Journal: Microbiology Spectrum

Article Title: Condition-dependent effects of Elexacaftor/Tezacaftor/Ivacaftor (Trikafta) on Aspergillus fumigatus growth

doi: 10.1128/spectrum.02275-24

Figure Lengend Snippet: High concentration of CFTR modulators (Elexacaftor [VX445], Tezacaftor [VX661], and Ivacaftor [VX770]) impairs macrophage control of A. fumigatus growth. ( A ) Measurement of A. fumigatus fungal length (Strain: DAL-DSred) infecting bone marrow-derived macrophages (BMDM) at a multiplicity of infection (MOI) of 5, using the IncuCyte live-cell analysis system in the presence of 3VX (VX445/VX661/VX770) treatment at [3VX] S , [3VX] E , or [3VX] 10μM , as well as with [Veh]. * P < 0.05, ** P < 0.01 (two-way ANOVA with Dunnett’s multiple comparisons test); data are presented as mean ± SEM, n = 3. ( B ) Release of TNF⍺ in BMDMs was assessed after 20 h with A. fumigatus (MOI of 15). ** P < 0.01 (one-way ANOVA with Dunnett’s multiple comparisons test); data are presented as mean ± SEM, n = 4. ( C ) Normalized phagocytosis to vehicle control ([Veh]) at 20 h time point. **P < 0.01 , ****P < 0.005 (one sample t test, theoretical mean of 1); data are presented as mean ± SEM, n = 4. ( D ) Representative measurement of zymosan phagocytosis by primed BMDMs in the presence of 3VX treatment at concentrations [3VX] S , [3VX] E or [3VX] 10μM , as well as in the presence of cytochalasine D or [Veh]. ( E ) Quantification of viable conidia following 4 h of BMDM infection in the presence of 3VX treatment at [3VX] S , [3VX] E , or [3VX] 10μM , as well as with [Veh]. ( F ) Immunoblot analysis of pro-caspase-1 ( P45 ), the caspase-1 subunit p20 ( P20 ), and β-actin of primed BMDMs left untreated (medium alone [Med]) or in the presence of 3VX treatment at [3VX] S , [3VX] E , or [3VX] 10μM , as well as with [Veh] during 20 h after infection with A. fumigatus (MOI of 15). ( G ) Release of IL-1β in BMDMs assessed after 20 h infection with A. fumigatus (MOI of 15) left untreated (medium alone [Med]) or in the presence of 3VX treatment at [3VX] S , [3VX] E , or [3VX] 10μM , as well as with [Veh]. *P < 0.05 , **P < 0.01 (one-way ANOVA with Dunnett’s multiple comparisons test); data are presented as mean ± SEM, n = 3.

Article Snippet: Cytokine levels were determined by ELISA using kits according to the manufacturer’s instructions: TNF⍺ DuoSet ELISA (DY410, R&D Systems) and IL-1β (DY401, R&D Systems).

Techniques: Concentration Assay, Control, Derivative Assay, Infection, Cell Analysis, Western Blot

Journal: bioRxiv

Article Title: Metformin inhibits PDGF signaling to suppress hyaluronan and IL-6 production in Thyroid Eye Disease

doi: 10.1101/2025.05.19.654298

Figure Lengend Snippet: A) non-TED (n=3) and TED (n=3) OFs were treated with or without PDGFβ for 72 hours. Secreted HA levels in media supernatant collected after treatment were analyzed on agarose gel electrophoresis and stained using Stains-All. 15 MDa and HiLadder were used as a size standard. B) Quantitative HA levels in both non-TED and TED after treatment were measured as described in the Methods section. C) and D) The relative production of IL6 and IL8 by OFs after PDGFβ treatment for 72 hours was measured with ELISA assay. The experiments were conducted in triplicate. The data represent mean ± SEM and were analyzed statistically using One-way ANOVA with Tukey’s multiple comparisons. Vehicle versus PDGFβ: * P ≤ 0.05, ** P = 0.001, *** P < 0.0008, **** P < 0.0001, and non-TED versus TED: #### P ≤ 0.0001. HA, hyaluronan.

Article Snippet: The Hyaluronan DuoSet ELISA kit was used for HA quantification (R&D Systems, Minneapolis, MN).

Techniques: Agarose Gel Electrophoresis, Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Metformin inhibits PDGF signaling to suppress hyaluronan and IL-6 production in Thyroid Eye Disease

doi: 10.1101/2025.05.19.654298

Figure Lengend Snippet: A) TED OFs were treated with different doses of metformin as indicated for 72 hours; thereafter, cells were analyzed by Western blot for pAMPKɑ Thr172 , total AMPKɑ, and β-tubulin (loading control). B) Relative levels of pAMPKɑ Thr172 normalized to total AMPKɑ in three strains with representative results. Band intensities were quantified using Image Lab software. C) and D) The relative production of IL6 and IL8 by OFs after metformin treatment for 72 hours was measured by ELISA. The experiments were conducted in triplicate on three different strains. The data represent mean ± SEM. One-way ANOVA with Dunnett’s multiple comparisons was used to compare vehicle versus metformin treatment. *p≤ 0.05, **p≤ 0.001.

Article Snippet: The Hyaluronan DuoSet ELISA kit was used for HA quantification (R&D Systems, Minneapolis, MN).

Techniques: Western Blot, Control, Software, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Metformin inhibits PDGF signaling to suppress hyaluronan and IL-6 production in Thyroid Eye Disease

doi: 10.1101/2025.05.19.654298

Figure Lengend Snippet: A) TED OFs were pretreated with AMPK activators metformin (1000 μM) or AICAR (400 μM) for 1 hour and then added PDGFβ (25 ng/mL) for the next 72 hours; thereafter, cells were analyzed by western blots for pAMPKɑ Thr172 , total AMPKɑ and β-tubulin as a loading control. B) Relative levels of pAMPKɑ Thr172 normalized to total AMPKɑ in three strains with representative results. Band intensities were quantified using Image Lab software. C) Quantitative HA levels in TED OFs after treatment with PDGFβ and AMPK activators, metformin/AICAR, were measured with the HA ELISA assay. D) and E) The relative production of IL6 and IL8 by OFs after treatment with PDGFβ and AMPK activators, metformin/AICAR, was measured by ELISA. The experiments were conducted in triplicate on three different strains. The data represent mean ± SEM. Student’s T-test and One-way ANOVA with Tukey’s multiple comparisons were used to compare PDGFβ and PDGFβ + metformin/AICAR. *P ≤ 0.05, **P ≤ 0.001, ***P < 0.0009, ****P < 0.0001, and vehicle versus metformin/AICAR treatment. # P ≤ 0.05, ### P ≤ 0.0005.

Article Snippet: The Hyaluronan DuoSet ELISA kit was used for HA quantification (R&D Systems, Minneapolis, MN).

Techniques: Western Blot, Control, Software, Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: Diminished cartilage-lubricating ability of human osteoarthritic synovial fluid deficient in proteoglycan 4: Restoration through proteoglycan 4 supplementation.

doi: 10.1002/art.34674

Figure Lengend Snippet: Figure 1. Characterization of the proteoglycan 4 (PRG4) enzyme- linked immunosorbent assay control by protein staining (A) and characterization of high molecular weight PRG4 immunoreactivity in PRG4 control, normal (NL) human synovial fluid (hSF), and osteo- arthritic (OA) SF samples by Western blotting using antipeptide antibody LPN (capture) (B) and horseradish peroxidase (HRP)– conjugated peanut agglutinin (PNA) (detection) (C). Samples were subjected to 3–8% sodium dodecyl sulfate–polyacrylamide gel electro- phoresis, followed by protein staining or Western blotting as described in Materials and Methods. PRG4 controls treated with neuraminidase and SF treated with hyaluronidase and neuraminidase were probed with LPN and with HRP–conjugated PNA.

Article Snippet: A hyaluronan DuoSet ELISA development kit was obtained from R&D Systems, proteinase K was from Roche Applied Science, and MegaLadder and HiLadder HA molecular weight markers were from Hyalose.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Staining, High Molecular Weight, Western Blot

Journal: Arthritis and rheumatism

Article Title: Diminished cartilage-lubricating ability of human osteoarthritic synovial fluid deficient in proteoglycan 4: Restoration through proteoglycan 4 supplementation.

doi: 10.1002/art.34674

Figure Lengend Snippet: Figure 3. Characterization of hyaluronan (HA) in normal and PRG4- deficient OA SF samples. A, Concentrations of HA in normal and PRG4-deficient OA SF samples. B, Molecular weight distribution of HA in normal SF samples (n 8) and in PRG4-deficient OA (OA-LO) SF samples (n 5). Values are the mean SEM. P 0.05. See Figure 1 for other definitions.

Article Snippet: A hyaluronan DuoSet ELISA development kit was obtained from R&D Systems, proteinase K was from Roche Applied Science, and MegaLadder and HiLadder HA molecular weight markers were from Hyalose.

Techniques: Molecular Weight

Journal: Arthritis and rheumatism

Article Title: Diminished cartilage-lubricating ability of human osteoarthritic synovial fluid deficient in proteoglycan 4: Restoration through proteoglycan 4 supplementation.

doi: 10.1002/art.34674

Figure Lengend Snippet: Figure 4. Effect of hyaluronan (HA) and proteoglycan 4 (PRG4) supplementation on the cartilage boundary–lubricating ability of PRG4-deficient osteoarthritic (OA) synovial fluid (SF) samples, as determined by cartilage-on-cartilage friction testing. Two friction coefficients (), static (static, Neq) (A) and kinetic (kinetic, Neq ; at a presliding duration of 1.2 seconds) (B), in phosphate buffered saline (PBS; negative control lubricant), PRG4-deficient OA (OA-LO) SF alone, PRG4-deficient OA SF plus PRG4, PRG4-deficient OA SF plus PRG4 and HA, and normal SF (NL; positive control lubricant) were calculated. Values are the mean SEM. P 0.05. Neq represents equilibrium axial load; angle brackets indicate that the value is an average.

Article Snippet: A hyaluronan DuoSet ELISA development kit was obtained from R&D Systems, proteinase K was from Roche Applied Science, and MegaLadder and HiLadder HA molecular weight markers were from Hyalose.

Techniques: Saline, Negative Control, Positive Control

Journal: Research Square

Article Title: Immune responses in COVID-19 respiratory tract and blood reveal mechanisms of disease severity

doi: 10.21203/rs.3.rs-802084/v1

Figure Lengend Snippet: Higher anti-RBD IgM seroconversion rate in respiratory samples compared to paired blood samples of COVID-19 patients. a ELISA titration curves against the SARS-CoV-2 receptor-binding domain (RBD) for IgM, IgG, and IgA in COVID-19 respiratory and paired blood samples and non-COVID-19 respiratory samples. Dotted lines within each graph indicates the cut-off used to determine end-point titres. b Endpoint titres of SARS-CoV-2 RBD antibodies between (i) respiratory samples of COVID-19 and non-COVID-19 patients, and (ii) plasma and respiratory samples of COVID-19 patients. (i) Bars indicate median with interquartile range. Dotted line indicates the detection level. (ii) Dotted lines connect the most closely matched plasma and respiratory samples from each patient. Statistical significance was determined with Mann-Whitney test. c ELISA titration curves against the SARS-CoV-2 RBD for 3 COVID-19 patients with serial respiratory samples. d Heatmap of percentage (%) inhibition tested by surrogate virus neutralization test (sVNT) and anti-RBD ELISA titres. e Correlation between anti-RBD antibody titres and (%) sVNT inhibition. Correlation was determined with Spearman’s correlation. f Number of (i) samples and (ii) patients with seroconverted anti-RBD IgM, IgG, IgA and positive % sVNT inhibition. Pink curved lines surrounding the donut graphs indicate the samples/patients with seroconverted IgM. Earliest samples were used for each patient when determining seroconversion which was defined as average titre +2xSD of non-COVID-19 samples. Positive % sVNT inhibition was defined as % sVNT inhibition ≥ 20%.

Article Snippet: DuoSet ELISA ancillary reagent kit (R&D Systems) was used for respiratory fluids and in-house reagents with the same composition were used for plasma samples.

Techniques: Enzyme-linked Immunosorbent Assay, Titration, Binding Assay, Clinical Proteomics, MANN-WHITNEY, Inhibition, Virus, Neutralization