Antifungal resistance due to upregulation of efflux pumps is prevalent in clinical Candida isolates. Potential efflux pump substrates (PEPSs), which are active against strains deficient in efflux pumps but inactive against wild-type strains, are usually missed in routine antifungal screening. Here we present a method for identification of PEPSs, and show that conjugation with mitochondria-targeting triphenylphosphonium cation (TPP⁺) can enhance or restore the compounds’ antifungal activity. The screening method involves co-culturing a wild-type C. albicans strain and a Cdr efflux pump-deficient strain, labelled with different fluorescent proteins. We identify several PEPSs from a library of natural terpenes, and restore their antifungal activity against wild-type and azole-resistant C. albicans by conjugation with TPP⁺. The most active conjugate (IS-2-Pi-TPP) kills C. albicans cells, prevents biofilm formation and eliminates preformed biofilms, without inducing significant resistance. The antifungal activity is accompanied by mitochondrial dysfunction and increased levels of intracellular reactive oxygen species. In addition, IS-2-Pi-TPP is effective against C. albicans in a mouse model of skin infection.

In the fields of medicine and health, traditional high-performance liquid chromatography or UV-visible spectrophotometry is generally used for substance quantification. However, over time, nuclear magnetic resonance spectroscopy (NMR) has gradually become more mature. Nuclear magnetic resonance spectroscopy has certain advantages in the quantitative analysis of substances, such as being nondestructive, having a high flux and short analysis time. Nuclear magnetic resonance spectroscopy has been included in the pharmacopoeiae of various countries. In this paper, the principle of nuclear magnetic resonance spectroscopy and the recent progress in the quantitative study of natural products by NMR are reviewed, and its application in the quantitative study of natural products is proposed. At the same time, the problems of using NMR alone to quantify natural products are summarized and corresponding suggestions are put forward.

The roles of nuclear receptor subfamily 1 group d member 1 (NR1D1) and the circadian clock in liver fibrosis remain unclear. Here, we showed that liver clock genes, especially NR1D1, were dysregulated in mice with carbon tetrachloride (CCl₄)-induced liver fibrosis. In turn, disruption of the circadian clock exacerbated experimental liver fibrosis. NR1D1-deficient mice were more sensitive to CCl₄-induced liver fibrosis, supporting a critical role of NR1D1 in liver fibrosis development. Validation at the tissue and cellular levels showed that NR1D1 was primarily degraded by N6-methyladenosine (m⁶A) methylation in a CCl₄-induced liver fibrosis model, and this result was also validated in rhythm-disordered mouse models. In addition, the degradation of NR1D1 further inhibited the phosphorylation of dynein-related protein 1-serine site 616 (DRP1^S616), resulting in weakened mitochondrial fission function and increased mitochondrial DNA (mtDNA) release in hepatic stellate cell (HSC), which in turn activated the cGMP-AMP synthase (cGAS) pathway. Activation of the cGAS pathway induced a local inflammatory microenvironment that further stimulated liver fibrosis progression. Interestingly, in the NR1D1 overexpression model, we observed that DRP1^S616 phosphorylation was restored, and cGAS pathway was also inhibited in HSCs, resulting in improved liver fibrosis. Taken together, our results suggest that targeting NR1D1 may be an effective approach to liver fibrosis prevention and management.