Journal: Scientific Reports

Article Title: A wild boar cathelicidin peptide derivative inhibits severe acute respiratory syndrome coronavirus-2 and its drifted variants

doi: 10.1038/s41598-023-41850-7

Figure Lengend Snippet: A wild boar cathelicidin PMAP-36R inhibits SARS-CoV-2 infection of Vero hACE2 cells. ( A ) 3-dimensional structure projection of PMAP-36R (left) and schematic of PMAP-36R variants (right). A 3D structure of PMAP-36R was determined by AlphaFold (P49931) and rendered in PyMOL , . Positively charged residues are depicted in blue while hydrophobic residues are depicted in red. Labeled residues along the N-terminal α-helix form a non-polar sector including the weakly hydrophilic threonine. ( B – E ) Inhibition of SARS-CoV-2 infection by PMAP-36R and its derivatives. PMAP-36R was compared against LL-37 ( B ), N-terminal truncations ( C ), C-terminal truncations ( D ), or a cysteine mutant ( E ) of PMAP-36R. Peptides at the labeled concentrations were pre-incubated with 100pfu of live SARS-CoV-2 virus (nCoV/USA_WA1/2020) for 1 h at 37 °C before addition to confluent Vero hACE2 cells in a 96-well plate. Infected cells were fixed and quantified by focus forming assay after 48 h. Inhibition of viral infection was calculated based on the percent area of each well staining positively for viral spike protein compared to control wells without peptide inhibitor treatment. Results are representative of 3 independent experiments performed in triplicate. Significance calculated by two-way ANOVA with Bonferonni’s correction comparing peptide derivatives against control PMAP-36R (* < .05, ** < .01, ** < .001, * < .0001).

Article Snippet: Vero E6 (ATCC CRL-1586) and HEK293T (ATCC CRL-1573) were purchased from ATCC.

Techniques: Infection, Labeling, Inhibition, Mutagenesis, Incubation, Virus, Focus Forming Assay, Staining, Control

Journal: Scientific Reports

Article Title: A wild boar cathelicidin peptide derivative inhibits severe acute respiratory syndrome coronavirus-2 and its drifted variants

doi: 10.1038/s41598-023-41850-7

Figure Lengend Snippet: Calculated IC 50 and TD 50 values for PMAP-36R derivatives and control LL-37 or OVA peptides.

Article Snippet: Vero E6 (ATCC CRL-1586) and HEK293T (ATCC CRL-1573) were purchased from ATCC.

Techniques: Control

Journal: Scientific Reports

Article Title: A wild boar cathelicidin peptide derivative inhibits severe acute respiratory syndrome coronavirus-2 and its drifted variants

doi: 10.1038/s41598-023-41850-7

Figure Lengend Snippet: The PMAP-36R cathelicidin derivative pSer possesses reduced cytotoxicity. ( A ) Human RBC hemolysis assay showing reduced lytic activity of pSer compared to PMAP-36R or LL-37. Peptides at the labeled concentrations were incubated with human RBCs for 1 h at 37 °C in 1X PBS buffer. RBC lysis was determined by the spectral absorbance (490 nm) of cell supernatants relative to PBS-only and TritonX-100 containing controls. ( B-C ) MTS formazan formation assay with ( B ) Vero hACE2, and ( C ) HEK-293 T cell lines showing reduced cell death in wells receiving pSer compared to PMAP36-R. Peptides at the labeled concentrations were incubated with cells in 1% FBS containing DMEM for 48 h prior to the addition of MTS substrate. The percentage of cell death was calculated relative to untreated and NP-40 receiving control wells. Results are representative of 3 independent experiments performed in triplicate. ( D ) MTS formazan formation assay with the Vero A2T2 cell line showing saturation of toxic effect at 100 µM. Peptides at the labeled concentrations were incubated with cells in 1% FBS containing DMEM for 24 h prior to the addition of MTS substrate. The percentage of cell death was calculated relative to untreated and NP-40 receiving control wells. Results are representative of 2 independent experiments performed in duplicate. Significance calculated by two-way ANOVA with Bonferonni’s correction comparing pSer against control PMAP-36R (* < .05, ** < .01, ** < .001, * < .0001).

Article Snippet: Vero E6 (ATCC CRL-1586) and HEK293T (ATCC CRL-1573) were purchased from ATCC.

Techniques: Hemolysis Assay, Activity Assay, Labeling, Incubation, Red Blood Cell Lysis, Tube Formation Assay, Control

Journal: Scientific Reports

Article Title: A wild boar cathelicidin peptide derivative inhibits severe acute respiratory syndrome coronavirus-2 and its drifted variants

doi: 10.1038/s41598-023-41850-7

Figure Lengend Snippet: The PMAP-36R derivative Yongshi (pSer) mediates SARS-CoV-2 inhibition via acting on both virions and cells. Inhibition of SARS-CoV-2 infection of Vero A2T2 cells. Peptides at the labeled concentrations were pre-incubated with virus for 1 h or added directly to cells 1 h before, at the time of, or 1 h after infection with 100pfu of SARS-CoV-2 (nCoV/USA_WA1/2020). For pre-treatment groups, the added peptide was removed immediately prior to infection and replaced with control medium. Infected cells were fixed and quantified by focus forming assay after 48 h. Inhibition of viral infection was calculated based on the percent area of each well staining positively for viral spike protein compared to control wells without peptide inhibitor treatment. Results are representative of 2 independent experiments performed in triplicate. Significance calculated by two-way ANOVA with Bonferonni’s correction comparing each group against pre-incubated virus (* < .05, ** < .01, ** < .001, * < .0001).

Article Snippet: Vero E6 (ATCC CRL-1586) and HEK293T (ATCC CRL-1573) were purchased from ATCC.

Techniques: Inhibition, Infection, Labeling, Incubation, Virus, Control, Focus Forming Assay, Staining

Journal: Scientific Reports

Article Title: A wild boar cathelicidin peptide derivative inhibits severe acute respiratory syndrome coronavirus-2 and its drifted variants

doi: 10.1038/s41598-023-41850-7

Figure Lengend Snippet: The D-enantiomer of Yongshi loses SARS-CoV-2 specificity. ( A ) Toxicity towards Vero hACE2 cells was evaluated by MTS formazan formation assay. Peptides at the labeled concentrations were incubated with cells in 1% FBS containing DMEM for 48 h prior to the addition of MTS substrate. The percentage of cell death was calculated relative to untreated and NP-40 receiving control wells. ( B ) Inhibition of SARS-CoV-2 infection of Vero hACE2 cells. Peptides at the labeled concentrations were pre-incubated with 100pfu of live SARS-CoV-2 virus (nCoV/USA_WA1/2020) for 1 h at 37 °C before addition to confluent Vero hACE2 cells in a 96-well plate. Infected cells were fixed and quantified by focus forming assay after 48 h. Inhibition of viral infection was calculated based on the percent area of each well staining positively for viral spike protein compared to control wells without peptide inhibitor treatment. Results are representative of 3 independent experiments performed in triplicate. Data for L-Yongshi reproduced from Figs. and . Significance calculated by two-way ANOVA with Bonferonni’s correction comparing D-Yongshi against control L-Yongshi (* < .05, ** < .01, ** < .001, * < .0001).

Article Snippet: Vero E6 (ATCC CRL-1586) and HEK293T (ATCC CRL-1573) were purchased from ATCC.

Techniques: Tube Formation Assay, Labeling, Incubation, Control, Inhibition, Infection, Virus, Focus Forming Assay, Staining

Journal: Scientific Reports

Article Title: A wild boar cathelicidin peptide derivative inhibits severe acute respiratory syndrome coronavirus-2 and its drifted variants

doi: 10.1038/s41598-023-41850-7

Figure Lengend Snippet: Yongshi retains inhibitory activity against emergent SARS-CoV-2 variants alpha, beta, gamma, kappa, and delta. Dilutions of the Yongshi peptide were tested for inhibition of SARS-CoV-2 and its drifted variants. Peptides at the labeled concentrations were pre-incubated with 100pfu of the indicated SARS-CoV-2 variant virus for 1 h at 37 °C before addition to confluent Vero hACE2 cells in a 96-well plate. Infected cells were fixed and quantified by focus forming assay after 48 h. Inhibition of viral infection was calculated based on the percent area of each well staining positively for viral spike protein compared to control wells without peptide inhibitor treatment. Results are representative of 2 to 3 independent experiments performed in triplicate. Significance calculated by two-way ANOVA with Bonferonni’s correction comparing A.1 inhibition against each variant (* < .05, ** < .01, ** < .001, * < .0001).

Article Snippet: Vero E6 (ATCC CRL-1586) and HEK293T (ATCC CRL-1573) were purchased from ATCC.

Techniques: Activity Assay, Inhibition, Labeling, Incubation, Variant Assay, Virus, Infection, Focus Forming Assay, Staining, Control

Journal: Scientific Reports

Article Title: A wild boar cathelicidin peptide derivative inhibits severe acute respiratory syndrome coronavirus-2 and its drifted variants

doi: 10.1038/s41598-023-41850-7

Figure Lengend Snippet: Yongshi remains active against the SARS-CoV-2 Omicron variant. Peptides at the labeled concentrations were pre-incubated with 100pfu of the SARS-CoV-2 XBB.1.16 variant virus for 1 h at 37 °C before addition to confluent Vero hACE2 cells in a 96-well plate. Infected cells were fixed and quantified by focus forming assay after 24 h. Inhibition of viral infection was calculated based on the percent area of each well staining positively for viral spike protein compared to control wells without peptide inhibitor treatment. Results are representative of 2 independent experiments performed in triplicate.

Article Snippet: Vero E6 (ATCC CRL-1586) and HEK293T (ATCC CRL-1573) were purchased from ATCC.

Techniques: Variant Assay, Labeling, Incubation, Virus, Infection, Focus Forming Assay, Inhibition, Staining, Control

Journal: bioRxiv

Article Title: Regulation of Interorganellar Ca 2+ Transfer and NFAT Activation by the Mitochondrial Ca 2+ Uniporter

doi: 10.1101/2021.04.07.438854

Figure Lengend Snippet: Western blot documenting MCU protein knockout in two independent CRISPR/Cas9 MCU-KO clones of HEK293 cells and quantification of MCU protein band densitometry relative to GAPDH from three independent experiments ( A ). Ca 2+ measurements in HEK293 parental cells and MCU-KO clones in response to stimulation with 2 µM thapsigargin (Tg) in Ca 2+ -free buffer followed by restoration of 2 mM extracellular Ca 2+ to determine the magnitude of SOCE ( B ). Quantification of SOCE (Δ SOCE) from at least three independent experiments are also shown ( B ). Identical experiments to ( A, B ) were performed in human Jurkat T-cells ( C, D ), mouse A20 B-cells ( E, F ), rat RBL-1 mast cells ( G, H ), human DLD1 colorectal carcinoma cells ( I, J ) and human HCT116 colorectal carcinoma cells ( K, L ).

Article Snippet: Parental HEK293, Jurkat E6-1, RBL-1, DLD1, HCT116 and A20 cell lines were purchased directly from ATCC (Catalog # CRL-1573, TIB-152, CRL-1378, CCL-221,CCL-247, and TIB-208).

Techniques: Western Blot, Knock-Out, CRISPR, Clone Assay

Journal: bioRxiv

Article Title: Regulation of Interorganellar Ca 2+ Transfer and NFAT Activation by the Mitochondrial Ca 2+ Uniporter

doi: 10.1101/2021.04.07.438854

Figure Lengend Snippet: (A) , Simultaneous measurements of mitochondrial Ca 2+ uptake and membrane potential in permeabilized cell populations of wildtype and MCU-KO HEK293 cells. ( B-D ), Similar measurements to ( A ) in RBL-1, Jurkat and A20 cells, respectively. ( E-H ), Quantification of mitochondrial Ca 2+ uptake in wildtype and MCU-KO of HEK293, RBL-1, Jurkat and A20 cells, respectively.

Article Snippet: Parental HEK293, Jurkat E6-1, RBL-1, DLD1, HCT116 and A20 cell lines were purchased directly from ATCC (Catalog # CRL-1573, TIB-152, CRL-1378, CCL-221,CCL-247, and TIB-208).

Techniques: Membrane

Journal: bioRxiv

Article Title: Regulation of Interorganellar Ca 2+ Transfer and NFAT Activation by the Mitochondrial Ca 2+ Uniporter

doi: 10.1101/2021.04.07.438854

Figure Lengend Snippet: (A) , Representative three-dimensional rendering of mitochondrial volume (red) relative to total cell volume in wildtype Jurkat, A20, RBL-1 and HEK293 cells. Cytosolic ( B ), cell ( C ) and mitochondrial ( D ) volumes, and mitochondrial/cell volume ratio ( E ) were calculated and statistically analyzed. Mitochondrial Ca 2+ extrusion was determined in HEK293 cells by comparison to Jurkat ( F ), A20 ( G ) and RBL-1 ( H ) cells in the permeabilized cell preparation. Bolus 10 µM Ca 2+ was added to allow Ca 2+ uptake, followed by addition of the MCU inhibitor RU360 (1 µM) to determine the rate of mitochondrial Ca 2+ extrusion. The NCLX inhibitor, CGP37157 (10 µM) was added at the end of the recordings. Mitochondrial Ca 2+ extrusion was calculated and statistically reported in ( I ).

Article Snippet: Parental HEK293, Jurkat E6-1, RBL-1, DLD1, HCT116 and A20 cell lines were purchased directly from ATCC (Catalog # CRL-1573, TIB-152, CRL-1378, CCL-221,CCL-247, and TIB-208).

Techniques: Comparison