fluorescence based usp30 enzyme activity assay recombinant human usp30  (R&D Systems)

Journal: Infection and Immunity

Article Title: Elevated vaginal heparan sulfate correlates with impaired neutrophil killing of Candida albicans in women with vulvovaginal candidiasis

doi: 10.1128/iai.00709-25

Figure Lengend Snippet: PMN antifungal activity in VCM spiked with HS or treated with HPSEs. Vaginal swab samples were collected from three groups: women with RVVC experiencing a symptomatic acute episode of VVC (symptomatic group, n = 31), those in asymptomatic remission (asymptomatic group, n = 14), and healthy women without a history of frequent VVC (control group, n = 16). The data sets included an additional five follow-up swabs collected from women in the symptomatic and asymptomatic groups. Detached swab tips were suspended individually in 1 mL RPMI 1640 medium to release vaginal secretions and cellular content. Supernatants were sterile-filtered as VCM. PMNs (5 × 10 5 ) isolated from peripheral blood of healthy volunteers were incubated with C. albicans cells (1 × 10 5 ) in 100 µL of VCM or RPMI medium for 3 h at 37°C with 5% CO 2 . Controls consisted of C. albicans cultured alone in VCM or RPMI medium and were used to calculate % killing. ( A ) VCM samples with normal killing activity (non-inhibitory VCM, gray bar) were spiked with purified porcine HS (500 µg/mL) and reevaluated for PMN killing activity (blue bar). Spiked non-inhibitory VCM was then pretreated with r Ph HPSE (3 ng/mL) and reevaluated for PMN killing activity (red bar). ( B ) VCM samples with low killing activity (inhibitory VCM, solid blue bar) were pretreated with rhHPSE (4 µg/mL, red-dotted bar) or r Ph HPSE (3 ng/mL, red-striped bar) and reevaluated for PMN killing activity. After coculture, viable C. albicans cells were enumerated by quantitative plate counts. Data were analyzed using the one-way ANOVA and Bonferroni’s post-test. Bar heights and error bars represent group means ± SEM for % killing values across independent replicates of unique VCM samples. The graphs represent cumulative data from three to four experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Article Snippet: To degrade HS, symptomatic (inhibitory) VCM was pretreated with either recombinant human HPSE (rhHPSE, 4 μg/mL, R&D Systems) or recombinant Pedobacter heparinus heparinase III (r Ph HPSE, 3 ng/mL, R&D Systems) for 1 h at 37°C.

Techniques: Activity Assay, Control, Sterility, Isolation, Incubation, Cell Culture, Purification