Journal: PLoS ONE

Article Title: Targeting Homologous Recombination in Notch-Driven C . elegans Stem Cell and Human Tumors

doi: 10.1371/journal.pone.0127862

Figure Lengend Snippet: RNAi of DDR orthologs detects genes that enhance ar202 tumor radiosensitivity.

Article Snippet: The IDs of C . elegans gene expression assay are: mre-11 —Ce02480998_g1; rad-51 —Ce02458920_g1; atl-1 —Ce02479867_g1; mus-101 —Ce02413322_g1; cku-80 —Ce02445546_g1; lig-4 —Ce02449042_g1; hsr-9 —Ce02412427_g1; rad-50 —Ce02482582_g1; mec-7— Ce02497588_g1.

Techniques: Ubiquitin Proteomics, Histone Deacetylase Assay

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: a Schematic diagram of the CRISPR-Cas9 screen process. b Genes identified by NGS were analyzed with the MAGeCK program and plotted based on the log 2 value of fold change of NGS reads and statistical significance (shown as log 10 value of RRA p -value and plotted as the y -axis). The genes involved in the GPI (glycosylphosphatidylinositol) biosynthetic pathway are colored red. c Schematic diagram of human TFPIβ (TFPI), a GPI-anchored protein with two BPTI/Kunitz protease inhibitor domains (K1 and K2). N, N-termini; C, C-termini. HeLa ( d ) or A549 ( e ) KO cells lacking TFPI, TFPI2 (a homolog of TFPI), PIGS, or PIGV were generated via the CRISPR-Cas9 approach. UGP2-KO cells were also analyzed as a control. Cells were exposed to recombinant TcdB4.2 for 24 h. Their CR 50 values are normalized to WT and plotted in a bar-chart ( f ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). g – i HeLa ( g ) or 5637 ( h ) cells overexpressing triple-HA-tagged TFPI, TFPI2, or mouse TFPI (mTFPI) via lentiviral transduction were exposed to TcdB4.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations. Their CR 50 values are normalized to WT and plotted in a bar-chart ( i ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). j Binding of TcdB4.2 (500 nM) to Fc-tagged TFPI, TFPI2, and mTFPI (immobilized onto capture biosensors) was examined using biolayer interferometry (BLI) assays. Fc-tagged extracellular domains of FZD2 (CRD2), SEMA6A, and IgG were used as controls. Representative sensorgrams from one of three independent experiments are shown. k HeLa cells were exposed to either TcdB4.2 alone (4 pM) or TcdB4.2 pre-incubated with Fc-tagged TFPI, TFPI2, or mTFPI at the indicated molar ratios (1:250 ~ 1:20,000) on ice for 1 h. The percentage of cell-rounding at 6 h incubation was plotted. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). l HeLa-WT, TFPI-KO, and TFPI2-KO cells were exposed to recombinant TcdB2.1, TcdB7.2, TcdB12.1, TcsL, or culture supernatants of C. difficile strains expressing TcdB10.1 or TcdB11.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations are shown in Supplementary Fig. . Their CR 50 values were normalized to WT and plotted here. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments). m – p HeLa-WT, two CSPG4 KO single clones (CSPG4#1 and CSPG4#8), and two CSPG4/TFPI double KO cells (CSPG4#1-TFPI-KO and CSPG4#8-TFPI-KO) were exposed to TcdB4.2 ( m ), TcdB2.1 ( n ), or TcdB2.2 ( o ) for 24 h. The percentages of rounded cells were plotted over toxin concentrations. Their relative CR 50 values are plotted in a bar-chart ( p ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). q HeLa cells overexpressing HA-tagged TFPI, TFPI2, or mTFPI via lentiviral transduction were exposed to recombinant TcdB2.1, TcdB2.2, TcdB7.2, TcdB12.1, TcsL, or culture supernatants of C. difficile strains expressingTcdB10.1 or TcdB11.2 for 24 h. The percentages of rounded cells were plotted over toxin concentrations and are shown in Supplementary Fig. . Their CR 50 values are normalized to WT and plotted here. Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: CRISPR, Protease Inhibitor, Generated, Recombinant, Transduction, Binding Assay, Incubation, Expressing, Clone Assay

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: a Schematic diagrams of TcdB4.2, TcdB4.2 1286–1805 , TcdB4.2 1835–2367 , TcdB1.1-FBD. TcdB4.1(B1.1) and TcdB1.1(B4.2) represent two mutant fragments exchanging 29 residues in the region 1432– 1600 that differ between TcdB4.1 and TcdB1.1. The numbers indicate the position of amino acid residues. GTD, glucosyltransferase domain; CPD, cysteine protease domain; DRBD, delivery/receptor-binding domain; CROPs, combined repetitive oligopeptides. Binding of 500 nM TcdB4.2 1286– 1805 ( b ) or TcdB4.2 1835– 2367 ( c ) to Fc-tagged TFPI and mTFPI was examined using BLI assays. Fc-tagged TFPI2, CRD2, SEMA6A, and IgG were used as controls. Representative sensorgrams from one of three independent experiments are shown. d HeLa cells transiently transfected with TFPI, TFPI2, mTFPI, SEMA6A, or FZD2 were exposed to FLAG-tagged TcdB4.2 1286– 1805 (5 µg/mL) on ice for 60 min, washed, fixed, permeabilized, and subjected to immunostaining analysis. Expression of exogenous proteins was confirmed by detecting fused HA or 1D4 tag. Nuclei were labeled with DAPI (blue). Scale bar, 5 µm. Representative images were from one of three independent experiments. Binding of 500 nM TcdB1.1-FBD, TcdB4.2 1286– 1805 , TcdB4.2(B1.1), and TcdB1.1(B4.2) to Fc-tagged CRD2 ( e ) and TFPI ( f ) was examined using BLI assays. Representative sensorgrams from one of three independent experiments are shown. g Schematic diagram of TFPIβ, TFPI-K1, and TFPI-K2 fragments. Sig, signal peptide; N, N-terminal domain; K1, BPTI/Kunitz inhibitor domain 1; L1, loop 1; K2, BPTI/Kunitz inhibitor domain 2; L2, loop 2; β, GPI anchor sequence for TFPIβ. h Binding of 500 nM TcdB4.2 1286-1805 to Fc-tagged TFPI, TFPI-K1, and TFPI-K2 was examined using BLI assays. Representative sensorgrams from one of three independent experiments are shown. i , j TcdB4.2 binding to TFPI-K2 prevents TFPI-K2 binding to its natural ligand coagulation factor Xa (FXa). FXa (0.5 ng/mL) cleaves its fluorescently labeled substrate and generates increasing fluorescent signal (measured as relative light unit, RLU, y -axis) over time ( x -axis). FXa’s enzymatic activity was inhibited by TFPI-K2 ( i , 7.5 ng/mL). The inhibitory effect of TFPI was blocked by adding TcdB4.2 1286– 1805 in a dose-dependent manner (1:1, 1:3, or 1:10 molar ratio). Representative curve from one of three independent experiments is shown. FXa activity was quantified by measuring the slope of RLU curves (1–10 min) and plotted as a bar-chart ( j ). The same experiments were also carried out for TFPI-Fc and mTFPI-Fc, with their curves shown in Supplementary Fig. and quantification in ( j ). Error bars indicate mean ± s.d.; N = 3; *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: Mutagenesis, Binding Assay, Transfection, Immunostaining, Expressing, Labeling, Sequencing, Coagulation, Activity Assay

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: a Cultured undifferentiated human enteroids (in growth medium), differentiated human rectoids (in differentiation medium), and mouse intestinal organoids were exposed to either TcdB4.2 alone (10 pM) or TcdB4.2 pre-incubated with Fc-tagged TFPI, TFPI2, or mTFPI (100 nM) for 8 h. PBS was used as control (Ctrl). Stars indicate the dissociated organoids with released luminal contents; arrows indicate shrunken organoids; scale bar, 50 µm. Representative images are from one of three independent experiments. b Experiments were carried out as described in panel ( a ). After exposure to toxin for 3 days, cell viability was measured using the MTT assay and plotted as a bar-chart. Error bars indicate ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). c , d Accumulation of fluid in the thoracic cavity occurred within 15 h after intraperitoneal injection of TcdB4.2 into mice (50 ng per 25 g bodyweight). Injection of TcdB4.2 pre-incubated with Fc-tagged TFPI or mTFPI at 1:2000 molar ratios showed less fluid accumulation. Co-injection of TcdB4.2 with Fc-tagged TFPI2 at 1:2000 molar ratio did not affect fluid accumulation. Injection of saline was included as a control. The range of boxes indicates ± s.e.m.; whiskers indicate ± s.d.; percentiles indicate median; **, p < 0.01 (Student’s t -test, two-sided). e Experiments were carried out as described in panel ( c ) and the edema in lung tissues was evaluated by calculating dry-to-wet weight ratios. TcdB4.2 reduced dry-to-wet weight ratio of lung tissue more than in the saline group. Co-injection of TcdB4.2 with TFPI-Fc or mTFPI-Fc prevented this reduction, whereas co-injection with TFPI2-Fc showed no protection from TcdB4.2. The range of boxes indicates ± s.e.m.; whiskers indicate ± s.d.; percentiles indicate median; *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). f , g The sensitivity of HUVECs transfected with siRNAs targeting TFPI to TcdB1.1 or TcdB4.2 was analyzed using the 24 h cell-rounding assay. HUVECs transfected with non-targeting scrambled siRNAs served as a control. Dose-response curves are plotted in ( f ), and their relative CR 50 are plotted in a bar chart ( g ). Error bars indicate ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01; NS not significant (Student’s t -test, two-sided). Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: Cell Culture, Incubation, MTT Assay, Injection, Transfection

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: a Amino acids across all 206 known TcdB sequences and 6 TcsL sequences were aligned and visualized using a haplotype coloring algorithm we recently developed , showing variation patterns across TcdB members. The first sequence (TcdB1.1) is assigned black color, and all other sequences colored black if they share the same residues. Unique residues in the second sequence (TcdB2.1) are colored green, followed by unique residues in the third sequence (TcdB4.2) colored red. The region 1460 to 1626 is enlarged for TcdB2/4/7 members. A unique B4/B7-haplotype can be visualized with residues in red color, with their position marked. Residue A1518 is unique in TcdB7 members and is colored gray. b The sequences of TcdB2.11, TcdB2.22, TcdB7.2, and TcdB7.5 were analyzed using a sliding window comparison with TcdB1.1, 2.1, 4.2, and 7.1, revealing their recombination patterns. Below each sliding window plot is a graphical summary depicting the recombination pattern. The location surrounding the TFPI/FZD-binding site (specificity-determining region) is marked. HeLa-WT, TFPI-KO, and TFPI2-KO cells were exposed to recombinant TcdB7.1 ( c ) or the culture supernatant of C. difficile strain expressing TcdB7.9 ( d ) for 24 h. The percentages of round-shaped cells were plotted over toxin or supernatant dilutions. Their CR values are normalized to WT and plotted in a bar-chart ( e ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). HeLa cells overexpressing HA-tagged TFPI, TFPI2, or mTFPI via lentiviral transduction were exposed to recombinant TcdB7.1 ( f ) or the culture supernatant of C. difficile strain expressing TcdB7.9 ( g ) for 24 h. The percentages of round-shaped cells were plotted over toxin or supernatant dilutions. Their CR values were normalized to WT and plotted in a bar-chart ( h ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); **, p < 0.01 (Student’s t -test, two-sided). Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: Sequencing, Binding Assay, Recombinant, Expressing, Transduction

Journal: Nature Communications

Article Title: Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants

doi: 10.1038/s41467-022-33964-9

Figure Lengend Snippet: Binding of 500 nM TcdB1.1-FBD, TcdB2.1 1285– 1804 , TcdB2.11 1286– 1805 , TcdB4.2 1286– 1805 , TcdB7.2 1286– 1805 , TcdB7.5 1286– 1805 , TcdB10.1 1285– 1804 , TcdB11.2 1285– 1804 , TcdB12.1 1285– 1804 , and TcsL 1285– 1804 to Fc-tagged TFPI ( a ), mTFPI ( b ), or CRD2 ( c ) was examined using BLI assays. Representative sensorgrams from one of three independent experiments are shown. d , e HeLa-WT, FZD1/2/7-KO, CSPG4-KO, and UGP2-KO cells were exposed to the culture supernatant of a C. difficile strain expressing TcdB2.22 for 24 h. The percentages of round-shaped cells were plotted over supernatant dilution ( d ). The relative CR 50 values in different cell lines were normalized to the WT and plotted as a bar-chart ( e ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments); *, p < 0.05; **, p < 0.01 (Student’s t -test, two-sided). f , g HeLa-WT, TFPI-KO, and TFPI2-KO cells were exposed to the culture supernatant of a C. difficile strain expressing TcdB2.22 for 24 h. The percentages of round-shaped cells were plotted over supernatant dilutions ( f ). The relative CR50 values in different cell lines were normalized to the WT and plotted as bar-chart ( g ). Error bars indicate mean ± s.d.; N = 3 (biologically independent experiments). h There are six residues that are different between TFPI-binding TcdB4.2, 2.11, 7.9 versus TcdB2.1 and 7.2 (1495, 1505, 1509, 1547, 1596, and 1599, marked as MT site 1–6). Mutagenesis studies were performed to replace the indicated residues on TcdB2.1 1285-1804 with the corresponding residues found in TcdB4.2. The binding of the indicated mutant proteins (500 nM) to immobilized TFPI-Fc was analyzed using BLI assays. TcdB2.11 1286– 1805 and TcdB2.1 1285– 1804 were analyzed as controls. Representative sensorgrams from one of three independent experiments are shown. i – k A TcdB1.1-FBD-5M mutant proteins were generated by replacing five residues in TcdB1.1 with the corresponding residues found in TcdB4.2 (positions 1495, 1505, 1547, 1596, and 1599). The binding of this mutant protein to immobilized TFPI-Fc ( i ), mTFPI-Fc ( j ), and CRD2 ( k ) was analyzed using BLI assays. TcdB1.1-FBD and TcdB4.2 1285– 1804 were analyzed as controls. Source data are provided as a Source Data file.

Article Snippet: The cDNAs of TFPI were obtained from the indicated vendors: TFPI (Horizon Discovery, MHS6278-202756867), TFPI2 (Horizon Discovery, MHS6278-202839472), and mouse TFPI (Sino Bio, MG50131-M).

Techniques: Binding Assay, Expressing, Mutagenesis, Generated

Journal: Gene Therapy

Article Title: First use of gene therapy to treat growth hormone resistant dwarfism in a mouse model

doi: 10.1038/s41434-022-00313-w

Figure Lengend Snippet: a RT-PCR gel of mGHR mRNA expression. 18S rRNA housekeeping gene was used as loading control. b Western blot of mGHR protein expression. Actin housekeeping gene was used as loading control. c Immunofluorescence staining of mGHR expression, scale bars are 50 µm and nuclei were stained with DAPI. HepG2 cells were transfected with pAAV-HLP-mGHR consisted of hybrid liver-specific promoter (HLP) driving the expression of mGHR. Untransfected control and cells transfected with pAAV-HLP-Luc expressing luciferase gene were used as negative control. Analyses were performed 48 h post-transfection.

Article Snippet: The mGHR PCR fragment was amplified from cloning vector MG50043-M (Sino Biological, Beijing, China) containing mouse growth hormone receptor/GHR/GHBP transcript variant 1 gene ORF cDNA clone using forward primer with introducing NotI site: 5’-ATAAGAATGCGGCCGCACCATGGATCTTTGTCAGGTCTTC and reverse primer with introducing XhoI site: 5’-CCGCGCTCGAGCTACTGCATGATTTTGTTCAGTTG.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Staining, Transfection, Luciferase, Negative Control