EMBO/soda-nel huggingface dataset:
Modelling, Optimization and Comparable Efficacy of T cells
and HSC Gene Editing for treating HIGM1 (2020)
Figure 2

Figure 2 - Edited cells enrichment and increased CD40L expression level by adding NGFR selector to the therapeutic gene.

A Schematics of donor DNA template used for edited cells enrichment.

B Percentage of NGFR+ cells or HDR at 17 days after CD40LG editing of male HD bulk edited, sorted NGFR+ or sorted NGFR- CD4+ T cells (n=7 for each group). Median ± IQR.

C Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by RFI (normalized to T0) on UT (n=2), edited (NGFR+) or unedited (NGFR-) HD or Pt derived CD4+ T cells (n=4 HD, 1 Pt). Median ± IQR.

D Representative plots showing CD40L and NGFR expression in UT or bulk edited (Treated) CD4+ T cells derived from male HD or Pt from (C) before and 8 hr after Pma/Ionomycin stimulation.

E Population composition in male HD bulk edited, sorted NGFR+ or sorted NGFR- CD4+ T cells from (B) (n=7 for each group). Friedman test (to account for the same donor) with p-values adjusted with Bonferroni's correction to account for multiple testing. Mean ± SEM.

F Human CD45+ cell engraftment in PB after transplantation of male HD UT (n=5), bulk edited (NGFR bulk; n=5) or sorted NGFR+ (n=5) CD4+ T cells. Longitudinal comparisons were performed with an LME model, followed by an appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups. Median ± IQR.

G Percentage of HDR within human cells over time, measured by ddPCR in PB of mice from (F) (n=5 for each group, measured in engrafted mice with sufficient blood material). Longitudinal comparisons were performed with an LME model with time treated as a continuous variable (see Appendix Supplementary Statistical Methods). UT group was not included in the analysis. Effect of the time and the eventual different effect of the time in the groups were not retained in the final model after backward variable selection, highlighting a constant (and significantly different, ****p<0.0001) behavior of the groups over time. Median ± IQR.

H Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by MFI on pooled CD4+ T cells retrieved from spleens of mice from (F) (n=1 for each group, except for NGFR- unedited group, n=3). Median ± IQR.

I Dot plot depicting the ratio between expression of CD40LG edited mRNA (codon-usage optimized, CO) and CD40LG wild-type mRNA (WT) in cells edited with three different donor templates from Fig EV2H. Three independent experiments (n=3 EF1a.GFP, 1 IRES.NGFR, 3 IRES.GFP). Median ± IQR.

J Time course of expression of WT-CD40LG mRNA or CO-CD40LG mRNA, measured as fold change (FC) on IPO8 housekeeping gene and normalized to T0. Sorted edited (+) and sorted unedited (-) CD4+ T cells were analyzed before and 3, 6, 12 hr after Actinomycin D treatment (n=1 for each group).
Modelling, Optimization and Comparable Efficacy of T cells
and HSC Gene Editing for treating HIGM1 (2020)
Figure 3

Figure 3 - Selection and depletion of the edited T cells by exploiting an optimized hEGFRt gene.

A Schematics of donor DNA templates carrying corrective cDNA coupled to IRES sequence and hEGFRt selector genes.

B Representative plots showing hEGFRt expression in bulk edited CD4+ T cells derived from male HD before and 8 hr after Pma/Ionomycin stimulation. Cells were edited with the three constructs depicted in (A).

C Representative plots showing EGFR expression in bulk edited CD4+ T cells derived from male HD at 3 days after treatment with immunotoxin (left), antibody (middle) or toxin (right).

D Bar plot showing percentage of EGFR+ T cells at 3 days after treatment with 5 nM or 1 nM of immunotoxin, antibody or toxin, measured by FACS Analysis. Friedman test with Dunn's multiple comparisons. P-values were adjusted with Bonferroni's correction to account for multiple comparisons (**p=0.0052 and **p=0.0024 for immunotoxin vs antibody and **p=0.0010 and **p=0.0024 for immunotoxin vs toxin, referring to EGFRmod1 and EGFRmod2 respectively). Different dose-conditions were used as a unified group for statistical analysis (n=10 for each group). Median ± IQR.
Modelling, Optimization and Comparable Efficacy of T cells
and HSC Gene Editing for treating HIGM1 (2020)
Figure 5

Figure 5 - Partial rescue of the IgG response by adoptive transfer of naive functional T cells in Cd40lg-/- mice.

A Schematics of adoptive transfer of spleen derived CD45.1.2+ CD3+ WT T cells (donor) into CD45.2 Cd40lg-/- mice (recipient) pre-conditioned with 300 mg/kg CPA or not (NC) and immune response studies.

B Total counts of recipient CD3+ cells gated within CD45.2+ cells in PB of experimental mice (n=4 CPA 300, 9 NC). Comparison between groups was performed by NLME model with an asymptotic model (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to day 1 (****p<0.0001). Mean ± SEM.

C Total counts of engrafted donor CD45.1.2+ cells in PB of mice from (B). Comparison between groups was performed by NLME model with an asymptotic model (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to plateau (***p=0.0001). Mean ± SEM.

D, E Percentage of donor CD4+ and CD8+ cells gated within CD3+CD45.1.2+ cells in PB of CPA treated (D) or NC mice (E) from (B). Mean ± SEM.

F TNP-KLH specific IgG in sera of transplanted mice collected at the times indicated in (A) (n=7 CPA 300, 13 NC for first boost and 4 CPA 300, 9 NC for second boost). Sera from vaccinated WT (n=5) and CD40lg-/- (n=5) mice were used as positive and negative controls, respectively. Two independent experiments. Comparisons of first boost data were performed with an LME model, accounting for multiple experiments, followed by appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups (****p<0.0001 in all comparisons). Median ± IQR.

G Average area of PNA+ foci in splenic sections of experimental mice from (B), calculated as ratio between total PNA+ area and number of PNA+ foci/cells. Kruskal-Wallis test followed by post-hoc analysis with Dunn's test (n=9 CD40LG, 6 WT, 18 NC, 8 CPA300). P-values were adjusted with Bonferroni's correction to account for multiple comparisons (*p=0.0149, **p=0.0065 and ****p<0.0001). Mean ± SEM.

H Detection of splenic TNP-KLH specific IgM (left) and IgG (right) -secreting cells by ELISPOT assay (n=7 NC, 4 CPA300, 3 WT, 2 CD40LG). Spots were counted by an ELISPOT Reader using a size range of 0.005-1 mm. Median ± IQR.
A metabolite binding protein moonlights as a bile-responsive chaperone (2020)
Figure 1

Figure 1. Genetic Screening Using Tn-Seq and a Protein Folding Sensor

(A) As a protein folding sensor, we constructed a tripartite fusion protein in which the test protein (shown in blue) is inserted between the N-terminal portion of the selectable marker, β-lactamase (shown in pink), and the C-terminal portion of β-lactamase (shown in peach). Instability of the test protein leads to either degradation or aggregation, resulting in lower ampicillin resistance (AmpS) of strains containing this biosensor compared to those in which the test protein is stable. If a host factor or chaperone such as Spy stabilizes the test protein, higher levels of ampicillin resistance (AmpR) will be exhibited. First, we generated a transposon (Tn) library in strains containing either a protein folding sensor or a β-lactamase glycine-serine linker construct (see Methods for details). Secondly, ampicillin resistance selections were performed, and colonies resistant to various levels of ampicillin were pooled and subjected to deep sequencing. This enabled the genomic mapping of the position and frequency of transposon insertion sites. Genes showing an altered transposon insertion frequency in the presence of a protein folding sensor were selected as candidates for host factors that affect the stability of that protein folding sensor. See also Appendix Figure S1A.

(B) When transposon insertion libraries subjected to ampicillin resistance selection, a substantial increase in transposon insertion frequency in the pstSCA genes was observed in strains containing the protein folding sensor (pBR322 bla::Im7 L53A I54A) relative to a strain containing just pBR322. Transposon insertion frequency increased 21-, 24-, and 8-fold in the pstS, pstA, and pstC genes, respectively, compared to the insertion frequency found with no ampicillin selection.
A metabolite binding protein moonlights as a bile-responsive chaperone (2020)
Figure 2

Figure 2. Deletion Mutants in the pstSCA Genes Act to Stabilize the β-lactamase-Im7 L53A I54A Folding Biosensor and the Im7 L53A I54A Protein Itself In Vivo

(A) To verify that disruption of the pst genes is sufficient to increase ampicillin resistance of strains containing the pBR322 bla::Im7 L53A I54A folding biosensor and to test which of the genes in the pst operon are sufficient for this effect, pBR322 and pBR322 bla::Im7 L53A I54A were transformed into strains containing in-frame deletion mutants of each of the pst genes (pstS, pstC, and pstA, respectively). The deletion mutants of each of these pst genes showed higher ampicillin resistance than did the WT strain MG1655 in the presence of pBR322 bla::Im7 L53A I54A, but did not shower higher ampicillin resistance in the presence of pBR322. See also Appendix Figure S2A.

(B) Levels of β-lactamase (Bla) and β-lactamase-Im7 L53A I54A were detected by Western blotting using a polyclonal antibody recognizing β-lactamase in the WT strain MG1655 and in isogenic strains containing insertions in the pstSCA genes (CL248 (pstS::Tn5), CL249 (pstC::Tn5), CL250 (pstA::Tn5)). Maltose binding protein (MBP) was used as a control.

(C) β-lactamase enzymatic activities present in periplasmic extracts of the Im7 folding biosensor were monitored in pst WT and pstSCA mutant strains using a spectrophotometric assay. Data are represented as mean ± SEM from three technical replicates (unpaired t-test,** P<0.01, *** P<0.001 comparison to MG1655; ns, not significant).

(D) To test if levels of the Im7 L53A I54A protein itself, in the absence of the fusion, were also higher in the pstA mutant, expression of the Im7 L53A I54A protein from the IPTG-inducible plasmid pCDFTrc was induced using IPTG concentrations ranging from 0-5 mM. Periplasmic fractions were isolated and examined by SDS-PAGE and subsequent Coomassie staining to determine overall protein expression patterns. Im7 L53A I54A levels were determined by Western blotting using an antibody for Im7. MBP was used as a loading control. Im7 L53A I54A was increased in the pstA strain relative to the WT control strain. Coomassie staining revealed that a number of periplasmic proteins, including UgpB, were also induced in the pstA strain.

Data information: These experiments were repeated twice with similar results and one representative is shown.
A metabolite binding protein moonlights as a bile-responsive chaperone (2020)
Figure 3

Figure 3. UgpB Confers High Ampicillin Resistance to Strains Containing the Destabilized Protein Im7 L53A I54A

(A) MG1655 WT, single mutants of pstA and ugpB, and a double mutant of pstA ugpB were transformed with pBR322 and various pBR322-based folding biosensor constructs. The transformants were examined by a spot titer assay on LB agar plates containing various ampicillin concentrations. Strains containing pBR322 bla::Im7 L53A I54A gain considerable ampicillin resistance in the presence of a pstA mutation (compare uppermost left and right panels). This resistance is completely lost upon transducing in a ugpB mutation (compare next panels down), showing that ugpB is responsible for the resistance gained by the pstA mutation.

(B) Ampicillin sensitivity conferred by a ugpB deletion mutant (CL543) is complemented by UgpB expression from the arabinose inducible expression vector pBAD33 ugpB (pCL1).

(C) UgpB expression is sufficient to lead to higher levels of Im7 L53A I54A protein. Im7 L53A I54A and UgpB were expressed in a ugpB mutant strain (CL543) from pCDFTrc and pTrc99a vectors, respectively, both of which have an IPTG-inducible promoter. Periplasmic fractions were isolated and examined by Western blotting. "-" indicates strains containing empty vector and "+" indicates strains containing a recombinant plasmid encoding either Im7 L53A I54A or UgpB. MBP was used as a loading control. Data information: These experiments were repeated twice with similar results and one representative is shown.
A metabolite binding protein moonlights as a bile-responsive chaperone (2020)
Figure 4

Figure 4. UgpB Prevents Bile-Induced Protein Aggregation

(A) UgpB overexpression confers bile salt resistance, and a ugpB deletion mutant is slightly CHO sensitive.

(B) CHO-induced MDH protein aggregation analyzed by monitoring light scattering.

(C) CHO-induced MDH protein aggregation analyzed by a solubility assay using centrifugation (cfg) at 16,000 g for 20 min. The supernatant (Sup) represents the soluble fraction and the pellet the insoluble fraction; 12 µM MDH was used as a model aggregation-prone substrate (see Methods). Addition of increasing quantities of UgpB increasingly solubilizes MDH. The molar ratios of MDH:UgpB that were used (left to right) are as follows: 1:0, 1:0.1, 1:0.2, 1:0.5, and 1:1. 5-fold more of the pellet fraction was loaded relative to the supernatant fraction.

(D) The supernatant/pellet assay was performed using a 1:1 MDH:chaperone ratio for Spy, SurA, and OsmY. The pellet fraction was concentrated 5-fold.

(E) 12 µM of MDH alone and a 1:1 ratio of either MDH:UgpB or MDH:MBP were incubated at 37°C for 60 min in the presence of CHO and then fractionated via centrifugation for use in a solubility assay as described above (C) . The pellet fraction was concentrated 5-fold. In some samples (+G3P), 120 µM of G3P was added prior to the addition of any other protein. Addition of G3P inactivates UgpB's ability to inhibit protein aggregation.

(F) 800 μg of E. coli total lysate and 60 µM of UgpB/MBP were used for the supernatant/pellet assay. 600 µM of G3P was added before adding proteins. The pellet fraction was concentrated 10-fold. As before, addition of UgpB inhibited aggregation, but only in the absence of G3P.

(G) The anti-aggregation activity of tryptophan single mutant (W169S) and double mutant (W169S/W172S) was examined by CHO-induced MDH aggregation assay. Light scattering values were monitored.

(H) Solubility of MDH is also monitored by supernatant/pellet assay. Tryptophan mutants exhibit constitutively active chaperone function independent of the presence of G3P. The pellet fraction was concentrated 5-fold. 12 µM of MDH and 1:1 ratio of MDH:UgpB proteins and 120 µM of G3P were used.

(I) The anti-aggregation activity of the UgpB variants (E66A and R374V) was examined by CHO-induced MDH aggregation assay. CHO-induced MDH protein aggregation analyzed by monitoring light scattering.

(J) Solubility of MDH is also monitored by supernatant/pellet assay. 12 µM of MDH alone and a 1:1 ratio of MDH:UgpB proteins, 120 µM of G3P were used.

15 mM CHO were used for all the aggregation and supernatant/pellet assays.

Data information: These experiments were repeated at least twice with similar results and one representative is shown.
A metabolite binding protein moonlights as a bile-responsive chaperone (2020)
Figure 6

Figure 6. pH Transition Can Activate UgpB's Chaperone Activity

(A) Acid denatures UgpB. The secondary structure of 2 µM UgpB was monitored by circular dichroism (CD) spectroscopy in 10 mM sodium phosphate buffer at pH 2 and pH 7.

(B) G3P binds to acid-stripped UgpB as observed by monitoring the tryptophan fluorescence spectrum, either at pH7 or after transition from pH 2 to 7 (see Methods). Transitions from pH 2 to 7 in the presence of G3P are accompanied by a blue shift, indicating G3P binding.

(C) The anti-aggregation activity of UgpB is monitored by CHO-induced MDH aggregation assay. Light scattering values were monitored. The anti-aggregation activity of UgpB is activated after transition from pH 2 to 7 and the addition of G3P (120 µM) can again inactivate its chaperone activity.

(D) Addition of G3P to UgpB inhibits its anti-aggregation chaperone activity. 12 µM of MDH and a 1:1 ratio of UgpB:MDH were used in a CHO-induced aggregation assay. At 30 min, the indicated concentrations of G3P were added to solutions containing MDH, and MDH aggregation was monitored by light scattering. Note that G3P binding to UgpB still occurs in the presence of 15 mM CHO as monitored by tryptophan fluorescence (inset and Appendix Figure S9).

Data information: These experiments were repeated twice with similar results and one representative is shown.

EXPANDED VIEW FIGURE LEGENDS
Metronomic  chemotherapy  offsets  HIFα  induction  upon maximum-tolerated dose in metastatic cancers (2020)
Figure 1

Figure 1: LDM chemotherapy selectively offsets HIF-1α levels in experimental colon cancers

A: HIF-1α levels in HT29 primary tumors.

Left: vehicle-treated controls

Middle/left: Doublet LDM cyclophosphamide + capecitabine (LDMCTX + LDMCPB)

Middle/right: LDM cyclophosphamide + MTD capecitabine (LDMCTX + MTDCPB). Inset, high-magnification image of the region marked with a green asterisk (*).

Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on HIF-1α+ area. F(5,12) = 8.791 and P = 0.001 for overall treatment by Brown-Forsythe ANOVA; *, P= 0.046 vs vehicle; #, P< 0.0001 vs LDMCTX + MTDCPB by Benjamini, Krieger and Yekuteli post hoc test.

B: HIF-2α levels in HT29 primary tumors.

Left: Example of immunostaining in vehicle-treated controls. Inset, high-magnification image of the region marked with a green asterisk (*).

Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on HIF-2α+ area. F(5,18) = 1.215 and P = 0.3424 (not significant) for overall treatment by Brown-Forsythe ANOVA.

C: CA9 levels in HT29 primary tumors.

Left: Example of immunostaining in vehicle-treated controls. Inset, high-magnification image of the region marked with a green asterisk (*).

Right: Quantification of the effect of monotherapies or doublet LDM/MTD regimens on CA9+ area. F(5,11) = 2.466 and P = 0.0961 (not significant) for overall treatment by Brown-Forsythe ANOVA.

D: Correlation between HIF-1α levels and proliferation indexes in HT29 primary tumors.

Left and middle/left: HIF-1α and Ki67 expression in vehicle-treated controls; consecutive sections are shown.

Middle/right: HIF-1α+ vs Ki67+ proliferation index scatterplot. Each point represents median values for each tumor. Regression line (red) and 95% CI (shaded blue area) are indicated. F(1,27) = 34.45 and P = 10-4; slope ≠ 0 by F-test

Right: Machine-learning quantification of the effect of monotherapies or doublet LDM/MTD regimens upon Ki67+ proliferative index. Median indexes per tumor are indicated. F(5,12) = 1.609 and P = 0.2312 (not significant) for overall treatment by Brown-Forsythe ANOVA.

Data information: Violin plots present 50th (red line), 25th and 75th percentiles (blue line); numbers in brackets indicate number of tumors per group. L, low-dose metronomic; M, maximum-tolerated dose; r, correlation coefficient. Low power magnification images of all experimental conditions can be found in Appendix Figure S1. Blue frames in D indicate consecutive sections stained for HIF-1α and Ki67.
Metronomic  chemotherapy  offsets  HIFα  induction  upon maximum-tolerated dose in metastatic cancers (2020)
Figure 3

Figure 3: LDM chemotherapy offsets HIF-1α levels in breast cancer metastases to the lung

A: EMT6-CDDP lung metastatic nodule size.

Left: Histogram of cross-sectional metastatic diameter (Ø).

Middle: Metastatic diameter (Ø) by chemotherapy regimen. F(5,99) = 0.9310 and P= 0.4644 (not significant) for overall treatment by Brown-Forsythe ANOVA.

Right: Dichotomized metastatic size at median diameter of vehicle-treated tumors. Lung nodule size was classified as small (blue) or large (red). Individual nodule counts per group and size category (small/large) are plotted on the right ordinate. χ2 (df=5) = 2.631; P= 0.7567 (not significant) for overall effects on size.

B: Hypoxic fraction in lung metastatic nodules. Pimonidazole adduct immunoreactivity was automatically quantified and expressed as fractional positive areas per nodule (rightmost panel). F(5,110) = 7.544 and P< 0.0001 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 vs vehicle; #, P= 0.0111 doublet LDM cyclophosphamide + capecitabine vs LDM cyclophosphamide + MTD capecitabine by Benjamini, Krieger and Yekuteli post hoc test.

C: HIF-1α levels in lung metastatic nodules. Automatic quantification of HIF-1α levels measured as fractional positive areas per nodule (rightmost panel). F(5,114) = 5.325 and P= 0.0004 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 vs vehicleby Benjamini, Krieger and Yekuteli post hoc test.

D: Microvessel density in lung metastatic nodules. Automatic quantification of CD31 fractional areas per nodule (rightmost panel). F(5,124) = 7.531 and P< 0.0001 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 (LDM capecitabine, cyclophosphamide, or doublet capecitabine + cyclophosphamide vs vehicle), #, P= 0.0417 (doublet LDM capecitabine + cyclophosphamide vs doublet LDM cyclophosphamide + MTD capecitabine) by Benjamini, Krieger and Yekuteli post hoc test.

Data information: Violin plots present 50th (blue line), 25th and 75th percentiles (red line); numbers in brackets indicate number of nodules (n) or animals (mice) per group. CPB, capecitabine; CTX, cyclophosphamide; L, low-dose metronomic; M, maximum-tolerated dose. Inset: high-magnification image of the region marked with an asterisk (*; C rightmost panel). Green, blue, and red frames in B, C and D indicate consecutive sections from the same lung metastatic nodule, stained for pimonidazole (hypoxia), HIF-1α or CD31 (microvascular density).