Journal: Cells

Article Title: Phospholipid Encapsulation of an Anti-Fibrotic Endopeptide to Enhance Cellular Uptake and Myocardial Retention

doi: 10.3390/cells12121589

Figure Lengend Snippet: L-Ac-SDKP bioavailability after daily i.p. injection in mice with and without left chest radiation. Biodistribution of Ac-SDKP in the serum and heart was evaluated after four i.p. injections given daily one day after thoracic irradiation. ( a ), representative IVIS spectrum images ( b ), and biofluorescence quantification ( c ) of the tissues demonstrating fluorescence intensity in animals receiving L-Ac-SDKP with and without radiation normalized to that in PBS-treated (vehicle) controls. The coloring corresponds to the spectrum gradient bar for the radiant efficiency unit (p/s/cm 2 /sr). Scale bar, 5 mm.

Article Snippet: The biodistribution of the fluorescent compound was analyzed using the IVIS Spectrum in vivo imaging system (PerkinElmer) in a smaller subset of animals exposed to single dose chest radiation as described before [ ].

Techniques: Injection, Irradiation, Fluorescence

Journal: Cell Reports

Article Title: Cdc14 and PP2A Phosphatases Cooperate to Shape Phosphoproteome Dynamics during Mitotic Exit

doi: 10.1016/j.celrep.2019.10.041

Figure Lengend Snippet:

Article Snippet: ECL Prime Western Blotting Detection Regent , GE Healthcare , Cat# RPN2232.

Techniques: Recombinant, Protease Inhibitor, Staining, Clone Assay, Western Blot, Mass Spectrometry, Software

Journal: bioRxiv

Article Title: Discovery of five HIV nucleoside analog reverse-transcriptase inhibitors (NRTIs) as potent inhibitors against the RNA-dependent RNA polymerase (RdRp) of SARS-CoV and 2019-nCoV

doi: 10.1101/2020.11.01.363788

Figure Lengend Snippet: (A) Purified nsp12, nsp13, nsp7 and nsp8 of SARS-CoV and nsp12 of 2019-nCoV were determined by SDS-PAGE and indicated by stars (*) . Nsp8 alternative cleavage by factor Xa protease was indicated by (#) . Five impurities of SARS-CoV nsp12 (a-e) were determined by Mass spectrometry. (B) The activity of SARS-CoV nsp12 on a double-stranded primer-template RNA complex was evaluated by primer extension assay at various concentrations of MgCl 2 or MnCl 2 combined with 50 mM either NaCl or KCl. The assay contained 25 mM Tris HCl pH 8.0, 1 mM DTT, 100 μM rNTPs, 32 nM nsp12 and 10 nM Cy5.5-labeled RNA primer which was annealed to its template. Reaction mix was incubated at 37°C for 2h and analyzed by denaturing Urea-PAGE. The gel was scanned and extended products were indicated by red (*) or (>). (C) The activity of nsp12 were also evaluated in combination with nsp7 and nsp8. Concentrations of nsp12, nsp7 and nsp8 were 16 nM, 400 nM and 400 nM, respectively. 50% Glycerol was used as control.

Article Snippet: In addition, the 24 nucleotide analogs were screened on SARS-CoV primase nsp8 which revealed stavudine and remdesivir were specific inhibitors to nsp12.

Techniques: Purification, SDS Page, Mass Spectrometry, Activity Assay, Primer Extension Assay, Labeling, Incubation

Journal: bioRxiv

Article Title: Discovery of five HIV nucleoside analog reverse-transcriptase inhibitors (NRTIs) as potent inhibitors against the RNA-dependent RNA polymerase (RdRp) of SARS-CoV and 2019-nCoV

doi: 10.1101/2020.11.01.363788

Figure Lengend Snippet: (A) Two RNA templates were used in this study. One perfectly paired with the Cy5.5-labeled RNA primer, which had been used in . The other one formed a stem-loop structure with the primer. The primer was annealed to template in a ratio of 1:3 to form a P/T complex. (B) The extension activity of nsp12 on the two primer-template complexes was determined with combination of SARS-CoV helicase nsp13. Two combination ratios were used: 1:13 and 1:26. The assay contained 25 mM Tris HCl pH 8.0, 50 mM KCl, 1 mM DTT, 1 mM MgCl2, 10 nM P/T complex, 16 nM nsp12, 210 or 420 nM nsp13, 100 μM rNTPs (25 μM each), 10% Glycerol, 6 mM NaCl and 0.02% triton X-100. The activities of nsp12, nsp13, nsp7 and nsp8 on the single-stranded primer without a template were also determined. 50% Glycerol was used as controls. Reaction mix was incubated at 37°C for 2h. (C) Back-priming mechanism was proposed. The sequence and secondary strucuture of the extended product was predicted. Primer was shown in black and the extension was shown in red.

Article Snippet: In addition, the 24 nucleotide analogs were screened on SARS-CoV primase nsp8 which revealed stavudine and remdesivir were specific inhibitors to nsp12.

Techniques: Labeling, Activity Assay, Incubation, Sequencing

Journal: bioRxiv

Article Title: Discovery of five HIV nucleoside analog reverse-transcriptase inhibitors (NRTIs) as potent inhibitors against the RNA-dependent RNA polymerase (RdRp) of SARS-CoV and 2019-nCoV

doi: 10.1101/2020.11.01.363788

Figure Lengend Snippet: (A) The nsp12 hits with >40% inhibition were verified at 1, 2, and 4 mM using the same assay as screening. Dose-dependent decrease of extended RNA product was observed. Remdesivir which had 28.44% inhibition on nsp12 was included as well. ATP was used as normalization control. Same concentration of primer was used as primer control. 100 μM rNTPs without adding extra nucleotide was used as rNTP control. (B) The inhibition on nsp8 of the 24 nucleotide analog triphosphates were also screened to identify nsp12-specific inhibitors and their inhibition percentages on nsp8 were compared to nsp12. Drugs with >20% differential inhibition between nsp12 and nsp8 were highlighted in bold.

Article Snippet: In addition, the 24 nucleotide analogs were screened on SARS-CoV primase nsp8 which revealed stavudine and remdesivir were specific inhibitors to nsp12.

Techniques: Inhibition, Concentration Assay

Journal: bioRxiv

Article Title: Discovery of five HIV nucleoside analog reverse-transcriptase inhibitors (NRTIs) as potent inhibitors against the RNA-dependent RNA polymerase (RdRp) of SARS-CoV and 2019-nCoV

doi: 10.1101/2020.11.01.363788

Figure Lengend Snippet: (A) The activity of 2019-nCoV nsp12 was determined at different time-points which showed similar activities to SARS-CoV. 50% Glycerol incubated for 120 min was used as control. (B) Clofarabine, stavudine, gemcitabine, tenofovir, ganciclovir and remdesivir from SARS-CoV screening were selected and their inhibition on 2019-nCoV nsp12 was evaluated at 4 mM which showed inhibition as well. The assay condition was same as SARS-CoV. The analogs were evaluated in their corresponding triphosphate forms. ATP at 4 mM was used as normalization control. Same concentration of primer was loaded as negative control. (C) showed the percent of inhibition as compared to ATP of which the activity was defined as 100% and inhibition as zero.

Article Snippet: In addition, the 24 nucleotide analogs were screened on SARS-CoV primase nsp8 which revealed stavudine and remdesivir were specific inhibitors to nsp12.

Techniques: Activity Assay, Incubation, Inhibition, Concentration Assay, Negative Control

Journal: bioRxiv

Article Title: Discovery of five HIV nucleoside analog reverse-transcriptase inhibitors (NRTIs) as potent inhibitors against the RNA-dependent RNA polymerase (RdRp) of SARS-CoV and 2019-nCoV

doi: 10.1101/2020.11.01.363788

Figure Lengend Snippet: Eight FDA-approved HIV NRTIs were evaluated for their inhibition on the nsp12 of SARS-CoV (A) and 2019-nCoV (B) . The assay contained 16 nM nsp12, 10 nM RNA primer, 100 μM rNTPs and 4 mM a NRTI, in its corresponding active triphosphate form. The extended product of the RNA primer by nsp12 upon NRTI treatment was analyzed by Urea-PAGE. Product was quantified and percent of inhibition was calculated. ATP was used as normalization control. Clofarabine, an anticancer drug, was used as treatment control. Assay with 50% glycerol instead of nsp12 was used as primer control. (C) showed the percent of inhibition of the 8 NRTIs, compared to ATP control. SARS-CoV was presented by white bar with slashes. 2019-nCoV was presented by gray bar with dots.

Article Snippet: In addition, the 24 nucleotide analogs were screened on SARS-CoV primase nsp8 which revealed stavudine and remdesivir were specific inhibitors to nsp12.

Techniques: Inhibition, Control Assay

Journal: bioRxiv

Article Title: Discovery of five HIV nucleoside analog reverse-transcriptase inhibitors (NRTIs) as potent inhibitors against the RNA-dependent RNA polymerase (RdRp) of SARS-CoV and 2019-nCoV

doi: 10.1101/2020.11.01.363788

Figure Lengend Snippet: The three newly identified NRTIs, abacavir (carbovir-TP), zidovudine and zalcitabine triphosphates, were verified at three concentrations (1, 2 and 4 mM) for their inhibition on the RNA extension by nsp12, using SARS-CoV as a model. The Cy5.5-labeled RNA primer was used and its extended product as well as pyrophosphorylation product by SARS-CoV nsp12 were visualized. ATP was used as treatment negative control. Tenofovir was used as treatment positive control. Reaction with 50% glycerol instead of nsp12 was used as primer control. For abacavir and zidovudine, inhibition was confirmed at 4 mM. For zalcitabine, inhibition was observed at both 2 and 4 mM.

Article Snippet: In addition, the 24 nucleotide analogs were screened on SARS-CoV primase nsp8 which revealed stavudine and remdesivir were specific inhibitors to nsp12.

Techniques: Inhibition, Labeling, Negative Control, Positive Control