portion of the Northport-Huntington Bay complex, located on the north shore of Long Island, NY, USA ( ).

Techniques: Standard Deviation