Dehydroepiandrosterone

The following substrates were purchased from Sigma-Aldrich: dehydroepiandrosterone (DHEA), androstenediol (androst-5-ene-3β,17β-diol), androstenedione (androst-4-ene-3,17-dione, AD), adrenosterone (androst-4-ene-3,11,17-trione, ADR), progesterone (P), 17α-methyltestosterone (17-mT) (Fig. 1.). 17β-Hydroxyandrost-1,4,6-triene-3-one (1,4,6-triene-T) was taken from the resources of the Department of Chemistry, Wrocław University of Environmental and Life Sciences.Fig. 1

Structures of substates

Microorganisms: Twelve Isaria farinosa strains named KCh J1.1, KCh J1.2, KCh J1.3, KCh J1.4, KCh J1.6, KCh J2.2, KCh J2.3, KCh J2.4, KCh J2.6, KCh KW1.1, KCh KW1.2 and KCh RJ1.1 were obtained from the collection of the Department of Chemistry, Wrocław University of Environmental and Life Sciences, Wrocław, Poland. The strains were maintained on Sabouraud 4% dextrose-agar slopes and freshly subcultured before being used in the transformation experiments.

Progesterone (PROG), dehydroepiandrosterone (DHEA), testosterone (TS), aldosterone (ALDO), cortisol (COR) and corticosterone (COS) were all purchased from Sigma-Aldrich, Glostrup, Denmark, with a purity > 96%. Deuterated analogues were applied as internal standards (IS); d8-corticosterone (COSd8) was obtained from CDN isotopes (Pointe-Claire, QC, Canada), while d9-Progesterone (PROGd9) d3-testosterone (TSd3), and d4-cortisol (CORd4) were purchased from TRC, all with a deuterated purity above 98%. d6-dehydroepiandrosterone (DHEAd6) was obtained from Sigma-Aldrich with a deuterated purity > 98%. All utilized solvents were of analytical grade. Methanol was obtained from Fisher Scientific (Leics, UK), acetone and n-heptane were obtained from Fisher Scientific (Slangerup, Denmark). Formic acid 98–100% was purchased from Merck (Merck KGaA, Darmstadt, Germany). All H₂O used was ultrapure water produced by a Milli-Q system (Millipak 40).

The effect of HI0628 or HI0629 mutation or the addition of 50 μM of various steroids [beclomethasone dipropionate, prednisone, mometasone, dehydroepiandrosterone (Sigma-Aldrich)] to H. influenzae strains (including clinical isolates) on expression of genes was monitored by qRT–PCR. RNA was isolated from cultures of wild-type strain, wild-type supplemented with 50 μM steroid or mutant grown to an OD600 of 0.7, converted to cDNA using AMV Reverse Transcriptase (Promega) using conditions specified by the manufacturer. The quantitative PCR was carried out using Platinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen). The primers used to amplify the genes are listed in Supplementary Table S2. As a control, RT–PCR was similarly applied to analyze expression of the 16S rRNA gene. The reverse transcription and PCR protocols were performed as described previously. The amplified products were subjected to electrophoresis on 2.5% agarose gels and stained with ethidium bromide. RT–PCR analysis was repeated at least twice for each gene tested.

All the animal experiments were approved by Shanghai East Hospital and were carried out in accordance with the regulations set by Shanghai East Hospital. Ethical clearance was obtained from ethics committee of Shanghai East Hospital. Female SD rats (JRDUN Biotechnology, Shanghai, China) aged 3 weeks and weighing 50–55 g was maintained in a pathogen-free environment at 22°C with 33% humidity in 12 h light/12 h dark cycles. To establish the PCOS model, DHEA (Dehydroepiandrosterone, Sigma, Shanghai, China) (60 mg/kg BW s.c.) was injected daily for 21 days (Kim et al., 2018 (link); Wen et al., 2020 (link)). PBS was substituted for DHEA in the control group.

Dehydroepiandrosterone (Sigma Chemicals; St Louis, MO) was dissolved in 0.3 ml of dimethylsulfoxide (DMSO) and then diluted to 1 mg/ml of saline (final DMSO concentration was 1%). Rats were injected i.p. with DHEA (2 mg/kg) or vehicle (saline containing 1% of DMSO) 90 min prior to exposure to the operant chamber. Based on previous research showing that cocaine addicts that had a twofold increase in plasma DHEA-S levels compared to other addicts were less prone to relapse and based on prior evaluation of the effect of DHEA injections on DHEA-S plasma levels in rats, we found that the optimal DHEA dose to achieve a twofold increase in DHEA-S plasma levels was 2 mg/kg in cocaine self-administrating rats (Everitt et al., 2001 (link)).