Heat stress-induced NO enhanced perylenequinone biosynthesis of Shiraia sp. via calcium signaling pathway.

Perylenequinones (PQs) are natural photosensitizing compounds used as photodynamic therapy, and heat stress (HS) is the main limiting factor of mycelial growth and secondary metabolism of fungi. This study aimed to unravel the impact of HS-induced Ca2+ and the calcium signaling pathway on PQ biosynthesis of Shiraia sp. Slf14(w). Meanwhile, the intricate interplay between HS-induced NO and Ca2+ and the calcium signaling pathway was investigated. The outcomes disclosed that Ca2+ and the calcium signaling pathway activated by HS could effectively enhance the production of PQs in Shiraia sp. Slf14(w). Further investigations elucidated the specific mechanism through which NO signaling molecules induced by HS act upon the Ca2+/CaM (calmodulin) signaling pathway, thus propelling PQ biosynthesis in Shiraia sp. Slf14(w). This was substantiated by decoding the downstream positioning of the CaM/CaN (calcineurin) pathway in relation to NO through comprehensive analyses encompassing transcript levels, enzyme assays, and the introduction of chemical agents. Concurrently, the engagement of Ca2+ and the calcium signaling pathway in heat shock signaling was also evidenced. The implications of our study underscore the pivotal role of HS-induced Ca2+ and the calcium signaling pathway, which not only participate in heat shock signal transduction but also play an instrumental role in promoting PQ biosynthesis. Consequently, our study not only enriches our comprehension of the mechanisms driving HS signaling transduction in fungi but also offers novel insights into the PQ synthesis paradigm within Shiraia sp. Slf14(w). KEY POINTS: • The calcium signaling pathway was proposed to participate in PQ biosynthesis under HS. • HS-induced NO was revealed to act upon the calcium signaling pathway for the first time.

Heat stress enhanced perylenequinones biosynthesis of Shiraia sp. Slf14(w) through nitric oxide formation.

Perylenequinones (PQs) are a class of natural polyketides used as photodynamic therapeutics. Heat stress (HS) is an important environmental factor affecting secondary metabolism of fungi. This study investigated the effects of HS treatment on PQs biosynthesis of Shiraia sp. Slf14(w) and the underlying molecular mechanism. After the optimization of HS treatment conditions, the total PQs amount reached 577 ± 34.56 mg/L, which was 20.89-fold improvement over the control. Also, HS treatment stimulated the formation of intracellular nitric oxide (NO). Genome-wide analysis of Shiraia sp. Slf14(w) revealed iNOSL and cNOSL encoding inducible and constitutive NOS-like proteins (iNOSL and cNOSL), respectively. Cloned iNOSL in Escherichia coli BL21 showed higher nitric oxide synthase (NOS) activity than cNOSL, and the expression level of iNOSL under HS treatment was observably higher than that of cNOSL, suggesting that iNOSL is more responsible for NO production in the HS-treated strain Slf14(w) and may play an important role in regulating PQs biosynthesis. Moreover, the putative biosynthetic gene clusters for PQs and genes encoding iNOSL and nitrate reductase (NR) in the HS-treated strain Slf14(w) were obviously upregulated. PQs biosynthesis and efflux stimulated by HS treatment were significantly inhibited upon the addition of NO scavenger, NOS inhibitor, and NR inhibitor, indicating that HS-induced NO, as a signaling molecule, triggered promoted PQs biosynthesis and efflux. Our results provide an effective strategy for PQs production and contribute to the understanding of heat shock signal transduction studies of other fungi.Key points• PQs titer of Shiraia sp. Slf14(w) was significantly enhanced by HS treatment.• HS-induced NO was first reported to participate in PQs biosynthetic regulation.• Novel inducible and constitutive NOS-like proteins (iNOSL and cNOSL) were obtained and their NOS activities were determined.

L-Arginine enhanced perylenequinone production in the endophytic fungus Shiraia sp. Slf14(w) via NO signaling pathway.

Perylenequinones (PQ) are natural polyketides used as anti-microbial, -cancers, and -viral photodynamic therapy agents. Herein, the effects of L-arginine (Arg) on PQ biosynthesis of Shiraia sp. Slf14(w) and the underlying molecular mechanism were investigated. The total content of PQ reached 817.64 ± 72.53 mg/L under optimal conditions of Arg addition, indicating a 30.52-fold improvement over controls. Comparative transcriptome analysis demonstrated that Arg supplement promoted PQ precursors biosynthesis of Slf14(w) by upregulating the expression of critical genes associated with the glycolysis pathway, and acetyl-CoA and malonyl-CoA synthesis. By downregulating the expression of genes related to the glyoxylate cycle pathway and succinate dehydrogenase, more acetyl-CoA flow into the formation of PQ. Arg supplement upregulated the putative biosynthetic gene clusters for PQ and activated the transporter proteins (MFS and ABC) for exudation of PQ. Further studies showed that Arg increased the gene transcription levels of nitric oxide synthase (NOS) and nitrate reductase (NR), and activated NOS and NR, thus promoting the formation of nitric oxide (NO). A supplement of NO donor sodium nitroprusside (SNP) also confirmed that NO triggered promoted biosynthesis and efflux of PQ. PQ production stimulated by Arg or/and SNP can be significantly inhibited upon the addition of NO scavenger carboxy-PTIO, NOS inhibitor Nω-nitro-L-arginine, or soluble guanylate cyclase inhibitor NS-2028. These results showed that Arg-derived NO, as a signaling molecule, is involved in the biosynthesis and regulation of PQ in Slf14(W) through the NO-cGMP-PKG signaling pathway. Our results provide a valuable strategy for large-scale PQ production and contribute to further understanding of NO signaling in the fungal metabolite biosynthesis. KEY POINTS: • PQ production of Shiraia sp. Slf14(w) was significantly improved by L-arginine addition. • Arginine-derived NO was firstly reported to be involved in the biosynthesis and regulation of PQ. • The NO-cGMP-PKG signaling pathway was proposed for the first time to participate in PQ biosynthesis.

Chemotherapy response and recurrence-free survival in neoadjuvant breast cancer depends on biomarker profiles: results from the I-SPY 1 TRIAL (CALGB 150007/150012; ACRIN 6657).

Neoadjuvant chemotherapy for breast cancer allows individual tumor response to be assessed depending on molecular subtype, and to judge the impact of response to therapy on recurrence-free survival (RFS). The multicenter I-SPY 1 TRIAL evaluated patients with ≥ 3 cm tumors by using early imaging and molecular signatures, with outcomes of pathologic complete response (pCR) and RFS. The current analysis was performed using data from patients who had molecular profiles and did not receive trastuzumab. The various molecular classifiers tested were highly correlated. Categorization of breast cancer by molecular signatures enhanced the ability of pCR to predict improvement in RFS compared to the population as a whole. In multivariate analysis, the molecular signatures that added to the ability of HR and HER2 receptors, clinical stage, and pCR in predicting RFS included 70-gene signature, wound healing signature, p53 mutation signature, and PAM50 risk of recurrence. The low risk signatures were associated with significantly better prognosis, and also identified additional patients with a good prognosis within the no pCR group, primarily in the hormone receptor positive, HER-2 negative subgroup. The I-SPY 1 population is enriched for tumors with a poor prognosis but is still heterogeneous in terms of rates of pCR and RFS. The ability of pCR to predict RFS is better by subset than it is for the whole group. Molecular markers improve prediction of RFS by identifying additional patients with excellent prognosis within the no pCR group.