Journal: Cell Reports

Article Title: Modeling Type 1 Diabetes In Vitro Using Human Pluripotent Stem Cells

doi: 10.1016/j.celrep.2020.107894

Figure Lengend Snippet: Activation and Killing by T Cells Is Selective for iPSC-β (A) Experimental design: PBMCs co-cultured with autologous iPSC-derived cells. (B, C, and F) CD25 or CD69 expression shown as MFI. (B) PBMCs (gated on CD3 + , CD4 + , and CD8 + populations) co-cultured for 48 h with autologous iPSC-β (purple) or iPSC-α (red). (n = 3 T1D and n = 1 ND donor, n = 3 differentiation batches per donor line). T1D 1 , T1D 2 , and T1D 3 were pooled together. (C) Donor-matched PBMCs (CD3 + gated) (n = 1 T1D, n = 3 differentiation batches per donor line) co-cultured for 48 h with autologous iPSC-β (purple) or iPSC-cardiomyocytes (orange). (D) Percentage of live iPSC-β (C-peptide + /glucagon − ) or iPSC-α (C-peptide − /glucagon + ) from iPSC-β or iPSC-α differentiations, respectively (n = 3 T1D donors, n = 3 differentiation batches per donor line). T1D 1 , T1D 2 , and T1D 3 were pooled together. (F) Representative flow cytometry histograms after 48 h of co-culture. Dashed histogram represents the control (untreated target cells). (E) Percentage of apoptotic (apopxin + ) iPSC-β (CD49a + /CD26 − ) or iPSC-α (CD49a − /CD26 + ) from iPSC-β or iPSC-α differentiations (n = 1 T1D iPSC donor, n = 3 differentiation batches per donor line). (G) Unmatched PBMCs (CD3 + gated cells) co-cultured for 48 h with iPSC-α (n = 3 T1D donors, n = 3 differentiation batches per donor line). T1D 1 , T1D 2 , and T1D 3 were pooled together. (H) Donor-matched PBMCs (gated on CD3 + , CD4 + , and CD8 + populations) co-cultured for 48 h with autologous enriched iPSC-β or iPSC-α. Data are means ± SEMs, 2-way ANOVA. ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.0005, and ∗∗∗∗ p < 0.0001; ns, non-significant. See also Figure S4 .

Article Snippet: For the apoptosis assay, clusters were dissociated, and single cells were stained at room temperature for 30 min using a 1:100 dilution of recently reported stem cell derived β-cell marker, anti-human CD49a ( ) PE-conjugated (BD Biosciences, 559596), α-cell marker anti-human CD26 APC-conjugated (Miltenyi Biotech, 130-120-769), and Apopxin green indicator (Abcam, ab176750).

Techniques: Activation Assay, Cell Culture, Derivative Assay, Expressing, Flow Cytometry, Co-Culture Assay, Control

Journal: Cell Reports

Article Title: Modeling Type 1 Diabetes In Vitro Using Human Pluripotent Stem Cells

doi: 10.1016/j.celrep.2020.107894

Figure Lengend Snippet:

Article Snippet: For the apoptosis assay, clusters were dissociated, and single cells were stained at room temperature for 30 min using a 1:100 dilution of recently reported stem cell derived β-cell marker, anti-human CD49a ( ) PE-conjugated (BD Biosciences, 559596), α-cell marker anti-human CD26 APC-conjugated (Miltenyi Biotech, 130-120-769), and Apopxin green indicator (Abcam, ab176750).

Techniques: Virus, Recombinant, Software, Real-time Polymerase Chain Reaction

Journal: Viruses

Article Title: Unveiling the Role of TMPRSS2 in the Proteolytic Activation of Pandemic and Zoonotic Influenza Viruses and Coronaviruses in Human Airway Cells

doi: 10.3390/v16111798

Figure Lengend Snippet: Activation of SARS-CoV and MERS-CoV S by TMPRSS2 and further host proteases. ( A , B ) HeLa cells were co-transfected with plasmids coding for MERS-CoV S ( A ) or SARS-CoV ( B ) with a C-terminal FLAG-tag and plasmids coding for human TMPRSS2 or HAT or murine TMPRSS4 or TMPRSS13. Cell lysates were analysed via SDS-PAGE at 48 h p.t. Western blot analysis was performed using an antibody targeting the C-terminal FLAG-tag. Tubulin was used as a loading control. The data shown are representative of three individual experiments. EV = empty vector. ( C , E ) HeLa cells co-expressing MERS-CoV ( C ) or SARS-CoV ( E ) S, as well as the respective receptors DPP4 or ACE2, were incubated for 48 h. After fixation, the cells were stained with a primary antibody targeting the C-terminal FLAG-tag of S and a fluorescence-coupled secondary antibody. DAPI was used to stain the nuclei. Representative images of three independent experiments are shown. The scale bar represents 100 µM. ( D , F ) Quantification of MERS-CoV S ( D ) or SARS-CoV ( F ) mediated cell–cell fusion. For each condition, ten randomly taken images were analysed by counting the nuclei per syncytium (with at least three nuclei). The data shown are the mean values + SEM of three independent experiments. Statistical significance was determined with a one-way ANOVA followed by Šídák’s multiple comparisons test. ns = not significant, ** = p < 0.01, **** = p < 0.0001.

Article Snippet: The expression vectors coding for SARS- or MERS-CoV S with a C-terminal FLAG-tag, pCMV3-MERS-Spike-cFLAG (VG40069-CF) and pCMV3-SARS-Spike-cFLAG (VG40150-CF), as well as a vector encoding human dipeptidyl peptidase 4 (DPP4) with a C-terminal HA-tag, pCMV3-hDPP4-cHA (HG10688-CY), were purchased from Sino Biological, Houston, TX, USA.

Techniques: Activation Assay, Transfection, FLAG-tag, SDS Page, Western Blot, Control, Plasmid Preparation, Expressing, Incubation, Staining, Fluorescence

Journal: Molecular Cancer Therapeutics

Article Title: PhAc-ALGP-Dox, a Novel Anticancer Prodrug with Targeted Activation and Improved Therapeutic Index

doi: 10.1158/1535-7163.mct-21-0518

Figure Lengend Snippet: Figure 1. PhAc-ALGP-Dox is activated sequentially by extracellular THOP1 and cytoplasmic DPP4/FAPa in the tumor microenvironment. A–C, Generation of PhAc-ALGP-Dox cleavage products in the presence of recombinant enzymes (THOP1, 0.1 mg/mL; FAPa, 0.04 mg/mL; or DPP4, 0.04 mg/mL). Data are represented as mean SD of triplicate analysis, LoQ, 3 nmol/L. D, Quantification of Dox autofluorescence following exposure to 20 mmol/L PhAc-ALGP-Dox for 5 hours. Nuclear/cytoplasmic fluorescence was assessed in HUVEC cells in the presence of recombinant THOP1 (0.1 mg/mL) alone or together with either recombinant FAPa (0.04 mg/mL) or PT-100 (300 nmol/L), an inhibitor of FAPa/DPP4. Bars represent mean þ SD of n ¼ 8–12. Asterisks indicate significant differences versus no enzyme or as specified by brackets. , P < 0.001, , P < 0.0001 as defined by one-way ANOVA corrected for multiple comparisons (Holm–Sidak). E–L, Representative images of HUVEC cells stimulated with PhAc-ALGP-Dox alone (E, F), together with THOP1 (G, H), THOP1 and FAPa (I–J) or THOP1, and PT-100 (K, L). Top panels are merged images of cytoplasmic (Agglutinin, green), nuclear (DAPI, blue), or Dox (autofluorescence, red). Bottom panels are black-white images of Dox autofluorescence. Scale bar, 20 mm. M, Quantification of ELISA data of human THOP1 protein levels in cell lysates and conditioned media of cancer cells (MDA-MB-231 and LS 174T) compared with normal (HME-1) cells. Bars are mean þ SD of three independent ELISAs run in duplicate. Asterisks indicate significant differences versus no enzyme or as specified by brackets. , P < 0.001, , P < 0.0001 as defined by one-way ANOVA corrected for multiple comparisons (Holm–Sidak). N, Kinetic activation of Dnp(k)-ALGP-CouAla by extracellular peptidases in normal (HME-1, blue) or tumor (LS 174T, purple) conditioned medium. Dashed purple line represents the signal in tumor conditioned media, preincubated with a THOP1 inhibitor (THOPi, 10 mmol/L). O, Quantification of Dnp(k)-ALGP-CouAla conversion after 180 minutes. Bars are mean þ SD, pooled from three independent experiments (n ¼ 6). , P < 0.01 as defined by one-way ANOVA.

Article Snippet: Following inhibitors were used to block activity of endogenous peptidases: THOP1 was specifically inhibited with Cpp-AAF-pAB (100 mmol/L Bachem), THOP1 and CD10 were blocked with JMV-390 (10 mmol/L, Tocris #2575), DPP4 and FAPa was constrained using talabostat (1⁄4PT-100, 10 mmol/L, Tocris #3719), whereas FAPi (MedChemExpress, HY100684) was used to specifically inhibit FAPa.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: bioRxiv

Article Title: Simultaneous and sequential multi-species coronavirus vaccination

doi: 10.1101/2022.05.07.491038

Figure Lengend Snippet: (A) Schematics of mRNA vaccine construct design against pathogenic human coronavirus species. Each construct has regulatory elements (5’UTR, 3’UTR and polyA) and spike ORF. The domain structures as well as engineered mutations of translated spike proteins of SARS-CoV-2 Delta variant (Delta), SARS-CoV (SARS) and MERS-CoV (MERS). (B) Engineered mutations in spike protein structures of SARS-CoV-2 Delta, SARS-CoV and MERS-CoV. The N-terminal domain (NTD, blue), receptor binding domain (RBD, green) and S2 subunit (orange) of one protomer along with homologous HexaPro mutations (pink) and Delta variant mutations (red) were highlighted in the spike trimer structures. (C) Schematics of characterization of LNP-mRNA vaccine formulations. Assembly procedure of LNP-mRNA vaccine on NanoAssemblr Ignite and downstream biophysical characterization assays. (D) Histogram displaying radius distribution of LNP-mRNA formulations of SARS-CoV-2 Delta and a Triplex (Delta + SARS + MERS) (abbreviated as Triplex-CoV or Triplex), measured by dynamic light scattering (DLS). The polydispersity index and mean radius of each LNP sample were shown at top left corner. (E) Transmission electron microscope (TEM) images of Delta and Triplex-CoV LNP-mRNAs. (F) Surface expression of functional spike proteins in 293T cells after electroporation of corresponding mRNA, as detected by human ACE2 or human DPP4 Fc fusion protein bound to PE anti-Fc antibody. (G) Schematics of vaccination schedule of the Triplex LNP-mRNA formulations, as well as downstream assays to evaluate the antibody responses and other immunological profiles. (H) Binding antibody titers of plasma samples from mice administered with PBS or different LNP-mRNAs (n = 9) against RBD or ectodomain (ECD) of SARS-CoV-2 wild type (WT, Wuhan/WA-1), Delta variant, SARS and MERS spikes. The binding antibody titers were quantified by area under curve of log 10 -transformed titration curve (log 10 AUC) in . The mice were intramuscularly injected with two doses (x2, 2 weeks apart) of PBS, 1μg SARS-CoV-2 Delta variant LNP-mRNA (delta), 1μg or 3μg equal mass mixture of Delta, SARS and MERS LNP-mRNA (Triplex-CoV). Notes: In the dot-box plots of this figure, each dot represents data from one mouse. Data are shown as mean ± s.e.m. plus individual data points in plots. Two-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance. Statistical significance labels: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Non-significant comparisons are not shown, unless otherwise noted as n.s., not significant.

Article Snippet: To detect surface-protein expression, the cells were stained with ACE2–Fc chimera (Genscript, Z03484) or DPP4-Fc (Sino Biological, 10688-H01H) in MACS buffer (D-PBS with 2 mM EDTA and 0.5% BSA) for 30 min on ice.

Techniques: Construct, Variant Assay, Binding Assay, Transmission Assay, Microscopy, Expressing, Functional Assay, Electroporation, Transformation Assay, Titration, Injection

Journal: Viruses

Article Title: Phage-Displayed Mimotopes of SARS-CoV-2 Spike Protein Targeted to Authentic and Alternative Cellular Receptors

doi: 10.3390/v14020384

Figure Lengend Snippet: Characteristics of recombinant human cell receptors and viral spike proteins.

Article Snippet: hDPP4 , Sino Biological , 10688-H08H , D34 , P766 , 744 , 86.3.

Techniques: Recombinant, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Co-localization of the identified proteins with cell membrane-bound SARS-CoV-2 spike proteins. Representative images of co-localization with SARS-CoV-2 spike proteins and the identified membrane proteins. Caco-2 cells were co-stained for SARS-CoV-2 spike protein (green) and the antibodies recognizing ACE2, CD133, Cadherin 17, DPP4, and VAPA (Red). The resulting specimens were subsequently stained with appropriate secondary antibodies and DAPI (Blue), then observed using confocal microscopy (20× objective). Co-localization is indicated in yellow in the “Merge” images. White bar: 10 μm.

Article Snippet: The ORFs of the candidate protein CDH17 and DPP4 were obtained from pCMV3-human CDH17 (HG11360-UT; Sino Biological) and pCMV3-human DPP4 (HG10688-UT; Sino Biological) by KpnI and XbaI digestion.

Techniques: Staining, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Host cell membrane proteins located near SARS-CoV-2 spike protein attachment sites are identified using proximity labeling and proteomic analysis

doi: 10.1016/j.jbc.2022.102500

Figure Lengend Snippet: Candidate proteins located near SARS-CoV-2 spike proteins. ( A to D ) Morphological observation of SARS-CoV-2 spike proteins and the identified membrane proteins. Caco-2 cells observed using electron microscopy. Cultured Caco-2 cells were fixed and co-stained with the SARS-CoV-2 spike protein (indicated as 20 nm particles), and candidate molecules identified. CD133 ( A ), DPP4 ( B ), CDH17 ( C ), and VAPA ( D ) are indicated as 10 nm particles. Red arrows indicate the locations of SARS-CoV-2 spike proteins. Yellow arrow heads indicate the location of each candidate protein. Scale bar; 200 or 500 nm.

Article Snippet: The ORFs of the candidate protein CDH17 and DPP4 were obtained from pCMV3-human CDH17 (HG11360-UT; Sino Biological) and pCMV3-human DPP4 (HG10688-UT; Sino Biological) by KpnI and XbaI digestion.

Techniques: Electron Microscopy, Cell Culture, Staining