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BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is an aggressive and highly lethal cancer with an increasing incidence worldwide that lacks effective treatment regimens. Hypocrellin A (HA), a natural small compound isolated from S. bambusicola, has multiple biomedical activities, including antitumor activity. PURPOSE: We intended to investigate the therapeutic effects of HA on ICC and its potential mechanisms. METHODS: RBE and HuccT1 cell lines were utilized for in vitro experiments. CCK8 assay, colony formation analysis, RTCA, and immunofluorescence staining of ki67 were employed to evaluate the suppression effects of HA on proliferation. The inhibitory effects of HA on cell migration and invasion were evaluate through transwell and wound healing assays, and Hoechst 33,258 staining was performed to evaluate apoptosis. Additionally, we performed transcriptome sequencing and molecular docking for targeting identification, and immunoblotting and immunofluorescence of key molecules for validation. Two in vivo models, HuccT1 xenografts, and the primary ICC model (KRAS/P19/SB) established via hydrodynamic tail-vein injection were implemented. Multiplex immunohistochemistry (mIHC) was used to illustrate the multi-target inhibitory effects of HA. RESULTS: The IC50 values of HA against RBE and HuccT1 cells were 4.612 µM and 10.01 µM for 24 h, as determined through the CCK8 assay. Our results confirmed that HA significantly repressed the proliferation, migration, invasion, and promoted the apoptosis of ICC cells at low concentrations. Moreover, HA exerted its anti-cancer effects through multi-target inhibition of the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways. This inhibitory effect was rescued by Recilisib, an activator of the PI3K-AKT-mTOR pathway. Bioinformatics analysis of a multi-center RNA-Seq cohort (n = 90) demonstrated significant associations between these target pathways and the occurrence and poor prognosis of ICC. Animal studies suggested that HA strongly inhibited tumor growth in xenograft ICC models, and repressed the tumor number and size in the liver of primary ICC models by suppressing these three crucial pathways. CONCLUSION: HA, a novel natural small molecule, demonstrated promising therapeutic efficacy against ICC through its multi-target inhibitory effects on the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways. Moreover, it exhibited notable therapeutic benefits in a primary ICC model (KRAS/P19/SB), positioning it as a novel therapeutic agent for ICC.

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Hypocrellin A (HA), a fungal perylenequinone from bambusicolous Shiraia species, is a newly developed photosensitizer for photodynamic therapy in cancer and other infectious diseases. The lower yield of HA is an important bottleneck for its biomedical application. This study is the first report of the enhancement of HA production in mycelium culture of Shiraia sp. S9 by the polysaccharides from its host bamboo which serve as a strong elicitor. A purified bamboo polysaccharide (BPSE) with an average molecular weight of 34.2 kDa was found to be the most effective elicitor to enhance fungal HA production and characterized as a polysaccharide fraction mainly composed of arabinose and galactose (53.7: 36.9). When BPSE was added to the culture at 10 mg/L on day 3, the highest HA production of 422.8 mg/L was achieved on day 8, which was about 4.0-fold of the control. BPSE changed the gene expressions mainly responsible for central carbon metabolism and the cellular oxidative stress. The induced generation of H2O2 and nitric oxide was found to be involved in both the permeabilization of cell membrane and HA biosynthesis, leading to enhancements in both intra- and extracellular HA production. Our results indicated the roles of plant polysaccharides in host-fungal interactions and provided a new elicitation technique to improve fungal perylenequinone production in mycelium cultures.

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BACKGROUND: Influenza A viruses can be transmitted indirectly by surviving on the surface of an object. Photodynamic inactivation (PDI) is a promising approach for disinfection of pathogens. METHODS: PDI was generated using Hypocrellin A (HA) and red light emitting diode (625-635 nm, 280 W/m2). Effects of the HA-mediated PDI on influenza viruses H1N1 and H3N2 were evaluated by the reduction of viral titers compared to virus control. After selection of the HA concentrations and illumination times, the applicability of PDI was assessed on surgical masks. Reactive oxygen species (ROS) were determined using a 2'-7'-dichlorodihydrofluorescein diacetate fluorescence probe. RESULTS: In solution, 10 µM HA inactivated up to 5.11 ± 0.19 log10 TCID50 of H1N1 and 4.89 ± 0.38 log10 TCID50 of H3N2 by illumination for 5 and 30 min, respectively. When surgical masks were contaminated by virus before HA addition, PDI inactivated 99.99% (4.33 ± 0.34 log reduction) of H1N1 and 99.40% (2.22 ± 0.39 log reduction) of H3N2 under the selected condition. When the masks were pretreated with HA before virus addition, PDI decontaminated 99.92% (3.11 ± 0.19 log reduction) of H1N1 and 98.71% (1.89 ± 0.20 log reduction) of H3N2 virus. The fluorescence intensity of 2',7'-dichlorofluorescein in photoactivated HA was significantly higher than the cell control (P > 0.05), indicating that HA efficiently generated ROS. CONCLUSIONS: HA-mediated PDI is effective for the disinfection of influenza viruses H1N1 and H3N2. The approach could be an alternative to decontaminating influenza A viruses on the surfaces of objects.

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BACKGROUND: Perylenequinones from Shiraia fruiting bodies are excellent photosensitizers and widely used for anti-cancer photodynamic therapy (PDT). The lower yield of Shiraia perylenequinones becomes a significant bottleneck for their medical application. Branched-chain amino acids (BCAAs) not only serve as important precursors for protein synthesis, but also are involved in signaling pathway in cell growth and development. However, there are few reports concerning their regulation of fungal secondary metabolism. In present study, the eliciting effects of BCAAs including L-isoleucine (L-Ile), L-leucine (L-Leu) and L-valine (L-Val) on Shiraia perylenequinone production were investigated. RESULTS: Based on the analysis of the transcriptome and amino acid contents of Shiraia in the production medium, we revealed the involvement of BCAAs in perylenequinone biosynthesis. The fungal conidiation was promoted by L-Val treatment at 1.5 g/L, but inhibited by L-Leu. The spore germination was promoted by both. The production of fungal perylenequinones including hypocrellins A (HA), HC and elsinochromes A-C (EA-EC) was stimulated significantly by L-Val at 1.5 g/L, but sharply suppressed by L-Leu. After L-Val treatment (1.5 g/L) in Shiraia mycelium cultures, HA, one of the main bioactive perylenequinones reached highest production 237.92 mg/L, about 2.12-fold than that of the control. Simultaneously, we found that the expression levels of key genes involved in the central carbon metabolism and in the late steps for perylenequinone biosynthesis were up-regulated significantly by L-Val, but most of them were down-regulated by L-Leu. CONCLUSIONS: Our transcriptome analysis demonstrated that BCAA metabolism was involved in Shiraia perylenequinone biosynthesis. Exogenous BCAAs exhibit contrasting effects on Shiraia growth and perylenequinones production. L-Val could promote perylenequinone biosynthesis via not only enhancing the central carbon metabolism for more precursors, but also eliciting perylenequinone biosynthetic gene expressions. This is the first report on the regulation of BCAAs on fungal perylenequinone production. These findings provided a basis for understanding physiological roles of BCAAs and a new avenue for increasing perylenequinone production in Shiraia mycelium cultures.

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BACKGROUND: Hypocrellin A (HA) is a perylene quinone pigment with high medicinal value that is produced by Shiraia bambusicola Henn. (S. bambusicola) and Hypocrella bambusae (Berk. & Broome) Sacc. (Ascomycetes) with great potential in clinical photodynamic therapy. Submerged cultivation of S. bambusicola is a popular technique for HA production. However, there is not much research on how temperature changes lead to differential yields of HA production. RESULTS: The temperature regulation of submerged fermentation is an efficient approach to promote HA productivity. After a 32 °C fermentation, the HA content in the mycelia S. bambusicola (GDMCC 60438) was increased by more than three- and fivefold when compared to that at 28 °C and 26 °C, respectively. RNA sequencing (RNA-seq) analysis showed that the regulation of the expression of transcription factors and genes essential for HA biosynthesis could be induced by high temperature. Among the 496 differentially expressed genes (DEGs) explicitly expressed at 32 °C, the hub genes MH01c06g0046321 and MH01c11g0073001 in the coexpression network may affect HA biosynthesis and cytoarchitecture, respectively. Moreover, five genes, i.e., MH01c01g0006641, MH01c03g0017691, MH01c04g0029531, MH01c04g0030701 and MH01c22g0111101, potentially related to HA synthesis also exhibited significantly higher expression levels. Morphological observation showed that the autolysis inside the mycelial pellets tightly composted intertwined mycelia without apparent holes. CONCLUSIONS: The obtained results provide an effective strategy in the submerged fermentation of S. bambusicola for improved HA production and reveal an alternative regulatory network responsive to the biosynthesis metabolism of HA in response to environmental signals.

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BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer, which is the deadliest form of cancer worldwide. Recent studies have shown that genes in the fibroblast growth factor (FGF) family are highly mutated in lung cancer, and fibroblast growth factor receptor 1 (FGFR1) has been found to be involved in various cancers, including lung cancer, suggesting that FGFR1 is a valid therapeutic target. Hypocrellin A (HA), a molecule with multiple biological activities, has been shown to influence cancer growth, but the specific mechanisms of its antitumor action have not been fully explored. METHODS: MTT, colony formation, wound healing, transwell cell invasion and EdU cell proliferation assays were performed upon HA treatment of three NSCLC cell lines, H460, PC-9 and H1975. Hoechst 33258 staining and caspase 3 activity assays were carried out to investigate the impact of HA on apoptosis in these cells. Molecular docking and surface plasmon resonance were conducted to assess binding of HA to FGFR1. A mouse tumor model was used to detect the NSCLC-inhibitory ability of HA in vivo. RESULTS: Through in vitro assays, HA was shown to negatively impact cell viability, migration, invasion and promote apoptosis in three human NSCLC cell line models. HA was shown to bind to FGFR1 and to inhibit its autophosphorylation and the phosphorylation of downstream signaling molecules. Inhibition of tumor growth was also demonstrated in a mouse xenograft tumor model, and no toxic effects of HA treatment were observed. CONCLUSIONS: HA inhibits the activity of the FGFR1 and STAT3 signaling pathways. HA thus represents a potential new FGFR1-targeted treatment for NSCLC.

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Keloids are characterized by abnormal proliferation of fibroblasts and continuous deposition of extracellular matrix (ECM) components. In the field of dermopathy, photodynamic therapy (PDT) with visible light has been increasingly investigated. The natural photosensitizer Hypocrellin A (HA) was shown to have excellent light induced anticancer, antimicrobial and antiviral activities. In this experiment, we investigated the impacts of HA united light-emitting diode (LED) red light irradiation on human keloid fibroblast cells (KFs). Our results showed that HA combined with red light irradiation treatment (HA-R-PDT) decreased KF viability, reduced KF collagen production and ECM accumulation, inhibited cell proliferation, suppressed cell invasion and induced cell apoptosis. Moreover, our observations demonstrated that the TGF-ß/Smad signalling pathway and autophagy were restrained by HA-R-PDT. TGF-ß1 could promote autophagy in KFs through both the Smad and ERK pathways, while inhibition of autophagy altered the TGF-ß1 levels through negative feedback. Therefore, HA-R-PDT suppressed cell hyperproliferation, collagen synthesis and ECM accumulation of KFs by regulating the TGF-ß1-ERK-autophagy-apoptosis signalling pathway. HA-R-PDT deserves systematic investigation as a potential therapeutic strategy for keloids, and autophagy might be a promising candidate in the treatment of KFs.

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Shiraia mycelial culture is a promising biotechnological alternative for the production of hypocrellin A (HA), a new photosensitizer for anticancer photodynamic therapy (PDT). The extractive fermentation of intracellular HA in the nonionic surfactant Triton X-100 (TX100) aqueous solution was studied in the present work. The addition of 25 g/L TX100 at 36 h of the fermentation not only enhanced HA exudation to the broth by 15.6-fold, but stimulated HA content in mycelia by 5.1-fold, leading to the higher production 206.2 mg/L, a 5.4-fold of the control on day 9. After the induced cell membrane permeabilization by TX100 addition, a rapid generation of nitric oxide (NO) and hydrogen peroxide (H2O2) was observed. The increase of NO level was suppressed by the scavenger vitamin C (VC) of reactive oxygen species (ROS), whereas the induced H2O2 production could not be prevented by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), suggesting that NO production may occur downstream of ROS in the extractive fermentation. Both NO and H2O2 were proved to be involved in the expressions of HA biosynthetic genes (Mono, PKS and Omef) and HA production. NO was found to be able to up-regulate the expression of transporter genes (MFS and ABC) for HA exudation. Our results indicated the integrated role of NO and ROS in the extractive fermentation and provided a practical biotechnological process for HA production.

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Cutaneous squamous cell carcinoma (cSCC) is a type of malignant skin tumor derived from epidermal Malpighian cells. Photodynamic therapy is regarded as a crucial method in oncology. Hypocrellin A (HA), an efficient natural photosensitizer, has been reported to exert excellent light induced antiviral, antimicrobial and anticancer activity through mediating multiple signaling pathways. The purpose of the present study is to examine the effects of HA united red light irradiation on human squamous carcinoma A431 cells and further reveal the underlying regulatory mechanisms. The results showed that synergistic treatment of HA and red light irradiation inhibited cell proliferation and induced cell apoptosis and autophagy. Moreover, HA united red light irradiation caused a significant accumulation of reactive oxygen species (ROS), and induced the activation of c-Jun NH 2 terminal kinases (JNKs) which was inhibited by the antioxidant N-Acetyl-cysteine (NAC). Furthermore, HA united red light irradiation activated the nuclear factor-kappa B (NF-κB) pathway, and inhibition of NF-κB activity exacerbated HA united red light irradiation-induced apoptosis but suppressed cell autophagy. In addition, the inhibition of autophagy promoted HA united red light irradiation-induced apoptosis and facilitated the NF-κB activity. Over all, our results revealed that HA united red light irradiation could inhibit A431 cell proliferation by inducing apoptosis and autophagy via the activation of the ROS mediated JNK and NF-κB pathways, providing prospective for HA as a potential therapeutic for the treatment of cSCC.

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There is an urgent need for new antibacterial strategies to overcome the emergence of antibiotic resistance. Antibacterial photodynamic therapy (APDT) may be an effective method to deliver photosensitizers for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report that the photosensitizer hypocrellin A (HA) loaded into lipase-sensitive methoxy poly (ethylene glycol)-block-poly(ε-caprolactone) (mPEG-PCL) micelles showed high anti-MRSA activity in vitro and in vivo by PDT. Once the micelles come into contact with bacteria that secrete lipase, the PCL is degraded to release HA. Our results showed that the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of mPEG-PCL/HA micelles after light irradiation were 0.69 and 1.38 mg/L (HA concentration), respectively. In the dark, the MIC and MBC of the micelles were 250 and 500 mg/L (HA concentration), respectively. The fluorescent stain results further demonstrated the photodynamic antibacterial activity of mPEG-PCL/HA micelles. The survival rate of mice subjected to experimental acute peritonitis increased to 86% after treated with the micelles. The polymeric micelles showed low hemolytic activity and biocompatibility, simultaneously preventing aggregation in vivo and enhancing the water solubility of HA. Thus, the photosensitizer HA loaded micelles could be used as APDT for infections caused by bacteria without antibiotic resistance.

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Hypocrellin A (HA) is a natural red perylenequinone pigment from Shiraia fruiting body, which was used clinically on various skin diseases and developed as a photodynamic therapy agent against cancers. The fruiting bodies may harbor a diverse but poorly understood microbial community. In this study, we characterized the bacterial community of Shiraia fruiting body using a combination of culture-based method and Illumina high-throughput sequencing, and tested the involvement of some companion bacteria in fungal HA production using the fungal-bacterial confrontation assay. Our results revealed that the bacterial community in the fruiting body was dominated by Bacillus and Pseudomonas. Some Pseudomonas isolates such as P. fulva, P. putida, and P. parafulva could stimulate fungal HA accumulation by Shiraia sp. S9. The bacterial treatment of P. fulva SB1 up-regulated the expression of polyketide synthase (PKS) for HA biosynthesis and transporter genes including ATP-binding cassette (ABC) and major facilitator superfamily transporter (MFS) for HA exudation. After the addition of live P. fulva SB1, the mycelium cultures of Shiraia sp. S9 presented a higher HA production (225.34 mg/L), about 3.25-fold over the mono-culture. On the other hand, B. cereus was capable of alleviating fungal self-toxicity from HA via down-regulation of HA biosynthetic genes or possible biodegradation on HA. To our knowledge, this is the first report on the diversified species of bacteria associated with Shiraia fruiting bodies and the regulation roles of the companion bacteria on fungal HA biosynthesis. Furthermore, the bacterial co-culture provided a good strategy for the enhanced HA production by Shiraia.

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Many studies have reported that hypocrellin A (HA) exhibits effective antimicrobial activities with proper irradiation. However, its antifungal activity and the involved mechanism have not been fully defined. In this study, HA-mediated cytotoxicity in Candida albicans cells was evaluated after antimicrobial photodynamic therapy (aPDT). The results showed that 1.0 µg/ml HA significantly decreased the survival rate of C. albicans cells with light illumination. Moreover, the ROS levels were also remarkably elevated by HA. Further study found that HA combined with illumination led to cell membrane potential depolarization and cell membrane integrity damage. To investigate the form of cell death, a series of apoptosis-related parameters, including mitochondrial transmembrane potential, metacaspase activity, DNA fragmentation, nuclear condensation, and cytosolic and mitochondrial calcium, were analyzed. Data showed that all the above mentioned apoptosis hallmarks were affected after treatment with HA, indicating that HA induced C. albicans cell apoptosis. Finally, HA-mediated aPDT was demonstrated to be low-toxic and effective in treating cutaneous C. albicans infections. This study highlights the antifungal effect and mechanism of HA-mediated aPDT against C. albicans and provides a promising photodynamic antifungal candidate for C. albicans skin infections.

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BACKGROUND: Fungal perylenequinonoid (PQ) pigments from Shiraia fruiting body have been well known as excellent photosensitizers for medical and agricultural uses. The fruiting bodies are colonized by a diverse bacterial community of unknown function. We screened the companion bacteria from the fruiting body of Shiraia sp. S9 and explored the bacterial elicitation on fungal PQ production. RESULTS: A bacterium Pseudomonas fulva SB1 isolated from the fruiting body was found to stimulate the production of fungal PQs including hypocrellins A, C (HA and HC), and elsinochromes A-C (EA, EB and EC). After 2 days of co-cultures, Shiraia mycelium cultures presented the highest production of HA (325.87 mg/L), about 3.20-fold of that in axenic culture. The co-culture resulted in the induction of fungal conidiation and the formation of more compact fungal pellets. Furthermore, the bacterial treatment up-regulated the expression of polyketide synthase gene (PKS), and activated transporter genes of ATP-binding cassette (ABC) and major facilitator superfamily transporter (MFS) for PQ exudation. CONCLUSIONS: We have established a bacterial co-culture with a host Shiraia fungus to induce PQ biosynthesis. Our results provide a basis for understanding bacterial-fungal interaction in fruiting bodies and a practical co-culture process to enhance PQ production for photodynamic therapy medicine.

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Over recent decades, many studies have reported that hypocrellin A (HA) can eliminate cancer cells with proper irradiation in several cancer cell lines. However, the precise molecular mechanism underlying its anticancer effect has not been fully defined. HA-mediated cytotoxicity and apoptosis in human lung adenocarcinoma A549 cells were evaluated after photodynamic therapy (PDT). A temporal quantitative proteomics approach by isobaric tag for relative and absolute quantitation (iTRAQ) 2D liquid chromatography with tandem mass spectrometric (LC-MS/MS) was introduced to help clarify molecular cytotoxic mechanisms and identify candidate targets of HA-induced apoptotic cell death. Specific caspase inhibitors were used to further elucidate the molecular pathway underlying apoptosis in PDT-treated A549 cells. Finally, down-stream apoptosis-related protein was evaluated. Apoptosis induced by HA was associated with cell shrinkage, externalization of cell membrane phosphatidylserine, DNA fragmentation, and mitochondrial disruption, which were preceded by increased intracellular reactive oxygen species (ROS) generations. Further studies showed that PDT treatment with 0.08 µmol/L HA resulted in mitochondrial disruption, pronounced release of cytochrome c, and activation of caspase-3, -9, and -7. Together, HA may be a possible therapeutic agent directed toward mitochondria and a promising photodynamic anticancer candidate for further evaluation.

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Hepatocellular carcinoma (HCC) is one of the most frequently occurring cancer worldwide and the fifth most common malignancy. The Hippo pathway has been found to play a critical role in cancer development. YAP, a transcriptional coactivator, is the major downstream effector of the Hippo signaling pathway. Hypocrellin A (HA), a natural perylenequinone light-sensitive compound, is usually used for the therapy of eukoplakia of the vulva and keloids in China. Oleanolic acid (OA), a pentacyclic triterpene compound, is prevalent in the treatment of human liver diseases such as viral hepatitis. In this study, we aimed to explore the mechanism by which HA modulates the Hippo/YAP signaling pathway in HCC cells and the anticancer effect of HA combined with OA. Treatment with HA resulted in a decrease in cell viability and migration in HCC cells. Furthermore, we found that HA decreased the YAP and TEAD protein levels of the Hippo pathway. Next we demonstrated that HA could potentiate OA's effect on HCC cells. Our results indicated that HA could inhibit the growth of HCC cells in darkness through Hippo-YAP signaling and HA combined with OA had a better effect than HA or OA alone.Thus, our results provide an alternative combinational inhibitor to combat hepatocellular carcinoma diseases.

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Hypocrellin A (HA) is a major bioactive perylenequinone from the fruiting body of Shiraia bambusicola used for the treatment of skin diseases and developed as a photodynamic therapy (PDT) agent against cancers and viruses. The mycelial culture of S. bambusicola under dark is a biotechnological alternative for HA production but with low yield. In this study, light and dark conditions were investigated to develop effective elicitation on HA production in the cultures. Our results showed the constant light at 200 lx stimulated HA production without any growth retardation of mycelia. A light/dark shift (24: 24 h) not only increased HA content in mycelia by 65%, but stimulated HA release into the medium with the highest total HA production 181.67 mg/L on day 8, about 73% increase over the dark control. Moreover, light/dark shifting induced the formation of smaller and more compact fungal pellets, suggesting a new effective strategy for large-scale production of HA in mycelium cultures. The light/dark shift up-regulated the expression levels of two reactive oxygen species (ROS) related genes including superoxide-generating NADPH oxidase (Nox) and cytochrome c peroxidase (CCP), and induced the generation of ROS. With the treatment of vitamin C, we found that ROS was involved in the up-regulated expression of key biosynthetical genes for hypocrellins and improved HA production. These results provide a basis for understanding the influence of light/dark shift on fungal metabolism and the application of a novel strategy for enhancing HA production in submerged Shiraia cultures.

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The addition of surfactant is a useful strategy to enhance the product yield in submerged fermentation process. In this study, we sought to explore the mechanism for the elicitation of Triton X-100 on production of hypocrellin A (HA) in cultures of Shiraia bambusicola through transcriptomic analysis. Triton X-100 at 2.5% (w/v) not only induced HA biosynthesis in mycelia, but also stimulated the release of HA into the medium. We found 23 of 2463 transcripts, possible candidate genes for HA biosynthesis under Triton X-100 induction. Gene ontology (GO) analysis showed Triton X-100 treatment changed expression of genes involved in transmembrane transport and oxidation-reduction process, indicating that enhanced HA production was mainly due to both elicited biosynthesis in mycelium and the increased membrane permeability for HA release. These data provided new insights into elicitation of surfactants in submerged cultures of fungi.

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Hypocrellin A (HA), a naturally occurring fungal perylenequinone, is widely used in clinic to treat skin diseases and developed as a photodynamic therapy (PDT) agent against cancers. In this study, a low intensity ultrasound (US, 0.28W/cm2 at 40kHz) was conducted thrice of repeated US exposure (5-min) with an interval of 12h to stimulate HA production of Shiraia bambusicola after 72h of the initial submerged cultures. US not only increased the content of HA by 177.2% in mycelia, but stimulated the release of HA into the medium with the highest total production of HA (247.67mg/L) on day 8. US could result in the decreased pellet diameter, the enhanced membrane permeability, the alternation of membrane compounds and reactive oxygen species (ROS) generation. Furthermore, the ultrasonic treatment up-regulated the expression of some HA biosynthetic genes including polyketide synthase gene (PKS), O-methyltransferase gene (Omef), O-methyltransferase/FAD-dependent monooxygenase (Mono) and FAD/FMN-dependent oxidoreductase gene (FAD), and activated major facilitator superfamily transporter gene (MFS) for HA exudation. The enhancement of HA production was mainly due to both the stimulated cellular biosynthesis and the enhanced fungal exudation of HA. These results provide a basis for understanding the US elicitation and a valuable strategy for enhancing HA production in submerged Shiraia cultures.

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Multifunctional nanocomposite has a huge potential for cell imaging, drug delivery, and improving therapeutic effect with less side effects. To date, diverse approaches have been demonstrated to endow a single nanostructure with multifunctionality. Herein, we report the synthesis and application of core-shell nanoparticles composed with upconversion nanoparticle (UCNP) as a core and a graphene oxide quantum dot (GOQD) as a shell. The UCNP was prepared and applied for imaging-guided analyses of upconversion luminescence. GOQD was prepared and employed as promising drug delivery vehicles to improve anti-tumor therapy effect in this study. Unique properties of UCNPs and GOQDs were incorporated into a single nanostructure to provide desirable functions for cell imaging and drug delivery. In addition, hypocrellin A (HA) was loaded on GOQDs for photo-dynamic therapy (PDT). HA, a commonly used chemotherapy drug and a photo-sensitizer, was conjugated with GOQD by π-π interaction and loaded on PEGylated UCNP without complicated synthetic process, which can break structure of HA. Applying these core-shell nanoparticles to MTT assay, we demonstrated that the UCNPs with GOQD shell loaded with HA could be excellent candidates as multifunctional agents for cell imaging, drug delivery and cell therapy.

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Hypocrellin A (HA), well known as one of the best natural pigments and bioactive agent to treat skin diseases, is further anticipated to play a vital role in photodynamic therapy (PDT) in anticancer and antiviral treatments. In this study, an HA-producing strain ZZZ816 (Shiraia sp.) was isolated from the moso bamboo (Phyllostachys edulis) seeds, and gamma irradiation was used to mutagenize spores of the original strain. After treatment with cobalt-60 gamma ((60)Coγ) with different doses (20, 50, 80, 100, 150, 180, 300, and 500 Gy), the 100 Gy was selected as the optimal condition, which led to 77.2 % lethality of spores and 35 % positive mutant frequency. The extracted compound of the most excellent HA-producing strain (H-4-2) was precisely analyzed by a combination of seven detection methods, and the maximum HA content was shown to reach 2018.3 mg/L. HA production in H-4-2 increased by 414.9 % compared to that of original strain ZZZ816 (392 mg/L) and was significantly higher than all the other industrial HA-producing strains in published reports.

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