Journal: bioRxiv

Article Title: Ratiometric Fluorescent Protein Biosensors Reveal Citrate Dynamics and Cellular Heterogeneity

doi: 10.64898/2026.04.16.718871

Figure Lengend Snippet: A-B. Pseudo-colored images of HeLa cells expressing ratioCitron1Low ( A ) and ratioCitron1High ( B ) in cytosol before and after addition of citrate. Scale bar: 100 µm. C-D. Δ R / R and Δ F / F values of ratioCitron1Low ( C ) and ratioCitron1High ( D ) versus time upon the addition of citrate. HeLa cells were incubated with 4 μM digitonin for 10 mins before the experiment and a final concentration of 20 mM citrate was added at t = 0 (ratioCitron1Low n = 16, ratioCitron1High n = 19, mean ± SD). E. Time courses of Δ R / R ₀ of ratioCitron1 variants (ratioCitron1Low ( n = 20), ratioCitron1High ( n = 10), ratioCitron1Low-con ( n = 5), ratioCitron1High-con ( n = 5)) expressed in HeLa cells upon the citrate titration. HeLa cells expressing ratioCitron1 variants were treated with digitonin and citrate titration was carried out. The final concentration of citrate is 100 μM at 0 min, 1 mM at 5 min, 10 mM at 10 min (mean ± SD). F-G. Δ R / R values of ratioCitron1 variants expressed in cytosol ( F ) and in mitochondria (mito-ratioCiton1) ( G ) of HeLa cells upon the addition of the ACLY inhibitor of BMS-303141. H. Δ R / R values of ratioCitron1 variants expressed in mitochondria (mito-ratioCitron1) of HeLa cells upon the addition of MPC inhibitor UK-5099 (final concentration: 25 μM). Δ F/F = ( F on - F off )/ F off , and F with excitation 405 nm or 470 nm and emission 518/45 nm. Δ R / R = ( R on - R off )/ R off , and R = ( F with excitation 470 nm and emission 518/45 nm) / ( F with excitation 405 nm and emission 518/45 nm).

Article Snippet: For imaging the treatment with MPC inhibitor UK-5099 (MedChemExpress) and ACLY inhibitor BMS-303141 (MedChemExpress), Hank’s balanced salt solution (HBSS; Nacalai Tesque, 09735-75) and 10 mM HEPES (Nacalai Tesque, 177557-94) was used as imaging buffer.

Techniques: Expressing, Incubation, Concentration Assay, Titration

Journal: bioRxiv

Article Title: Atlas of HIV cis-regulatory elements reveals extensive transcriptional variation across clades, isolates, and within individuals

doi: 10.64898/2026.04.03.716403

Figure Lengend Snippet: (A) Schematic of MPRAs tiling through HIV genomes, saturation mutagenesis, and evaluation of different isolates. (B) Saturation mutagenesis MPRA experiments in unstimulated Jurkat cells for four tiles in the HIV-1 LTR from the clade B REJO strain that show transcriptional activity. Region coordinates are provided using standardized HXB2 genomic coordinates. TF motifs that contribute to activity are outlined. Inset shows MPRA activity tiling through the HIV-1 LTR. Blue indicates activity of tiles in the sense, and red in the antisense (opposite orientation) strand. (C) CASCADE-derived motifs for different cofactors at various regions of the HIV-1 LTR. (D) Regional distribution of HIV-1 isolates tested by MPRAs. (E) Violin plots showing the distribution of activity in unstimulated Jurkat cells across 5,569 isolates for different regions of the HIV-1 LTR across clades. The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. The dots heatmaps below indicate the fold activation by stimulation of Jurkat cells with αCD3+PMA, TNFα, or IFNγ. The color indicates the median fold activation across isolates, whereas the size of the dots reflects the fold activity differences between the 5th and 95th percentiles. (F) Pearson correlation between the activity of different regions of the HIV-1 LTR across isolates. (G) Schematic of chimeric proviral constructs and reactivation experimental approach. (H-I) Fold change in GFP+ cells relative to DMSO control (H) or geometric mean fluorescence intensity of GFP+ cells (I) in Jurkat cells infected with chimeric proviruses carrying the U3 region of the indicated isolates from clades A, B, and C, and stimulated with TNFα for 2 days. (J) Comparison between fold activation of MPRA data from tile HXB2:250-431 in Jurkat cells activated with TNFα, and provirus reactivation by TNFα measured as fold change in GFP+ cells relative to DMSO control or geometric mean fluorescence intensity of GFP+ cells. Pearson correlation coefficients and one-tailed p-values are indicated for each comparison to MPRA data. (K) Dot heatmaps of recombinant HIV-1 viruses derived from clades B and C. Median baseline activity in tile HXB2:250-431 is shown in shades of green, whereas the median fold activation by αCD3+PMA, TNFα, or IFNγ is shown blue-white-red gradient. The size for the dots reflects the fold activity differences between the 5th and 95th percentiles.

Article Snippet: Jurkat cells (ATCC-TIB-15) were cultured in RPMI media (Fisher Scientific, Catalogue # A1049101) with 10% Fetal Bovine Serum (R&D Systems, Catalog # S12450H) and 1% Antibiotic-antimicotic (Thermofisher Scientific, Catalogue #15240062) up to a density of 1 million cells per mL prior to transfection.

Techniques: Mutagenesis, Activity Assay, Derivative Assay, Activation Assay, Construct, Control, Fluorescence, Infection, Comparison, One-tailed Test, Recombinant

Journal: bioRxiv

Article Title: Atlas of HIV cis-regulatory elements reveals extensive transcriptional variation across clades, isolates, and within individuals

doi: 10.64898/2026.04.03.716403

Figure Lengend Snippet: (A) Distribution of the number of TF binding sites across HIV-1 isolates in tiles HXB2:250-431 and HXB2:581-780. Each pie chart shows the proportion of isolates with different number of binding sites for the indicated TFs. (B-C) Violin plots of baseline activity or fold activation by αCD3+PMA or TNFα for tile HXB2:250-431 across HIV-1 isolates based on the number of NF-κB (B) or SP/KLF sites (C). The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. (D, H) Activity distribution in Jurkat cells across HIV-1 isolates with different TF configurations in tiles HXB2:250-431 (D) and HXB2:581-780 (H). The left boxes represent the TF configurations based on aligned TF positions. The distribution of isolates from different clades across TF configurations is shown as pie chats. Violin plots indicate the distributions of activity in unstimulated Jurkat cells (baseline), and cells stimulated with αCD3+PMA, TNFα, or IFNγ. In the case tile HXB2:250-431 only configurations with at least 10 isolates are shown. (E) Alphafold3 model of the HIV-1 REJO LTR including three SP1 and two sets of NF-κB (p65 and p50) proteins. (F-G) Violin plots of baseline activity or fold activation by IFNγ for tile HXB2:581-780 across HIV-1 isolates based on the number of IRF (F) or SP/KLF sites (G). The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. (I) Violin plots of F-statistic showing the variability in activity for each isolate for tile HXB2:581-780 across four donors in CD4+ T cells. The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. (J) Activity distribution and donor variability (F-statistic) in CD4+ T cells in Jurkat cells across HIV-1 isolates with different TF configurations in tile HXB2:581-780. The left boxes represent the TF configurations based on aligned TF positions. The distribution of isolates from different clades across TF configurations is shown as pie charts. (K) Distribution of donor variability (F-statistic) in tile HXB2:581-780 for isolates that contain or lack an SP/KLF site across four CD4+T cell donors or five replicates of Jurkat cells. Statistical significance determined by two-tailed Brunner-Munzel test.

Article Snippet: Jurkat cells (ATCC-TIB-15) were cultured in RPMI media (Fisher Scientific, Catalogue # A1049101) with 10% Fetal Bovine Serum (R&D Systems, Catalog # S12450H) and 1% Antibiotic-antimicotic (Thermofisher Scientific, Catalogue #15240062) up to a density of 1 million cells per mL prior to transfection.

Techniques: Binding Assay, Activity Assay, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Atlas of HIV cis-regulatory elements reveals extensive transcriptional variation across clades, isolates, and within individuals

doi: 10.64898/2026.04.03.716403

Figure Lengend Snippet: (A) Schematic of CREST and LARM models. CREST predicts baseline activity in Jurkat cells from DNA sequence. LARM predicts fold-activation by αCD3+PMA or TNFα for sequences corresponding to tile HXB2:250-431. (B) Pearson correlation between baseline activity in Jurkat cells measured by MPRAs and predicted using CREST for the validation and test sets. (C) Pearson correlation between TNFα-induced fold activation of tile HXB2:250-431 isolates in Jurkat cells measured by MPRAs and the corresponding values predicted by LARM for the validation and test sets. (D-E) Baseline activity predicted by CREST in tile HXB2:250-431 for isolates heterosexual (D) or mother-to-infant (E) transmission pairs. , The activity of the predicted founder sequence is indicated for infants. (F) Baseline activity predicted by CREST in tile HXB2:250-431 for isolates from 7 PWH from samples obtained at different days post infection. Error bars indicate the standard deviation, dots indicate the average value. (G) Baseline activity predicted by CREST for tile HXB2:250-431 of two PWH. Isolates are shown within their respective evolutionary trees. Node color represents the activity and node size indicates the average day post infection for the corresponding haplotype. Square nodes correspond to predicted ancestral sequences. (H) Baseline activity predicted by CREST for tile HXB2:250-431 for samples from Los Alamos database plotted versus the date of sample collection. Isolates are colored by clade. (I) Fold activation in Jurkat cells stimulated with TNFα predicted by LARM in tile HXB2:250-431 for isolates from 7 PWH from samples obtained at different days post infection. Error bars indicate the standard deviation, dots indicate the average value.

Article Snippet: Jurkat cells (ATCC-TIB-15) were cultured in RPMI media (Fisher Scientific, Catalogue # A1049101) with 10% Fetal Bovine Serum (R&D Systems, Catalog # S12450H) and 1% Antibiotic-antimicotic (Thermofisher Scientific, Catalogue #15240062) up to a density of 1 million cells per mL prior to transfection.

Techniques: Activity Assay, Sequencing, Activation Assay, Biomarker Discovery, Transmission Assay, Infection, Standard Deviation

Journal: bioRxiv

Article Title: Atlas of HIV cis-regulatory elements reveals extensive transcriptional variation across clades, isolates, and within individuals

doi: 10.64898/2026.04.03.716403

Figure Lengend Snippet: (A) MPRA activity map across the genome of HIV-1 clade B REJO strain in unstimulated Jurkat cells. The genome organization is shown below. (B) Saturation mutagenesis MPRA experiments in unstimulated Jurkat cells for HIV-1 intragenic CREs at HXB2:1330-1530 and HXB2:7784-7984. TF motifs that contribute to activity are outlined. (C-D) Violin plots showing the distribution of activity across isolates for intragenic CREs from HIV-1 across clades: HXB2:1330-1530 (C) and HXB2:7784-7984 (D). The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. (E) Entropy decomposition across the ETS–bZIP region in HIV-1 Gag aligned to HXB2 coordinates (codon start positions). Codon entropy (H(codon)), amino-acid entropy (H(a.a.)), and conditional synonymous entropy (H(codon|a.a.)) are shown; the motifs are outlined. (F) Violin plots showing the distribution of Δ median entropy (ROI − rest of window) from circular-shift permutation tests for H(codon), H(a.a.), and H(codon|a.a.). P values are indicated; n.s. denotes not significant. (G) Los Alamos National Laboratory (LANL) genome alignments of HIV-1 strains using the HXB2 genome as a reference. Transcriptional activity predicted using CREST is shown in shades of green. (H) Scatter plot showing the activity predicted using CREST for the two CREs in HIV-1 env . Dots are colored by clade. The top diagram shows the location of the CREs within the env protein coding sequence. C1-5 = conserved regions, V1-5 = variable regions, FP = fusion peptide. (I) TF motif profiles determined using CREST-based saturation mutagenesis across HIV-1 isolates from LANL. Regions were aligned to the HXB2 genome. (J-K) Baseline activity in Jurkat cells for two PWH predicted using CREST corresponding to HXB2 regions 7432-7632 (J) and 7712-7912 (K). Isolates are shown within their respective evolutionary trees. The color of the nodes represents activity levels. The size of the nodes indicate the day post infection of the corresponding sample. Square nodes represent predicted ancestral sequences.

Article Snippet: Jurkat cells (ATCC-TIB-15) were cultured in RPMI media (Fisher Scientific, Catalogue # A1049101) with 10% Fetal Bovine Serum (R&D Systems, Catalog # S12450H) and 1% Antibiotic-antimicotic (Thermofisher Scientific, Catalogue #15240062) up to a density of 1 million cells per mL prior to transfection.

Techniques: Activity Assay, Mutagenesis, Sequencing, Infection

Journal: bioRxiv

Article Title: Atlas of HIV cis-regulatory elements reveals extensive transcriptional variation across clades, isolates, and within individuals

doi: 10.64898/2026.04.03.716403

Figure Lengend Snippet: (A) MPRA activity in Jurkat cells tiling through the LTRs of HIV-1 and HIV-2. (B, G) Saturation mutagenesis MPRA experiments in unstimulated Jurkat cells for tiles in the LTR (B), gag/env (G) regions of HIV-2 ROD strain that show transcriptional activity. Region coordinates are provided using standardized SIVmac239 genomic coordinates. TF motifs that contribute to activity are outlined. (C) Saturation mutagenesis MPRA experiments in unstimulated and stimulated Jurkat cells for tile SIVmac239:379-571 in the HIV-2 LTR. (D) Violin plots showing the distribution of LTR activity in unstimulated Jurkat cells across 41 HIV-2 isolates with full-length sequences in NCBI. The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. The dots heatmaps below indicate the fold activation by stimulation of Jurkat cells with αCD3+PMA, TNFα, or IFNγ. The color indicates the median fold activation across isolates, whereas the size of the dots reflects the fold activity differences between the 5th and 95th percentiles. (E) CASCADE-derived motifs for different cofactors in the LTR and gag regions of HIV-2 ROD strain. (F) MPRA activity map across the genome of HIV-2 ROD strain in unstimulated Jurkat cells. The genome organization is shown below. (H-I) Los Alamos National Laboratory (LANL) genome alignments of HIV-2 strains using the SIVmac239 genome as a reference. (H) Transcriptional activity predicted using CREST is shown in shades of green. (I) Transcription start sites predicted using Puffin trained on FANTOM CAGE data are shown in shades of red.

Article Snippet: Jurkat cells (ATCC-TIB-15) were cultured in RPMI media (Fisher Scientific, Catalogue # A1049101) with 10% Fetal Bovine Serum (R&D Systems, Catalog # S12450H) and 1% Antibiotic-antimicotic (Thermofisher Scientific, Catalogue #15240062) up to a density of 1 million cells per mL prior to transfection.

Techniques: Activity Assay, Mutagenesis, Activation Assay, Derivative Assay