Journal: Scientific Reports

Article Title: A screen for novel hepatitis C virus RdRp inhibitor identifies a broad-spectrum antiviral compound

doi: 10.1038/s41598-017-04449-3

Figure Lengend Snippet: Assays with HCV genotype 3a replicon. ( A ) G418 resistant HCV-3a replicon expressing Huh7.5 cells were treated with indicated compounds for 48 h and the firefly luciferase activity was plotted as relative luciferase units (RLU). DMSO treated HCV-3a replicon expressing Huh 7.5 cells was taken as 100%. ( B ) The toxicity of these compounds in the replicon expressing cells was measured using WST-1 assay reagent. The values are depicted as percentages with the DMSO treated cells taken as 100%. ( C ) The replicon expressing cells were treated with varying concentrations of 66E2 and relative luciferase unit is plotted against the concentration of 66E2. EC 50 is the compound concentration that inhibits 50% of viral replication (RLU). ( D ) Huh7.5 cells were treated with indicated concentrations of 66E2 and cytotoxicity determined using WST-1 assay reagent. The values are plotted as percentages with the DMSO treated cells taken as 100%. CC 50 is the compound concentration that produces 50% of cytotoxicity. ( E ) Western blot analysis. Replicon expressing Huh7.5 cells were treated with different concentrations of 66E2 and CMC for 48 hours and expression of NS5A monitored using anti NS5A antibody. Expression of GAPDH was determined for loading control. ( F ) Reverse-transcriptase quantitative PCR analysis was performed to determine the levels of positive and negative sense HCV-3a RNA upon treatment with 66E2 and CMC at 5 μM for 48 h. The % mean is shown above the bars and the error bars are standard deviations. All the assays in the figure were performed in triplicates and results presented are representative of at least three independent assays.

Article Snippet: Antibodies against HCV NS5A (ab13833) and DENV (ab26837 and ab9202) were from Abcam and anti GAPDH was purchased from Santa Cruz Biotechnology (USA).

Techniques: Expressing, Luciferase, Activity Assay, WST-1 Assay, Concentration Assay, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: British Journal of Pharmacology

Article Title: Role of H 2 O 2 in hypertension, renin-angiotensin system activation and renal medullary disfunction caused by angiotensin II

doi: 10.1111/j.1476-5381.2012.01957.x

Figure Lengend Snippet: Earlier (day 10) effects of Ang II, with or without PEG-catalase, on renal Nox1 protein expression on day 10. Data are expressed as representative immunoblots with summary bar graphs (mean ± SEM values), part III, n= 4–7.

Article Snippet: Proteins were transferred onto nitrocellulose membranes, blocked for 1 h and incubated overnight at 4°C with specific antibody against Nox4 (sc-21860, 1:200, Santa Cruz Biotechnology or ET3174-1, 1:200, Epitomics, Burlingame, CA, USA), Nox1 (sc-5821, 1:200, Santa Cruz Biotechnology) or AT 1 receptor (sc-1173, 1:500, Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot

Journal: BMC Infectious Diseases

Article Title: The PTAP sequence duplication in HIV-1 subtype C Gag p6 in drug-naive subjects of India and South Africa

doi: 10.1186/s12879-017-2184-4

Figure Lengend Snippet: a Schematic representation of HIV-1 Gag protein domains. The four major domains of Gag (MA, CA, p7, and p6) are depicted including the two linker sequences p1 and p2. HIV-1 Gag interacts with the ESCRT complex proteins Tsg101 and Alix to regulate viral budding. The sequence of subtype B NL4-3 gag p6 is presented and the sequence motifs PTAP and YPXnL, which serve as the binding motif for Tsg101 and Alix, respectively, are highlighted using the square boxes. Con_C represents the subtype C gag p6 consensus amino acid sequence. The dashes represent sequence identity and the dots sequence deletion. b A comparative analysis of the PTAP sequence duplication in subtypes B and C. In subtype B, a partial PTAP duplication consisting of three amino-acid residues (APP) is common. In contrast in subtype C, a sequence duplication of 14 amino acids is common. A 14 amino acid sequence duplication of subtype C derived from the primary clinical isolate T004 is presented. The amino acid sequences, the original and the duplicated sequences, in Gag and Gag-Pol are depicted. The arrows represent the length of sequence duplication and the direction of polymerization by the reverse transcriptase while synthesizing the cDNA from the viral RNA. The core PTAP motifs are highlighted using the square boxes. The sequences flanking the 3 or 14 aa residues are shown in gray

Article Snippet: RNA was extracted from 150 μl of plasma samples using a commercial Viral RNA isolation kit (NucleoSpin® RNA Virus, Ref. No. 740956.50, MACHEREY-NAGEL GmbH & Co. KG, Germany).

Techniques: Sequencing, Binding Assay, Derivative Assay, Reverse Transcription

Journal: BMC Infectious Diseases

Article Title: The PTAP sequence duplication in HIV-1 subtype C Gag p6 in drug-naive subjects of India and South Africa

doi: 10.1186/s12879-017-2184-4

Figure Lengend Snippet: Multiple sequence alignment of the PTAP sequences of eight primary viral isolates containing PTAP duplication. The sequences were derived from the plasma viral RNA collected at multiple longitudinal time-points and by sequencing the PCR fragments. In three subjects (T004, 2012, 2032), the PTAP motif duplication involved the duplication of 14 amino acids. In the other five subjects, the duplicated sequence length was shorter containing 12 (2018), 11(2020), 9 (2037) or 8 (T014, and 2006) residues. Of note, in all the sequences, the ‘PTAP’ core motif was intact in the original and the duplicated sequences. Note that in two subjects (2014 and 2018) a mixed infection of single- and double-PTAP viral strains could be seen by conventional sequencing. In subject T014, PTAP duplication was observed only at a single time-point M24. For subject 2006, the plasma sample was available only at the baseline. The vertical dashed line demarcates the original and the duplicated PTAP motifs. The arrows indicate the direction of the RT polymerization. Cons: The consensus subtype C gag sequence of 14 amino acids. The same 14 amino acid consensus sequence was used twice in the analysis for convenience. *For these five samples, only gag p6 , not the full-length gag , was amplified. These sequences have not been deposited in the Genbank and are not part of the phylogenetic tree presented in Fig. . D: the duplicated PTAP motif, O, the original PTAP motif

Article Snippet: RNA was extracted from 150 μl of plasma samples using a commercial Viral RNA isolation kit (NucleoSpin® RNA Virus, Ref. No. 740956.50, MACHEREY-NAGEL GmbH & Co. KG, Germany).

Techniques: Sequencing, Derivative Assay, Clinical Proteomics, Infection, Amplification

Journal: MethodsX

Article Title: An oligo-swapping method: preparation of mismatch repair-monitoring substrate using a nicking endonuclease

doi: 10.1016/j.mex.2025.103715

Figure Lengend Snippet: Aliquots from various steps of the purification were analyzed on 0.8 % agarose gel, and the DNA substrates were visualized via EtBr staining. A) Lane 1, pBET2 (Method details, step 1); lane 2, Nt. Bbv CI-treatment (Method details, step 2); lane 3, T4 DNA ligase-treatment (Method details, step 4); lane 4, Spe I-HF- treatment (Method details, step 5); and lane 5, T5 exonuclease-treatment (Method details, step 6). Open circular DNA (OC), linear DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows. B) DNA conformation in each lane. In lane 1, the purified pBET2 plasmid is a closed circular DNA. In lane 2, gapped pBET2 is an open circular DNA. In lane 3, gapped and non-reacted DNA are open circular DNA, while mismatch and non-mismatch DNA are closed circular DNA. In lane 4, nicked DNA and non-mismatch DNA are digested with Spe I-HF, resulting in linear DNA. In lane 5, the subsequent step entails the removal of linear and gapped DNA by T5 exonuclease to isolate mismatch DNA, pBET2 C/A. A single nick site for MMR s introduced. In lane 6, pBET2 C/A is purified using a standard PCR purification kit. In lane 7, the DNA is digested by a nicking endonuclease, forming open circular DNA.

Article Snippet: Use a PCR purification kit (MACHEREY-NAGEL) to purify the mismatched DNA substrate from the reaction mixtures ( C, lanes 6, 200 ng/lane).

Techniques: Purification, Agarose Gel Electrophoresis, Staining, Plasmid Preparation