Journal: eLife

Article Title: Establishment and stability of the latent HIV-1 DNA reservoir

doi: 10.7554/eLife.18889

Figure Lengend Snippet: Phylogenetic trees of plasma HIV-1 RNA and PBMC HIV-1 DNA sequences from all patients included in the study. DOI: http://dx.doi.org/10.7554/eLife.18889.005

Article Snippet: Total DNA was extracted from the PBMCs using the OMEGA E.Z.N.A Blood DNA Mini Kit (Omega Bio-Tek, Norcross, Georgia) or the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions.

Techniques:

Journal: eLife

Article Title: Establishment and stability of the latent HIV-1 DNA reservoir

doi: 10.7554/eLife.18889

Figure Lengend Snippet: Sampling times before and after start of suppressive antiretroviral therapy. For each study participant, the thick grey bar indicates the period of untreated HIV-1 replication. Circles indicate the collection times of the plasma samples used for whole genome deep sequencing of the HIV-1 RNA populations ( Zanini et al., 2015 ). Triangles and squares indicate the collection times of the PBMC samples used for p17gag deep sequencing of the HIV-1 DNA populations. All times are relative to start of therapy. DOI: http://dx.doi.org/10.7554/eLife.18889.002

Article Snippet: Total DNA was extracted from the PBMCs using the OMEGA E.Z.N.A Blood DNA Mini Kit (Omega Bio-Tek, Norcross, Georgia) or the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions.

Techniques: Sampling, Sequencing

Journal: eLife

Article Title: Establishment and stability of the latent HIV-1 DNA reservoir

doi: 10.7554/eLife.18889

Figure Lengend Snippet: The distribution of plausible seeding times of reservoir HIV-1 DNA sequences for all 10 patients. For each HIV-1 DNA read obtained from the PBMCs, the plasma sample and HIV-1 RNA variant from which the read was most likely derived was determined. The plots present the distributions of these most likely origin samples across all available RNA samples. DOI: http://dx.doi.org/10.7554/eLife.18889.014

Article Snippet: Total DNA was extracted from the PBMCs using the OMEGA E.Z.N.A Blood DNA Mini Kit (Omega Bio-Tek, Norcross, Georgia) or the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions.

Techniques: Variant Assay, Derivative Assay

Journal: eLife

Article Title: Establishment and stability of the latent HIV-1 DNA reservoir

doi: 10.7554/eLife.18889

Figure Lengend Snippet: Mean root-to-tip distances for plasma HIV-1 RNA sequences obtained before the start of antiretroviral therapy (ART) and PBMC HIV-1 DNA sequences obtained after the start of ART. This figure is analogous to Figure 3 in the main text. Instead of weighing each sequence by its number of reads, each sequence was counted only once. The two approaches yielded similar results. The grey cone in the upper right quadrant indicates the rate of evolution during suppressive therapy estimated by Lorenzo-Redondo et al. (2016) . DOI: http://dx.doi.org/10.7554/eLife.18889.010

Article Snippet: Total DNA was extracted from the PBMCs using the OMEGA E.Z.N.A Blood DNA Mini Kit (Omega Bio-Tek, Norcross, Georgia) or the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions.

Techniques: Sequencing

Journal: eLife

Article Title: Establishment and stability of the latent HIV-1 DNA reservoir

doi: 10.7554/eLife.18889

Figure Lengend Snippet: Mean root-to-tip distances for plasma HIV-1 RNA sequences obtained before the start of ART and PBMC HIV-1 DNA sequences obtained after the start of ART. This figure is analogous to Figure 3 in the main text, but presents root-to-tip distance values for DNA sequences classified as hypermutants. The root-to-tip distances of the hypermutant HIV-1 DNA sequences were approximately 2 and 4% greater than the non-hypermutant sequences from the same samples, but the root-to-tip distances did not change over time. DOI: http://dx.doi.org/10.7554/eLife.18889.009

Article Snippet: Total DNA was extracted from the PBMCs using the OMEGA E.Z.N.A Blood DNA Mini Kit (Omega Bio-Tek, Norcross, Georgia) or the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions.

Techniques:

Journal: Scientific Reports

Article Title: Riboflavin, vitamin B2, attenuates NLRP3, NLRC4, AIM2, and non-canonical inflammasomes by the inhibition of caspase-1 activity

doi: 10.1038/s41598-020-76251-7

Figure Lengend Snippet: Mechanistic study of the anti-inflammasome property of riboflavin. ( A ) LPS-primed BMDMs were treated with rotenone (Rot) with/without riboflavin (B2). The maturation and secretion of IL-1β and Casp1 were analyzed by immunoblotting and the secretion of IL-1β was analyzed by ELISA. Diphenyleneiodonium chloride (DPI) was used as a ROS scavenger. ( B ) LPS-primed BMDMs were treated with ATP in the presence of B2. The cytosolic release of mitochondrial DNA (mtDNA, cytochrome c oxygenase 1/18S rDNA) was measured by quantitative real-time PCR. ( C ) The activity of human recombinant caspase-1 (hrCasp1) in the presence of B2 was measured using a commercial kit, which is a modified luciferase assay. Briefly, hrCasp1 catalyzes Z-WEHD-aminoluciferin to aminoluciferin, a substrate of luciferase, resulting in the generation of light, which represents the activity of Casp1. Ac-YVAD-CHO (YVAD, the kit supplied) was utilized as a caspase-1 inhibitor. All immunoblot data shown are representative of at least two independent experiments. The bar graph presents the mean ± SD with at least two independent experiments.

Article Snippet: The cytosolic fraction was prepared by centrifugation at 15,000 rcf for 30 min at 4 °C, and the cytosolic DNA was harvested using a G-spin total DNA extraction kit (Intron Biotechnology, Gyeonggi-do, Republic of Korea) .

Techniques: Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Activity Assay, Recombinant, Modification, Luciferase

Journal: Journal of Virology

Article Title: Enhanced Autointegration in Hyperstable Simian Immunodeficiency Virus Capsid Mutants Blocked after Reverse Transcription

doi: 10.1128/JVI.03239-12

Figure Lengend Snippet: Production of 2-LTR circles in infected cells. Recombinant viruses were used to infect HeLa cells, and at the indicated times, the levels of 2-LTR circles were measured by real-time PCR. Both a standard probe (2-LTR), which detects all 2-LTR circles, and a probe (JNCT) specific for canonical 2-LTR junctions were used. Some infected cells were treated with 1 μM raltegravir (Ral). The results for infections by wild-type (WT) SIVmac239 (A) and the A87E (B) and A87D (C) mutants are shown. The values shown represent the percentages of the maximum wild-type SIVmac239 value (823 copies/100 ng DNA at 24 h postinfection after raltegravir treatment). The results shown represent the averages and standard deviations derived from three independent infections.

Article Snippet: For Alu PCR, we performed a primary amplification using 100 ng total DNA, 0.2 μM each primer, and Pfu UltraII Hotstart 2× Master Mix (catalog number 600850; Agilent) in a final volume of 20 μl.

Techniques: Infection, Recombinant, Real-time Polymerase Chain Reaction, Derivative Assay