EMBO/soda-nel huggingface dataset:
Coupling spatial segregation with synthetic circuits to control bacterial survival (2016)
Figure 6

Figure 6. Experimental control of microbial swarmbot safeguard by other circuits.

Demonstration of modular safeguard performance of swarmbots with different collective survival systems. For the swarmbots containing cells carrying the QS-CAT circuit, pulsing flow of medium containing 100µg/ml chloramphenicol established safeguard control but not for the static condition. For the swarmbots with the QS-BlaM circuit, the static condition of 100 µg/ml carbenicillin was sufficient for safeguard control. For all the conditions, safeguard did not occur in the absence of antibiotic (top panels from each condition). All the images were from the swarmbots cultured at 37˚C for 16 hours. The scale bar is 250 µm.
Soluble stroma-related biomarkers of pancreatic cancer (2018)
Figure 3

Figure 3. Biomarkers in PdxCre/LSL-KrasG12D (KC) and KrasG12D/Trp53R172H (KPC) GEM models.

A) Levels of TIMP1, MMP7 and TSP2 in plasma of KC mice (n=7-9) at 60, 120, 180, 240 and 330 days of age and KPC mice (n=3-8) at 30, 90 and 150 days of age (mean ± SEM). *p<0.05 (Mann-Whitney). The exact n and p-values are indicated in Appendix Table S3A

B) Levels of TIMP1, MMP7 and TSP2 in plasma of healthy mice (n=15), mice with chronic pancreatitis at 150 days of age (n=19), KC mice at 330 days of age (control PdxCre n=3-4; KC n=7-8), KPC mice at 150 days of age (control PdxCre n=4-7; KPC n=4). The exact n is indicated in Appendix Table S3B. Box plots extends from 25th to 75th percentiles, whiskers extends from min to max and horizontal lines indicate median. p-values were calculated with 1-way ANOVA with Tukey"s multiple comparison test.

C) Histological analysis of pancreas from PdxCre and KC mice with different grades of PanIN lesions at 330 days of age. Anti-TIMP1, anti-MMP7, anti-TSP2 and anti-CCN2 staining of a representative KC PanIN lesion (200X, scale bars: 100μm).
Soluble stroma-related biomarkers of pancreatic cancer (2018)
Figure 5

Figure 5. Murine circulating TIMP1, MMP7 and TSP2 as biomarkers of treatment response in PDAC-PDX models.

Responsiveness to gemcitabine (GEM), either alone or combined with Nab-paclitaxel (NAB-P), was evaluated in mice bearing orthotopic HuPa8 in the pancreas. Nab-paclitaxel (25mg/kg) and gemcitabine (150mg/kg) were given i.v. on days 1 and 8 of each 21-day cycle (for a total of 8 treatments).

A) Levels of TIMP1, MMP7 and TSP2 in plasma of mice bearing orthotopic HuPa8 at 30, 90 and 150 days after tumor transplantation (mean ± SEM; n≥4 for each group). *p<0.05, (Mann-Whitney). The exact n and p-values are indicated in Table S5A.

B) Magnetic resonance imaging of HuPa8. Representative images are shown, the white dotted lines indicate tumor masses.

C) Tumor growth over time measured by MRI; each black arrow indicates one treatment (mean ± SEM; n=4 for each group; 2-way ANOVA with Tukey's multiple comparison test).

D) Levels of the 3 selected biomarkers in plasma of HuPa8 bearing mice, prior to (day 80; at randomization), during (day 120) and after (day 165) the treatments (mean ± SEM; n≥4 for each group; 2-way ANOVA with Tukey's multiple comparison test). The exact n is indicated in Appendix Table S5B.
The GENDULF algorithm: mining transcriptomics to uncover modifier genes for monogenic diseases (2020)
Figure 4

Figure 4. Knock-down of U2AF1 enhances SMN expression.

(A)-(D) Expression of (A) HNRNPA0, (B) U2AF1, (C) SF3B3, or (D) NECAP1 72 hours post-transfection with either SMN2 SSO or knockdown of each target using siRNAs in HEK293T cells.

(E)-(G) Expression of (E) SMN2-FL, (F) SMN2∆7, or (G) SMN1-FL following SMN2 SSO or siRNA transfection (n=4).

(H)-(J) mRNA expression of (H) U2AF1, (I) SMN2-FL, or (J) SMN2∆7 in SMA patient-derived fibroblasts (red bars) 72 hours post-transfection with SMN2 SSO, scrambled siRNA, or U2AF1 siRNA at the indicated dose. Unaffected, SMA carrier fibroblasts (blue bars) treated with scrambled siRNA.

(K) Representative Western blot.

(L)-(M) Quantification of protein expression of (L) U2AF1 or (M) SMN normalized to GAPDH (n=3).

Data information: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; empirical p-values are determined by a simulation and sampling method described in Methods. The error bars show the Standard Error of Mean (SEM). Data from A-G represent 4 biological replicates. Data from H-M represent 3 biological replicates. Three technical replicates were performed during qPCR for each biological replicate and averaged. Scrambled siRNA condition for each experiment was set to 1.
Development of a small molecule that corrects misfolding and increases secretion of Z α1-antitrypsin (2020)
Figure 1

Figure 1

Figure 1. Characteristics of the lead series of chiral hydroxy-carboxamides identified from the ELT screen.

(A) The structure of GSK425, identified from the ELT screen, and the derived compound GSK716 obtained through a structure-based design pipeline.

(B) The degree of polymerisation of Z α1-antitrypsin after 72 hours at 37°C, as determined by an end-point immunoassay using the 2C1 monoclonal antibody, in varying concentrations of compound (shown in panel A). Modification of the phenyl and indole heterocycle of GSK425 (pIC50 6.5) resulted in an ~100 fold increase in potency and the discovery of the 2-oxindole GSK716 (pIC50 8.3). Data presented as mean ± SD, n = 2 (GSK425) and n = 25 (GSK716).

(C) GSK716 binds to Z α1-antitrypsin with a high affinity mean pKD of 8.5 ± 0.12 (n = 18) as determined by a competition binding assay with a fluorescently labelled derivative. There was a 50-fold lower affinity for plasma-purified wild-type M α1-antitrypsin, with a mean pKD of 6.8 ± 0.18 (n = 10). Data presented as mean ± SD.

(D) The compound bound to monomeric but not polymeric Z α1-antitrypsin (Z α1-AT) as reported by fluorescence polarisation of an Alexa-488-labelled variant of GSK716.

(E) Representative curves reporting the interaction of different concentrations of GSK716 with Z α1-antitrypsin based on changes in intrinsic tryptophan fluorescence. Based on the concentration dependence (inset), the second-order rate constant of association was found to be 4.1 x 104 M-1 s-1.

(F) The association of GSK716 with M α1-antitrypsin, giving a second-order rate constant of 2.1 x 102 M-1 s-1.
Development of a small molecule that corrects misfolding and increases secretion of Z α1-antitrypsin (2020)
Figure 4

Figure 4. Stabilisation of α1-antitrypsin by GSK716.

(A) A SYPRO Orange-based thermal stability assay, which reports the transition of M and Z α1-antitrypsin from the native to an intermediate state over a 1°C/min thermal ramp, in the presence and absence of 50µM GSK716.

(B) M (above) and Z (below) α1-antitrypsin, at a concentration of 0.2mg/ml in PBS+5% v/v glycerol, were heated at a range of temperatures between 48°C-65°C for 4 hours in the presence and absence of GSK716, as indicated. The oligomerisation state was determined by non-denaturing PAGE electrophoresis; the lane denoted 'M' contains the unheated monomeric control.

(C) M and Z α1-antitrypsin were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride (gdnhcl) in the presence and absence of GSK716, with bis-ANS dye added to report the presence of the unfolded intermediate. The normalised fluorescence intensity data were fitted with an equation describing a three-state unfolding curve. Values shown are the mean of three independent experiments and the error bars represent ±SEM.

(D) Top panel, the association of GSK716 induced a marked quenching and blue-shift of the α1-antitrypsin intrinsic tryptophan fluorescence spectrum with respect to unbound protein (inset graph). The association of 10µM GSK716 with four α1-antitrypsin variants that vary in their propensity to polymerise, ranging from inert (M), to mild (S), moderate (Baghdad denoted 'B') and severe (Z). Representative progress curves of the change in intrinsic tryptophan fluorescence at 330nm for 0.2mg/ml protein are shown. Bottom panel, the half-time of association calculated from three such independent experiments (error bars are ±SEM) show a correspondence with the polymerisation propensity of the four variants. The change in Z α1-antitrypsin fluorescence was faster than the dead-time of the apparatus (~10s) and scaling was with reference to unbound intensity.

(E) Non-denaturing PAGE characterisation of the conformational state of M and Z α1-antitrypsin, after rapidly refolding by snap dilution from 6M urea in the presence or absence of GSK716 at the molar ratios indicated. The migration of polymers (pol) and monomers (mon) are shown. Arrows indicate the misfolded by-product M* arising at sub-stoichiometric concentrations of compound.
Development of a small molecule that corrects misfolding and increases secretion of Z α1-antitrypsin (2020)
Figure 5

Figure. 5. The effect of GSK716 on serpin inhibitory activity.

(A) α1-Antitrypsin was incubated at varying molar ratios with the model protease bovine α-chymotrypsin, and the residual protease activity determined. The intercept of the regression with the abscissa reflects the number of molecules of α1-antitrypsin required to inhibit one molecule of chymotrypsin in the presence (+c) and absence (-c) of 50µM GSK716. Error bars reflect ±SEM of three independent experiments.

(B) M α1-antitrypsin was incubated with different molar ratios of chymotrypsin and resolved by SDS-PAGE. The position of covalent α1-antitrypsin-chymotrypsin complex (cpx), native (nat) and cleaved (clv) is shown.

(C) The inhibitory activity of α1-antichymotrypsin against chymotrypsin (ach+chym), neuroserpin against trypsin (neus+tryp), and antithrombin against thrombin (atiii+thrombin) was determined in the presence and absence of GSK716. Error bars reflect ±SD of two independent experiments.
Development of a small molecule that corrects misfolding and increases secretion of Z α1-antitrypsin (2020)
Figure 6

Figure. 6. GSK716 increases secretion of Z α1-antitrypsin in a transgenic mouse model of Z α1-antitrypsin deficiency. Z α1-antitrypsin transgenic animals were dosed with 10, 30 or 100 mg/kg GSK716 three times a day. Total concentrations of GSK716 were determined by LC-MS/MS (Table 2).

(A) 100mg/kg GSK716 resulted in a mean 7-fold increase in circulating monomeric Z α1-antitrypsin levels demonstrating robust target engagement in the liver. The observed steady state total and free compound levels of GSK716 in the transgenic Z α1-antitrypsin mouse were well-predicted by the in silico PK model built on: (1) in vitro metabolic clearance data, (2) plasma protein binding data, (3) in vivo PK data from wild type mice and (4) a term comprising a 5μM circulating sink for drug with an affinity of 1.5 nM, representing the Z α1-antitrypsin within blood. The target free drug concentration was selected based on the observed potency in the in vitro secretion assays in which the total drug approximates to the free drug in the assay. Interestingly, 30mg/kg and 10mg/kg groups also gave significant, dose-dependent increases in circulating Z α1-antitrypsin despite free concentrations being below the cellular EC50 for secretion for much or all of the dosing period. Significance at p<0.05 by a student's t test is denoted by *, shown as pairwise comparisons (each animal is compared with itself, treated vs pre-treatment) and between groups (compound treated vs vehicle), with n= 7, 8 and 8 per group for day 6 dose level 10, 30 and 100mg/Kg respectively; day 15, n=10; day 21, n=5. Data presented as mean ± SD.

(B) Representative example images of 2C1-stained livers from a 20-day vehicle (i and ii) and 100mg/kg TID GSK716 (iii and iv) treated animals. The asterisks indicate regions of hepatocytes that are negative on 2C1 immunostaining. Scale bar (i) and (iii) 2mm and (ii) and (iv) 100 µm.

(C) Quantification of 2C1 stained area in livers from vehicle and 100mg/kg TID GSK716 treated animals at day 15 (top) and 21 (bottom), shown as total area and low-only or high and mid-only intensity stained areas. There were no significant differences between groups by Mann-Whitney U test; graphs show median±interquartile ranges, with n=5 per group.

Expanded view
Requirement for YAP1 signaling in myxoid liposarcoma (2019)
Figure 1

Figure 1. Identification of genes required by FUS-DDIT3-expressing mesenchymal stem cells. (A) Schematic of RNAi screens. SCP-1 cells expressing FUS-DDIT3 or EV were transduced with Module 1 of the DECIPHER Pooled Lentiviral Human Genome‑Wide shRNA Library. Half of the cells were harvested on day 3 (baseline sample) and day 12 (drop-out sample), respectively, and shRNA abundance was determined by next-generation sequencing (NGS). (B) RIGER analysis to identify genes that are preferentially essential in FUS-DDIT3-expressing SCP-1 cells. EV-transduced SCP-1 cells and 20 FUS-DDIT3-negative cancer cell lines screened with the same shRNA library were used as reference set. Genes were ranked according to relative shRNA depletion, and YAP1 was identified as top FUS‑DDIT3‑specific essential gene. NES, normalized enrichment score. (C) LFC change in YAP1 shRNA representation in 20 cancer cell lines and SCP-1 cells transduced with FUS-DDIT3 or EV. Error bars represent the mean ± SD of LFC scores for six independent shRNAs. (D) Competition assays with SCP-1 cells transduced with RFP-labeled NTC or YAP1 shRNAs. Flow cytometric quantification of RFP-positive cells on day 9 relative to day 3 showed that YAP1 knockdown was preferentially toxic to FUS-DDIT3‑expressing cultures. Error bars represent the mean ± SD of two independent experiments, two-way ANOVA.
Requirement for YAP1 signaling in myxoid liposarcoma (2019)
Figure 2

Figure 2. Increased nuclear YAP1 levels in FUS-DDIT3-expressing mesenchymal stem cells, MLS cell lines, and MLS patient samples. (A) Expression of YAP1 in SCP‑1 cells transduced with FUS‑DDIT3 or EV and liposarcoma cell lines. One of at least two independent experiments with similar results is shown. FUS‑DDIT3‑expressing cell types are indicated in red. (B) Expression of YAP1 in cytoplasmic (yellow) and nuclear (blue) fractions from SCP-1 cells transduced with FUS-DDIT3 or EV and liposarcoma cell lines. One of at least two independent experiments with similar results is shown. FUS‑DDIT3‑expressing cell types are indicated in red. (C) Expression of FOXM1 and PLK1 in MLS cell lines. One of at least two independent experiments with similar results is shown. (D) Strong nuclear expression of YAP1, FOXM1, and PLK1 in MLS patient samples (original magnification, x10 [inset, x20]). (E) Intensity of nuclear YAP1 expression in liposarcoma patient samples. Immunoreactivity was assessed using a semi‑quantitative score (0, negative; 1, weak; 2, moderate; and 3, strong) defining the staining intensity in the positive control (hepatocellular carcinoma) as strong. Only tumors with at least moderate staining (semi‑quantitative score ≥2) and ≥30% YAP positive cells were considered positive for the purposes of the study. (F) Proportion of cells with nuclear YAP1 expression in liposarcoma patient samples. Boxes represent mean values and lower and upper quartiles. Whiskers represent minimum and maximum values.
Requirement for YAP1 signaling in myxoid liposarcoma (2019)
Figure 3

Figure 3. Requirement for YAP1 activity in MLS cell lines. (A) Competition assays with liposarcoma cell lines transduced with RFP-labeled NTC or YAP1 shRNAs. Flow cytometric quantification of RFP-positive cells on day 17 relative to day 3 showed that YAP1 knockdown was preferentially toxic to MLS cells. Error bars represent the mean ± SD of two independent experiments. (B) Aggregate data from competition assays shown in (A). Statistical significance was assessed using an unpaired t-test. LS, non-myxoid liposarcoma. (C) Competition assays with MLS 1765-92 cells transduced with an RFP‑labeled NTC shRNA or an RFP-labeled shRNA against the YAP1 3' UTR following transduction with EV or the YAP1 coding sequence. Flow cytometric quantification of RFP‑positive cells on day 17 relative to day 3 showed that cell viability was rescued by expression of the shRNA‑resistant YAP1 cDNA. Error bars represent the mean ± SD of two independent experiments. (D) Expression of total and exogenous V5‑tagged YAP1 in MLS 1765‑92 cells shown in (C). One of at least two independent experiments with similar results is shown. (E) Cell viability and expression of FOXM1 and PLK1 in MLS cell lines following siRNA-mediated YAP1 knockdown. Error bars represent the mean ± SD of three independent experiments, unpaired t-test. The blots represent one of at least three independent experiments with similar results. (F) Flow cytometric cell cycle analysis of MLS cell lines following shRNA‑mediated YAP1 knockdown. Error bars represent the mean ± SD of three independent experiments, two-way ANOVA; ns, not significant. (G) Senescence‑associated β‑galactosidase (SABG) staining intensity in MLS cell lines following shRNA-mediated YAP1 knockdown. Error bars represent the mean ± SD of ten random microscopic fields, two-way ANOVA). (H) Expression of CDKN1A, CDKN2A, total and phosphorylated RB1, and TP53 in MLS cell lines following shRNA‑mediated YAP1 knockdown. One of at least two independent experiments with similar results is shown. (I) Expression of cleaved PARP and cleaved caspase 3/8 in MLS cell lines following shRNA-mediated YAP1 knockdown. One of at least two independent experiments with similar results is shown.
Requirement for YAP1 signaling in myxoid liposarcoma (2019)
Figure 4

Figure 4. Causal relationship between FUS-DDIT3 expression and increased YAP1 activity. (A) Expression of YAP1 and downstream effectors in SCP-1 cells transduced with FUS‑DDIT3 or EV. One of at least three independent experiments with similar results is shown. (B) Expression of YAP1 and downstream effectors in cytoplasmic (yellow) and nuclear (blue) fractions from SCP-1 cells transduced with FUS‑DDIT3 or EV. One of at least two independent experiments with similar results is shown. (C) YAP1-responsive luciferase activity in SCP-1 cells transduced with FUS‑DDIT3. Relative luciferase activity is displayed as fold change relative to control. Error bars represent the mean ± SD of three independent experiments, unpaired t-test. (D) Localization of FUS-DDIT3 and YAP1 in MLS 1765-92. Nuclei were counterstained with DAPI. The original magnification was x63, and images were zoomed in four times for co-localization analysis. The correlation between red and green fluorescence was determined by Pearson coefficient analysis (square, area for signal acquisition). (E) Co‑IP of transiently expressed FUS‑DDIT3 and YAP1 from HEK293T cells. V5-tagged YAP1 was pulled down using an anti-V5 antibody, and interacting proteins were detected by immunoblotting. One of at least two independent experiments with similar results is shown. (F) Co-IP of endogenous FUS-DDIT3 and YAP1 from MLS 1765-92 cells. FUS-DDIT3 was pulled down using an anti-DDIT3 antibody, and interacting proteins were detected by immunoblotting. One of at least two independent experiments with similar results is shown.
Requirement for YAP1 signaling in myxoid liposarcoma (2019)
Figure 5

Figure 5. Sensitivity of MLS cell lines to pharmacologic YAP1 inhibition. (A) Viability and proliferation of MLS 402‑91, MLS 1765‑92, and MLS 2645-94 cells cultured in the presence of verteporfin. Error bars represent the mean ± SEM of one representative experiment performed in quintuplicate. (B) Flow cytometric analysis of apoptosis (cleaved PARP) and mitotic fraction (phosphorylated histone H3S10) in MLS cell lines cultured in the presence of 0.25 µM Verteporfin. One of two independent experiments with similar results is shown. (C) Expression of total YAP1 and downstream effectors (FOXM1 and PLK1) in MLS cell lines treated with 0.5 or 1 µM verteporfin for 15 hours. One of at least three independent experiments with similar results is shown. (D) YAP1-responsive luciferase activity in MLS cell lines transfected with a constitutively active YAP1S127A mutant and treated with 1 µM verteporfin. Relative luciferase activity is displayed relative to control. Error bars represent the mean ± SD of three independent experiments, unpaired t-test. (E) Tumor formation on chicken CAM of MLS cell lines following shRNA-mediated YAP1 knockdown. The number of tumors is given above each bar, Fisher exact test; ns, not significant. (F) Tumor growth on chicken CAM of MLS cell lines following treatment with 1 µM verteporfin. Shown are tumor volumes and representative photographs of tumors. Error bars represent the mean ± SEM of at least four tumors, unpaired t-test.
EGR2 phosphatase regulates OST1 kinase activity and freezing tolerance in Arabidopsis (2018)
Figure 1

Figure 1. Cold-induced OST1 kinase activity is independent of ABA and ABA receptors.

A, B In-gel kinase assay of OST1 activity in ABA receptor mutants under cold stress. Ten-day-old wild type, ost1-3, pyr1 pyl1,4, pyr1 pyl1,2,4, pyr1 pyl4,5,8 and pyr1 pyl1,4,5,8 mutants were treated at 4°C for 2 h. Total protein extracts were prepared and separated on SDS-PAGE gel containing 0.1 mg/mL GST-∆ABF2 as a substrate, and incubated with 60 μCi of [γ-32P]ATP. Representative pictures are shown in (A) and relative kinase activity is shown in (B).

C, D In-gel kinase assay of OST1 activity in ABI1-OE14 and abi1 abi2 hab1 mutants under cold stress. Representative pictures are shown in (C) and relative kinase activity is shown in (D).

Data information: In (A and C), Top, gel autoradiograph; bottom, Coomassie Brilliant Blue (CBB) staining of RuBisCO large subunit was used as a loading control. The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (B and D), each bar represents the mean ± SEM of three biological repeats. *P < 0.05 (two-tailed t-test).
EGR2 phosphatase regulates OST1 kinase activity and freezing tolerance in Arabidopsis (2018)
Figure 2

Figure 2. OST1 interacts with EGR2 and inhibits OST1 activity under cold stress.

A Interaction of OST1 and EGR2 in yeast. Yeast cells harboring different vector combinations were grown on SC/−Leu/−Trp medium for 3 d or SC/−Leu/−Trp/−His/−Ade medium for 6 d. OST1-BD/AD and BD/EGR2-AD were used as negative controls.

B OST1 interacts with EGR2 in vitro. Purified recombinant MBP-His-EGR2 or MBP-His proteins from E. coli were immunoprecipitated with MBP beads, and then incubated with purified recombinant GST-OST1. Precipitated proteins were detected with anti-His and anti-GST antibodies.

C Co-IP assay of OST1 with EGR2 in vivo. 35S:HF-OST1/Super:EGR2-Myc or 35S:HF-OST1/Super:Myc was expressed in N. benthamiana leaves. Total proteins were immunoprecipitated with anti-Myc agarose beads, and the co-immunoprecipitation products were subjected to immunoblot analysis. EGR2-Myc and HF-OST1 were detected with anti-Myc and anti-HA antibodies, respectively.

D Bimolecular fluorescence complementation (BiFC) assay. Wild-type Arabidopsis protoplasts transformed with OST1-YFPC and EGR2-YFPN proteins were incubated for 18 h. The interaction signal was detected by confocal microcopy. The combinations of OST1-YFPC/GUS-YFPN and GUS-YFPC/EGR2-YFPN were used as negative controls. Scale bars: 100 μm.

E EGR2 inhibits OST1 kinase activity in vitro. Proteins were incubated with 1 μCi [γ-32P]ATP in kinase reaction buffer for 30 min at 30°C, and then separated by SDS-PAGE. Top, autoradiograph; bottom, CBB staining.

F, G In-gel assay of OST1 activity in 10-d-old wild type, egr1 and egr2 single mutants, and the egr1 egr2 double mutant under cold stress.

H, I In-gel assay of OST1 activity in 10-d-old wild-type and EGR2-overexpressing plants under cold stress.

Data information: In (F-H): the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (F and H), representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In (G and I), relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).