Journal: PLoS Pathogens

Article Title: HIV infects astrocytes in vivo and egresses from the brain to the periphery

doi: 10.1371/journal.ppat.1008381

Figure Lengend Snippet: Neonate NSG mice were xenotransplanted with HIV- or HIV+ NHAs as described in and HIV detection outside of the brain was evaluated by Real-time PCR products (HIV DNA, A , or HIV RNA, B ) from brain, cervical lymph node, spleen, splenocytes outgrowth assay and splenocytes outgrowth assay supernatant added to fresh PBMCs analyzed by electrophoresis and human GAPDH. Each column indicates individual animal. HIV- animals are shown to left of ladder, HIV+ animals shown to right for all gels shown. No template control (NTC) is the negative control. M/F indicates sex and number is animal number from that group. ( C ) GFP expression in splenocytes at day 14 day of culturing the cells and ( D ) flow cytometry of cultured splenocytes stained for CD3+/CD4+/GFP; dot blots (left) and cumulative data on right ( p = 0.05, Mann-Whitney U-test). ( E ) Representative image of supernatant from splenocytes cultured for 14 days from neonates from HIV- animal (top) or HIV+ animal (bottom) on fresh PBMCs stimulated with soluble α-CD3 and α-CD28 IL-2. Egress for neonates was analyzed in n = 7 (HIV-) and 12 (HIV+) mice; n = 3 (HIV-) and 4 (HIV+) representative mice are shown here; n = 3 (HIV-) and 5 (HIV+) representative mice are shown here.

Article Snippet: Monocytes were depleted using RosetteSep Human Monocyte Depletion Cocktail and CD4+ T cells by RosetteSep Human CD4+ Depletion Cocktail (STEMCELL Technologies, Cambridge, MA) and were injected IP with 2 × 10 7 of the remaining PBMCs.

Techniques: Real-time Polymerase Chain Reaction, Electrophoresis, Control, Negative Control, Expressing, Flow Cytometry, Cell Culture, Staining, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: HIV infects astrocytes in vivo and egresses from the brain to the periphery

doi: 10.1371/journal.ppat.1008381

Figure Lengend Snippet: Adult NSG mice were xenotransplanted with HIV- or HIV+ NHAs as described in and HIV detection outside of the brain was evaluated by Real-time PCR products (HIV DNA, A , or HIV RNA, B ) from brain, cervical lymph node, spleen, peripheral lymph node, splenocytes outgrowth assay and splenocytes outgrowth assay supernatant added to fresh PBMCs analyzed by electrophoresis and human GAPDH. Each column indicates individual animal. HIV- animals are shown to left of ladder, HIV+ animals shown to right for all gels shown. No template control (NTC) is the negative control. ( C ) GFP expression in splenocytes at day 14 day of culturing the cells and ( D ) flow cytometry of cultured splenocytes stained for CD3+/CD4+/GFP; dot blots (left) and cumulative data on right ( p = 0.04, Mann-Whitney U-test). ( E ) Representative image of supernatant from splenocytes cultured for 14 days from neonates from HIV- animal (top) or HIV+ animal (bottom) on fresh PBMCs stimulated with soluble α-CD3 and α-CD28 IL-2. Egress was analyzed in n = 7 (HIV-) and 9 (HIV+) mice; n = 3 (HIV-) and 5 (HIV+) representative mice are shown here.

Article Snippet: Monocytes were depleted using RosetteSep Human Monocyte Depletion Cocktail and CD4+ T cells by RosetteSep Human CD4+ Depletion Cocktail (STEMCELL Technologies, Cambridge, MA) and were injected IP with 2 × 10 7 of the remaining PBMCs.

Techniques: Real-time Polymerase Chain Reaction, Electrophoresis, Control, Negative Control, Expressing, Flow Cytometry, Cell Culture, Staining, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: HIV infects astrocytes in vivo and egresses from the brain to the periphery

doi: 10.1371/journal.ppat.1008381

Figure Lengend Snippet: ( A ) Representative image of neonate hippocampus (HPC) immunostained for human T cells (CD3+, magenta), human astrocytes (huGFAP; red), HIV (green) and Nuclei (DAPI, blue) at week 10 post-xenotransplantation and week 4 reconstitution with huPBMCs. Arrows indicates detection of CD3+/HIV+ cell. Scale bar, 10μm. ( B ) Total PBMCs (HIV+) or monocyte/CD4+ T cell depleted PBMCs analyzed by flow cytometry for CD3+/CD4+ positive T cells (top) or CD14 positive or CD14/CD16 positive monocytes before injection into the adult mouse xenotransplanted with HIV VSVg + NHAs. HIV DNA ( C ) and RNA ( D ) from spleen from animals xenotransplanted with HIV VSVg + NHAs and reconstituted with total PBMCs (HIV+) or PBMCs depleted of monocytes and CD4+ T cells (Mono/CD4). No analysis was performed on DNA as Mono/CD4 group had undetectable HIV DNA. RNA is depicted on the right ( p = 0 . 08 , Mann-Whitney U-test). n = 5–7 per group.

Article Snippet: Monocytes were depleted using RosetteSep Human Monocyte Depletion Cocktail and CD4+ T cells by RosetteSep Human CD4+ Depletion Cocktail (STEMCELL Technologies, Cambridge, MA) and were injected IP with 2 × 10 7 of the remaining PBMCs.

Techniques: Flow Cytometry, Injection, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: HIV infects astrocytes in vivo and egresses from the brain to the periphery

doi: 10.1371/journal.ppat.1008381

Figure Lengend Snippet: Human post mortem analysis of HIV+ astrocytes. Post mortem analysis from cortical or hippocampal brain regions of HIV- or HIV+ individuals. Laser capture microdissection (LCM) of GFAP+ cells analyzed for Alu-gag HIV by PCR. RNAscope and staining determined GFAP+ co-localization with gag mRNA, nef DNA and p24. Staining for each patient included analysis of 12 different sections with 20–24 fields per section (~240 fields) from an average tissue size of 1.5±0.75x0.91±0.66 cm. M: male; F: Female; N.A.: not applicable; U.D.: undetectable. Anti-retrovirals: ATV, atazanavir (Reyataz); EFV, efavirenz (Sustiva); RTV, ritonavir (Norvir); TFV, PMPA; tenofivir DF (Viread); 3TC, lamivudine (Epivir); D4T, stavudine (Zerit FTV, SQV2 or FTV saquinavir-sgc (Fortovase);SQV, saquinavir (Invirase); DDI, didanosine (Videx); NFV, nelfinavir (Viracept). Representative staining images of data are shown in Fig 9 .

Article Snippet: Monocytes were depleted using RosetteSep Human Monocyte Depletion Cocktail and CD4+ T cells by RosetteSep Human CD4+ Depletion Cocktail (STEMCELL Technologies, Cambridge, MA) and were injected IP with 2 × 10 7 of the remaining PBMCs.

Techniques: Laser Capture Microdissection, RNAscope, Staining, Clinical Proteomics

Journal: Cell reports

Article Title: Abundant binary promoter switches in lineage-determining transcription factors indicate a digital component of cell fate determination

doi: 10.1016/j.celrep.2023.113454

Figure Lengend Snippet: (A) Lineage-determining TFs were examined in IGV loaded with 5′scRNA-seq data from 2-week CD34 + HPC cultures, ex vivo purified tonsil ILCs, or purified peripheral blood NK cell libraries. Blue arrows indicate sense strand TSS and red arrows indicate antisense TSS. Red boxes indicate antisense exons, and blue boxes indicate sense exons. The larger dark-blue boxes indicate coding regions. The number of nucleotides separating convergent (yellow boxes) or divergent (bold text) promoters is shown. (B) Read densities of divergent sense and antisense transcripts visualized in IGV. Sequence coverage tracks from four 50′scRNA-seq libraries (CD34-1 [blue], CD34-2 [green], ILC [brown], and NK [purple]) are shown. The separation in base pairs between the sense and antisense TSS is shown above each panel. The gene name and cartoon showing sense exons in blue and antisense exons in red is shown below each panel.

Article Snippet: Cells were resuspended in FBS (Sigma-Aldrich) with leukocyte-depleted RBCs and a custom human NK cell enrichment RosetteSep reagent containing a mixture of bivalent antibodies against glycophorin A and CD3, CD4, CD19, CD20, CD36, CD66b, and CD123 (STEMCELL Technologies).

Techniques: Ex Vivo, Purification, Sequencing

Journal: Cell reports

Article Title: Abundant binary promoter switches in lineage-determining transcription factors indicate a digital component of cell fate determination

doi: 10.1016/j.celrep.2023.113454

Figure Lengend Snippet: (A) IGV analysis of NK cell 5′scRNA-seq reads in the AHR 5′ region. Paired-end reads of spliced RNAs transcribed from the asPro1 promoter are boxed. The empty box at the top left indicates the low level of asPro2 transcripts. (B) Venn diagram summarizing the expression of AHR sense and antisense transcripts in individual cells. (C) UMAP generated with 35PCs at a resolution of 0.3 utilizing 157,864 features across 30,637 samples. (D) Feature plot of sense and antisense transcripts, with antisense shown in red and sense in blue. (E and F) In situ RNA hybridization with AHR sense and asPro1-specific probes was performed on sorted human CD56 bright (E) and CD56 dim (F) NK cells. The red spots represent binding of a probe that recognizes the asPro1 antisense transcript. The blue spots correspond to a probe recognizing the first exon of the Pro1-derived AHR mRNA. Scale bars represent 20 μm. (G) Human AHR sequence spanning the opposing AHR Pro2 and asPro1 promoters. The coding region is italicized, with amino acids shown above the sequence. Mutations altering ATG codons or the splice donor are indicated in bold red type underneath the sequence. Thick black bars indicate 10-bp deletions assayed in (J). (H) Relative luciferase activity of the 375-bp AHR opposing promoters fragment in five cell lines. Values represent the mean, and error bars indicate the SEM of at least three independent experiments. (I) Relative luciferase activity of the 375-bp AHR opposing promoters fragment with nucleotide substitutions to remove competing ATG codons and the sense strand splice donor site. Values represent the mean, and error bars indicate the SEM of at least three independent experiments. (J) The effect of 10-bp deletions of EGR1/WT1-binding sites. Values represent the mean, and error bars indicate the SEM of at least three independent experiments.

Article Snippet: Cells were resuspended in FBS (Sigma-Aldrich) with leukocyte-depleted RBCs and a custom human NK cell enrichment RosetteSep reagent containing a mixture of bivalent antibodies against glycophorin A and CD3, CD4, CD19, CD20, CD36, CD66b, and CD123 (STEMCELL Technologies).

Techniques: Expressing, Generated, In Situ, Hybridization, Binding Assay, Derivative Assay, Sequencing, Luciferase, Activity Assay

Journal: Cell reports

Article Title: Abundant binary promoter switches in lineage-determining transcription factors indicate a digital component of cell fate determination

doi: 10.1016/j.celrep.2023.113454

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cells were resuspended in FBS (Sigma-Aldrich) with leukocyte-depleted RBCs and a custom human NK cell enrichment RosetteSep reagent containing a mixture of bivalent antibodies against glycophorin A and CD3, CD4, CD19, CD20, CD36, CD66b, and CD123 (STEMCELL Technologies).

Techniques: Virus, Recombinant, Staining, Clone Assay, Reporter Assay, Cell Culture, Software, Fluorescence, Imaging