Highly conserved gsc1 gene of Pneumocystis jirovecii in patients with or without prior exposure to Echinocandins

Echinocandins are noncompetitive inhibitors of the GSC1 subunit of the enzymatic complex involved in synthesis of 1,3-beta-D-glucan, a cell wall component of most fungi, including Pneumocystis spp. Echinocandins are widely used for treating systemic candidiasis and rarely used for treating Pneumocystis pneumonia. Consequently, data on P. jirovecii gsc1 gene diversity are still scarce, compared to the homologous fks1 gene of Candida spp. In this study, we analyzed P. jirovecii gsc1 gene diversity and the putative selection pressure of echinocandins on P. jirovecii. Gsc1 gene sequences of P. jirovecii specimens from two patient groups were compared. One group of 27 patients had prior exposure to echinocandins whereas the second group of 24 patients did not, at the time of P. jirovecii infection diagnoses. Two portions of P. jirovecii gsc1 gene, HS1 and HS2, homologous to hot spots described in Candida spp., were sequenced. Three SNPs at positions 2204, 2243, and 2303 close to the HS1 region and another SNP at position 4540 more distant from the HS2 region were identified. These SNPs represent synonymous mutations. Three gsc1 HS1 alleles, A, B, and C, and two gsc1 HS2 alleles, a and b, and four haplotypes, Ca, Cb, Aa, and Ba, were defined, without significant difference in haplotype distribution in both patient groups ( p = 0.57). Considering the identical diversity of P. jirovecii gsc1 gene and the detection of synonymous mutations in both patient groups, no selection pressure of echinocandins among P. jirovecii microorganisms can be pointed out so far.

Iridal-Type Triterpenoids Displaying Human Neutrophil Elastase Inhibition and Anti-Inflammatory Effects from Belamcanda chinensis

The aim of this study is to explore anti-inflammatory phytochemicals from B. chinensis based on the inhibition of pro-inflammatory enzyme, human neutrophil elastase (HNE) and anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage. Three stereoisomers of iridal-type triterpenoids (1–3) were isolated from the roots of B. chinensis and their stereochemistries were completely identified by NOESY spectra. These compounds were confirmed as reversible noncompetitive inhibitors against HNE with IC50 values of 6.8–27.0 µM. The binding affinity experiment proved that iridal-type triterpenoids had only a single binding site to the HNE enzyme. Among them, isoiridogermanal (1) and iridobelamal A (2) displayed significant anti-inflammatory effects by suppressing the expressions of pro-inflammatory cytokines, such as iNOS, IL-1β, and TNF-α through the NF-κB pathway in LPS-stimulated RAW264.7 cells. This is the first report that iridal-type triterpenoids are considered responsible phytochemicals for anti-inflammatory effects of B. chinensis.

Beta-Glucuronidase Inhibition by Constituents of Mulberry Bark

Intestinal bacterial β-glucuronidases, the key enzymes responsible for the hydrolysis of various glucuronides into free aglycone, have been recognized as key targets for treating various intestinal diseases. This study aimed to investigate the inhibitory effects and mechanisms of the Mulberry bark constituents on E. coli β-glucuronidase (EcGUS), the most abundant β-glucuronidases produced by intestinal bacteria. The results showed that the flavonoids isolated from Mulberry bark could strongly inhibit E. coli β-glucuronidase, with IC50 values ranging from 1.12 µM to 10.63 µM, which were more potent than D-glucaric acid-1,4-lactone. Furthermore, the mode of inhibition of 5 flavonoids with strong E. coli β-glucuronidase inhibitory activity (IC50 ≤ 5 µM) was carefully investigated by a set of kinetic assays and in silico analyses. The results demonstrated that these flavonoids were noncompetitive inhibitors against E. coli β-glucuronidase-catalyzed 4-nitrophenyl β-D-glucuronide hydrolysis, with Ki values of 0.97 µM, 2.71 µM, 3.74 µM, 3.35 µM, and 4.03 µM for morin (1), sanggenon C (2), kuwanon G (3), sanggenol A (4), and kuwanon C (5), respectively. Additionally, molecular docking simulations showed that all identified flavonoid-type E. coli β-glucuronidase inhibitors could be well-docked into E. coli β-glucuronidase at nonsubstrate binding sites, which were highly consistent with these agentsʼ noncompetitive inhibition mode. Collectively, our findings demonstrated that the flavonoids in Mulberry bark displayed strong E. coli β-glucuronidase inhibition activity, suggesting that Mulberry bark might be a promising dietary supplement for ameliorating β-glucuronidase-mediated intestinal toxicity.