Journal: Viruses

Article Title: Single MVA-SARS-2-ST/N Vaccination Rapidly Protects K18-hACE2 Mice against a Lethal SARS-CoV-2 Challenge Infection

doi: 10.3390/v16030417

Figure Lengend Snippet: Construction and in vitro characterization of MVA-N and MVA-ST/N. ( A ) Schematic diagram of the MVA genome with the major deletion sites I to VI. MVA-SARS-2-N (MVA-N): The intergenomic region 069R and 070L of the MVA genome was targeted by inserting the gene sequence encoding the N-protein of SARS-CoV-2 from the virus isolate Wuhan HU-1 under the control of the vaccina virus promoter PmH5. Repetitive sequences served to remove the GFP-marker gene by intragenomic homologous recombination (marker gene deletion) to generate MVA-N. MVA-SARS-2-ST/N (MVA-ST/N): The site of deletion III was targeted by inserting the gene sequence encoding the stabilized S-protein (ST) of SARS-CoV-2 under transcriptional control of the vaccinia virus promoter PmH5. Repetitive sequences served to remove the mCherry marker gene by intragenomic homologous recombination (marker gene deletion) to generate MVA-SARS-2-ST (MVA-ST) . The N-protein of SARS-CoV-2 was inserted into MVA-ST as described for MVA-N to generate MVA-ST/N. ( B ) Multiple-step growth analysis of recombinant MVA-N and MVA-ST/N. CEF or DF-1 cells as well as human HaCat cells were infected at a multiplicity of infection (MOI) of 0.1 with MVA, MVA-N, or MVA-ST/N and samples were collected at the indicated time points. Samples were titrated on CEF monolayers, and plaque-forming units (PFUs) were determined. Differences between the groups were analyzed, determining the area under the curve (AUC) prior to analysis by Kruskal–Wallis Test. ( C , D ) Synthesis of the SARS-2-ST and SARS-2-N protein by MVA-N and MVA-ST/N. ( C ) Western blot analysis of SARS-2-S2 and SARS-2-N in lysates of MVA-ST/N-infected cells indicating synthesis of S- and N-protein and Western blot analysis of SARS-2-N in lysates of MVA-N-infected cells indicating synthesis of N-protein. DF-1 cells were infected with an MOI of 5 with mock control, MVA, MVA-N, or MVA-ST/N and collected 0, 4, 8, 12, 24, and 48 h post infection (hpi). Polypeptides in cell lysates were detected using a monoclonal antibody against SARS-2-S2 and SARS-2-N. ( D ) Western blot analysis of SARS-2-S2 and SARS-2-N in lysates of MVA-N- and MVA-ST/N-infected cells indicating synthesis of S- and N-protein. Analysis of Hsp90 served as positive control. DF-1 cells were infected with an MOI of 5 with mock control, MVA, MVA-N, or MVA-ST/N and collected 24 h post infection (hpi). Polypeptides in cell lysates were detected using a monoclonal antibody against SARS-2-S2 and SARS-2-N. ( E ) Double immunofluorescent staining for SARS-2-N and SARS-2-S2 in MVA-N- and MVA-ST/N-infected Vero cells (MOI = 0.1; 17 hpi). Non-infected Vero cells (mock) and cells infected with non-recombinant MVA (MVA) served as controls. Permeabilized infected cells were probed with a monoclonal antibody directed against SARS-2-N or the S2-domain of SARS-CoV-2. Polyclonal goat anti-mouse antibody or polyclonal goat anti-rabbit antibody served to visualize red and green fluorescence for N-specific (green) and S-specific (red) fluorescence staining. Cell nuclei were counterstained with DAPI (blue). The yellowish color in the merge image indicates the overlapping of the red (S-protein) and green (N-protein) colors. Scale bar: 50 μm.

Article Snippet: The blots were blocked in a phosphate buffered saline buffer (PBS) containing 5% non-fat dried milk powder (PanReac AppliChem, Darmstadt, Germany) and 0.1% Tween20 (Sigma-Aldrich, Taufkirchen, Germany) and were incubated overnight with primary antibodies targeting the S2-domain of the S-protein (1A9, 1:4000; GeneTex, Irvine, CA, USA) or the N-protein (HL249, 1:4000; GeneTex, Irvine, CA, USA) of SARS-CoV-2.

Techniques: In Vitro, Sequencing, Virus, Control, Marker, Homologous Recombination, Recombinant, Infection, Western Blot, Positive Control, Staining, Fluorescence

Journal: bioRxiv

Article Title: Glycan Profiling Identifies Chondroitin-4-sulfate as a Biomarker for Platinum Response and Therapeutic Target in Ovarian Cancer

doi: 10.1101/2025.10.06.675352

Figure Lengend Snippet: ( A ) Carboplatin and Triplatin chemical structures. ( B and C ) Cytotoxicity of carboplatin and Triplatin on human OVTOKO and JHOC5 cancer cell lines; 1h treatment. ( D ) Flow cytometry of 200 nM and 50 nM rVAR2-V5 binding to ES2 wt cells, ES2 wt cells + chABC treatment, or Xylt1/Xylt2 KO cells. ( E and F ) Cytotoxicity of carboplatin and Triplatin in ES2 wt and Xylt1/Xylt2 KO cell lines; 1h treatment. ( G and H ). Platinum cellular accumulation in ES2 wt and Xylt1/Xylt2 KO cells treated with 10 µM carboplatin or Triplatin for 1, 2, 4, and 8h. Platinum content was measured by inductively coupled plasma mass spectroscopy (ICP-MS) and normalized by number of cells. ( I and J ). Platinum-DNA adducts in ES2 wt and Xylt1/Xylt2 KO cells treated with 10 µM carboplatin or Triplatin 4, and 8h. ( K and M ) ES2-luc wt and Xylt1/Xylt2 KO tumors were implanted on the left and right flanks mice. Tumors were harvested after 24h treatment with 40 mg/kg i.p Carboplatin or 0.3 mg/kg i.p. Triplatin. Tumors were digested in nitric acid and platinum measured by ICP-MS.

Article Snippet: Bound peptide was detected with mouse anti-V5 antibody (R960-25, ThermoFisher; 1:700 in casein buffer, 45 min, room temperature), followed by Leica post-primary rabbit anti-mouse antibody (8 min) and goat anti-rabbit HRP conjugate (8 min), with PBS washes between each step.

Techniques: Flow Cytometry, Binding Assay, Clinical Proteomics, Mass Spectrometry

Journal: bioRxiv

Article Title: Glycan Profiling Identifies Chondroitin-4-sulfate as a Biomarker for Platinum Response and Therapeutic Target in Ovarian Cancer

doi: 10.1101/2025.10.06.675352

Figure Lengend Snippet: Representative images of rVAR2-V5 and H&E OC PDX staining and Qupath analysis. (C) Qupath segmentation of rVAR2-V5 (+), rVAR2-V5 (-), and necrotic tumor area in OC PDX models. (D) Sensitivity of OC PDX models to Triplatin and carboplatin. OC PDX models were treated i.p. with carboplatin (40 mg/kg) or Triplatin (0.3 mg/kg) on days 0, 4 and 8 (orange arrows). * * p<0.01, * * * * p<0.0001, 2-way ANOVA, Tukey

Article Snippet: Bound peptide was detected with mouse anti-V5 antibody (R960-25, ThermoFisher; 1:700 in casein buffer, 45 min, room temperature), followed by Leica post-primary rabbit anti-mouse antibody (8 min) and goat anti-rabbit HRP conjugate (8 min), with PBS washes between each step.

Techniques: Staining

Journal: bioRxiv

Article Title: Glycan Profiling Identifies Chondroitin-4-sulfate as a Biomarker for Platinum Response and Therapeutic Target in Ovarian Cancer

doi: 10.1101/2025.10.06.675352

Figure Lengend Snippet: (A) UVA CHTN OC TMA; Representative samples of patient OC subtypes (clinical history unknown) and (B) normal tissues stained with rVAR2-V5 protein. (C) UVA CHTN OC TMA; Percentage of sample area staining positively for rVAR2-V5. Values are representative of the mean of 4 cores per patient sample. (D) UVA CHTN OC TMA; Percentage of TMA samples above the cut-off score. (E) UPenn CCC TMA samples; Percentage of sample area staining positively for rVAR2-V5.

Article Snippet: Bound peptide was detected with mouse anti-V5 antibody (R960-25, ThermoFisher; 1:700 in casein buffer, 45 min, room temperature), followed by Leica post-primary rabbit anti-mouse antibody (8 min) and goat anti-rabbit HRP conjugate (8 min), with PBS washes between each step.

Techniques: Staining

Journal: medRxiv

Article Title: Systems biological assessment of the temporal dynamics of immunity to a viral infection in the first weeks and months of life

doi: 10.1101/2023.01.28.23285133

Figure Lengend Snippet: Memory B and T cell response, related to A) Gating strategy used for SARS-CoV-2 spike specific IgG+ memory B cell staining and single-cell sorting. Gating was on singlets that were CD20+ and CD3-CD14-IgM-IgD-CD27low/+ IgG+. Sorted cells were Wuhan spike-AlexaFluor 488+ and/or Omicron spike-BV421+. B) The percentage of SARS-CoV-2 spike-specific IgG+ memory B cells in healthy, acute, and convalescent infant individuals. The sample number for each group is indicated in brackets. C) As in (B), the percentage of SARS-CoV-2 spike-specific IgG+ memory B cells in adult individuals with mild, severe, and ICU symptoms and in adult convalescent individuals. The sample number for each group is indicated in brackets. D-F) T cells were stimulated with overlapping peptides against WT (D-F) and Omicron (F) variants. Cytokine production was determined via flow cytometry. D) Box plot showing the fraction of responding T cells at different infection stages. E) Box plot showing the fraction of multifunctional T cells at different infection stages. F) Comparison of the multifunctional T cell response after stimulation with WT and Omicron (Om) peptides. Statistical comparisons were conducted with Wilcoxon rank sum test. Solid line indicates median healthy response; dashed line indicates 3x median healthy response.

Article Snippet: The following antibodies were used: IgD PE (BD Biosciences, 555779), IgM PerCP-Cy5.5 (BioLegend, 314512), CD20 APC-H7 (BD Biosciences, 560734), CD27 PE-Cy7 (BioLegend, 302838), CD14 PE/Dazzle™ 594 (BioLegend, 301852), CD16 BV605 (BioLegend, 302040), IgG BV650 (BD Biosciences, 740596), CD3 BUV737 (BD Biosciences, 612750) and Alexa Fluor 488-labeled Wuhan spike (SinoBiological, 40589-V27B-B), and BV421 labeled Omicron Spike (SinoBiological™, 40589-V49H3-B).

Techniques: Staining, FACS, Flow Cytometry, Infection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Rapamycin inhibits transforming growth factor β-induced peritoneal angiogenesis by blocking the secondary hypoxic response

doi: 10.1111/j.1582-4934.2011.01493.x

Figure Lengend Snippet: (A–C) Sections of mouse peritoneal tissue stained for pimonidazole 10 days after injection ( N = 3/group). (A) Mouse treated with AdDL showed no submesothelial thickening and no hypoxic response. (B) Peritoneum of AdTGFβ1-treated mouse revealed increased hypoxia in the submesothelium. (C) Control section from a mouse receiving AdTGFβ1 but no pimonidazole. All sections were taken at 200× magnification. (D) HIF1α gene expression was increased by AdTGFβ1 10 days after infection. (E) HIF1α protein is up-regulated in peritoneal tissue from AdTGFβ1-treated animals at 21 days after adenoviral infection. HIF1α was significantly down-regulated by rapamycin treatment at both 10 and 21 days after adenovirus infection. (F) Representative blot is shown ( N = 4 animals/group for HIF1α analysis).

Article Snippet: One hour later, anterior abdominal wall tissue was taken and stained for pimonidazole using a mAb to pimonidazole (HPI, Inc.) and a secondary Fab antimouse antibody.

Techniques: Staining, Injection, Control, Gene Expression, Infection