Nitric oxide mediates red light-induced perylenequinone production in Shiraia mycelium culture.

Perylenequinones (PQs) from bambusicolous Shiraia fungi serve as excellent photosensitizers for photodynamic therapy. However, the lower yield of PQ production in mycelium cultures is an important bottleneck for their clinical application. Light has long been recognized as a pivotal regulatory signal for fungal secondary metabolite biosynthesis. In this study, we explored the role of nitric oxide (NO) in the growth and PQ biosynthesis in mycelium cultures of Shiraia sp. S9 exposed to red light. The continuous irradiation with red light (627 nm, 200 lx) suppressed fungal conidiation, promoted hyphal branching, and elicited a notable increase in PQ accumulation. Red light exposure induced NO generation, peaking to 81.7 µmol/g FW on day 8 of the culture, with the involvement of nitric oxide synthase (NOS)- or nitrate reductase (NR)-dependent pathways. The application of a NO donor sodium nitroprusside (SNP) restored conidiation of Shiraia sp. S9 under red light and stimulated PQ production, which was mitigated upon the introduction of NO scavenger carboxy-PTIO or soluble guanylate cyclase inhibitor NS-2028. These results showed that red light-induced NO, as a signaling molecule, was involved in the regulation of growth and PQ production in Shiraia sp. S9 through the NO-cGMP-PKG signaling pathway. While mycelial H2O2 content exhibited no significant alternations, a transient increase of intracellular Ca2+ and extracellular ATP (eATP) content was detected upon exposure to red light. The generation of NO was found to be interdependent on cytosolic Ca2+ and eATP concentration. These signal molecules cooperated synergistically to enhance membrane permeability and elevate the transcript levels of PQ biosynthetic genes in Shiraia sp. S9. Notably, the combined treatment of red light with 5 µM SNP yielded a synergistic effect, resulting in a substantially higher level of hypocrellin A (HA, 254 mg/L), about 3.0-fold over the dark control. Our findings provide valuable insights into the regulation of NO on fungal secondary metabolite biosynthesis and present a promising strategy involving the combined elicitation with SNP for enhanced production of photoactive PQs and other valuable secondary metabolites in fungi.

The alleviating effect of Scutellaria amoena extract on the regulation of gut microbiota and its metabolites in NASH rats by inhibiting the NLRP3/ASC/caspase-1 axis.

Background: Scutellaria amoena (SA) is the root of S. amoena C.H. Wright of Labiatae, also known as Scutellaria southwestern. This is mainly distributed in Sichuan, Yunnan, and Guizhou in China. In southwest China, SA is used as an alternative method to genuine medicine for the treatment of allergy, diarrhea, inflammation, hepatitis, and bronchitis. Thus far, studies on the effects of SA on non-alcoholic steatohepatitis (NASH) are lacking. This paper investigated the effect of SA on the regulation of gut microbiota and its metabolites in NASH rats by inhibiting the NOD-like receptor 3 (NLRP3)/apoptosis-associated speck-like protein (ASC)/caspase-1 axis. Methods: A NASH rat model was induced by a high-fat diet (HFD) for 12 weeks, and rats were orally given different doses of SA extracts (150 and 300 mg/kg/d) for 6 weeks. Changes in histological parameters, body weight, organ indexes, cytokines, and biochemical parameters related to NLRP3 in NASH rats were checked. 16S rRNA gene sequencing and UPLC-MS/MS technology were used to analyze the changes in the gut microbiota composition and its metabolites in NASH rats. Results: SA significantly inhibited the HFD-induced increase in body weight, lipid levels, and inflammatory infiltration. SA notably inhibited the HFD-induced increase in the upper and lower factors of NLRP3, such as transforming growth factor (TGF)-ß, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-18, pro-IL-18, IL-1ß, pro-IL-1ß, NLRP3, ASC, and caspase-1. Additionally, mRNA expressions of caspase-1, NLRP3, and ASC were significantly downregulated after SA treatment. The results of the intestinal flora showed that SA could increase the diversity of flora and change its structure and composition in NASH rats by reducing Firmicutes/Bacteroidetes (F/B) ratio, Blautia (genus), Lachospiraceae (family), and Christensenellaceae R-7 group (genus), and increasing Muribaculaceae (family) and Bacteroides (genus). The metabolomics revealed that 24 metabolites were possibly the key metabolites for SA to regulate the metabolic balance of NASH rats, including chenodeoxycholic acid, xanthine, and 9-OxoODE. Nine metabolic pathways were identified, including primary bile acid biosynthesis, bile secretion, purine metabolism, and secondary bile acid biosynthesis. Conclusion: SA can regulate the intestinal microbial balance and metabolic disorder by inhibiting the NLRP3/ASC/caspase-1 axis to relieve NASH.

Bamboo polysaccharides elicit hypocrellin A biosynthesis of a bambusicolous fungus Shiraia sp. S9.

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.

Volatiles of Shiraia fruiting body-associated Pseudomonas putida No.24 stimulate fungal hypocrellin production.

Hypocrellins are major bioactive perylenequinones from Shiraia fruiting bodies and have been developed as efficient photosensitizers for photodynamic therapy. Pseudomonas is the second dominant genus inside Shiraia fruiting bodies, but with less known actions on the host fungus. In this work, the effects of bacterial volatiles from the Shiraia-associated Pseudomonas on fungal hypocrellin production were investigated. Pseudomonas putida No.24 was the most active to promote significantly accumulation of Shiraia perylenequinones including hypocrellin A (HA), HC, elsinochrome A (EA) and EC. Headspace analysis of the emitted volatiles revealed dimethyl disulfide as one of active compounds to promote fungal hypocrellin production. The bacterial volatiles induced an apoptosis in Shiraia hyphal cell, which was associated with the generation of reactive oxygen species (ROS). ROS generation was proved to mediate the volatile-induced membrane permeability and up-regulation of gene expressions for hypocrellin biosynthesis. In the submerged volatile co-culture, the bacterial volatiles stimulated not only HA content in mycelia, but also HA secretion into the medium, leading to the enhanced HA production to 249.85 mg/L, about 2.07-fold over the control. This is the first report on the regulation of Pseudomonas volatiles on fungal perylenequinone production. These findings could be helpful to understand the roles of bacterial volatiles in fruiting bodies and also provide new elicitation method using bacterial volatiles to stimulate fungal secondary metabolite production.

Effects of branched-chain amino acids on Shiraia perylenequinone production in mycelium cultures.

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.

Effects of Tylophora yunnanensis Schltr on regulating the gut microbiota and its metabolites in non-alcoholic steatohepatitis rats by inhibiting the activation of NOD-like receptor protein 3.

ETHNOPHARMACOLOGICAL RELEVANCE: Tylophora yunnanensis Schltr (TYS) is widely distributed in Yunnan, Guizhou, and other places in China. It is commonly used by folks to treat hepatitis and other liver-related diseases; however, its mechanism of action is still unclear. AIM OF THE STUDY: This study aimed to determine the effects of TYS on regulating gut microbiota and its metabolites in non-alcoholic steatohepatitis (NASH) rats by inhibiting the activation of NOD-like receptor protein3 (NLRP3). MATERIAL AND METHODS: An HFD-induced rat model was established to investigate if the intragastric administration of TYS could mediate gut microbiota and their metabolites to ultimately improve the symptoms of NASH. The improving effects of TYS on NASH rats were assessed by measuring their body weight, lipid levels, histopathology, and inflammatory factor levels in the rat models. The regulatory effects of TYS on NLRP3 in the NASH rats were analyzed using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), which determined the levels of NLRP3-related factors. The changes in the composition of the gut microbiota of NASH rats were analyzed using 16S rRNA gene sequencing technology. Meanwhile, the Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for the non-targeted analysis of metabolites in the cecum contents. RESULTS: The results showed that TYS could improve NASH by decreasing the body weight and levels of lipid, AST, ALT, LPS, FFA, VLDL, IL-1ß, IL-6, TNF-α, TGF-ß, NLRP3, ASC, and Caspase-1 in the NASH rats. The analysis of gut microbiota showed that TYS could improve the diversity and abundance of gut microbiota and alter their composition by decreasing the Firmicutes/Bacteroidetes (F/B) ratio and relative abundances of Lachnospiraceae, Christensenellaceae, Blautia, etc. while increasing those of Muribaculaceae, Rumiaococcus, Ruminococcaceae, etc. The analysis of metabolites in the cecum contents suggested that the arachidonic acid metabolism, bile secretion, serotonergic synapse, Fc epsilon RI signaling pathway, etc. were regulated by TYS. The metabolites enriched in these pathways mainly included chenodeoxycholic acid, prostaglandin D2, TXB2, 9-OxoODE, and 13(S)-HOTrE. CONCLUSIONS: These findings suggested that TYS could alleviate the NASH symptoms by decreasing the body weight, regulating the lipid levels, reducing the inflammatory response, and inhibiting the expression levels of NLRP3, ASC, and Caspase-1 in the NASH rats. The changes in the composition of gut microbiota and their metabolic disorder were closely related to the activation of NLRP3. TYS could significantly inhibit the activation of NLRP3 and regulate the composition of gut microbiota and the disorder of metabolites during NASH modeling.

Structure characterization of an exopolysaccharide from a Shiraia-associated bacterium and its strong eliciting activity on the fungal hypocrellin production.

Hypocrellins are fungal perylenequinones (PQs) from Shiraia fruiting bodies and potential photosensitizers for cancer photodynamic therapy. Shiraia fruiting bodies harbor diverse bacterial communities dominated by Pseudomonas. The present study was to characterize the exopolysaccharide (EPS) of P. fulva SB1 which acted as an elicitor to stimulate the PQ accumulation of the host Shiraia. A bacterial EPS named EPS-1 was purified from the culture broth of P. fulva SB1, which consisted of mannose (Man) and glucose (Glc) with an average molecular weight of 9.213 × 104 Da. EPS-1 had (1 â†’ 2)-linked α-mannopyranose (Manp) backbone and side chains of α-D-Manp-(1→ and α-D-Manp-(1 â†’ 6)-ß-D-Glcp-(1 â†’ 6)-α-D-Manp(1 â†’ group attached to the O-6 positions of (1 â†’ 2)-α-D-Manp. EPS-1 at 30 mg/L stimulated both intracellular and extracellular hypocrellin A (HA) by about 3-fold of the control group. The EPS-1 treatment up-regulated the expression of key genes for HA biosynthesis. The elicitation of HA biosynthesis by EPS-1 was strongly dependent on the induced reactive oxygen species (ROS) generation. The results may provide new insights on the role of bacterial EPS in bacterium-fungus interactions and effective elicitation strategy for hypocrellin production in mycelial cultures.

All-cause mortality risk in older patients with femoral neck fracture.

INTRODUCTION: The mortality rate after hip fracture is high. However, the 1-year mortality rate after femoral intertrochanteric fracture and femoral neck fracture differs (Gibson-Smith D, Klop C, Elders PJ, Welsing PM, van Schoor N, Leufkens HG, et al., Osteoporos Int 25:2555-2563, 2014), although both are types of hip fracture. A previous real-world single-center prospective cohort study showed that older age and high Charlson comorbidity index score were risk factors for femoral intertrochanteric fracture. Additionally, therapy with zoledronic acid 5 mg (Aclasta) was a protective factor (Li XP, Zhang P, Zhu SW, Yang MH, Wu XB, Jiang XY, J Orthop Surg Res. 16:727, 2021). We wished to determine the risk factors for all-cause mortality in femoral neck fracture patients. AIM: To identify the risk factors for postoperative all-cause mortality in aged patients with femoral neck fracture. MATERIALS AND METHODS: We enrolled 307 aged patients with femoral neck fracture; 38 were lost to follow-up after 2-3 years. The patients' general characteristics, bone mineral density, and anti-osteoporosis treatment after operation were recorded as potential risk factors. Kaplan-Meier curves and multivariate Cox proportional hazards models were constructed to analyze the influence of each factor on all-cause mortality. RESULTS: This was a real-world single-center prospective cohort study showing that (1) most of the patients who died were male, older (mean age of the patients who died: 84.8 years vs. 77.9 years for survivors), and had more comorbidities compared with surviving patients. Previous fracture history, body mass index, femoral neck T score, hemoglobin and 25-hydroxy vitamin D levels did not differ significantly between patients who died vs. survived. (2) Differing from patients with intertrochanteric fractures, older patients with femoral neck fracture experienced no reduction in all-cause mortality with treatment with zoledronic acid. CONCLUSION: In Chinese patients with femoral neck fracture, physicians should pay careful attention to male patients, older patients, and those with high numbers of comorbidities.

Contrasting regulation of live Bacillus cereus No.1 and its volatiles on Shiraia perylenequinone production.

BACKGROUND: Fungal perylenequinones (PQs) are a class of photoactivated polyketide mycotoxins produced by plant-associated fungi. Hypocrellins, the effective anticancer photodynamic therapy (PDT) agents are main bioactive PQs isolated from a bambusicolous Shiraia fruiting bodies. We found previously that bacterial communities inhabiting fungal fruiting bodies are diverse, but with unknown functions. Bacillus is the most dominant genus inside Shiraia fruiting body. To understand the regulation role of the dominant Bacillus isolates on host fungus, we continued our work on co-culture of the dominant bacterium B. cereus No.1 with host fungus Shiraia sp. S9 to elucidate bacterial regulation on fungal hypocrellin production. RESULTS: Results from "donut" plate tests indicated that the bacterial culture could promote significantly fungal PQ production including hypocrellin A (HA), HC and elsinochrome A-C through bacterial volatiles. After analysis by gas chromatograph/mass spectrometer and confirmation with commercial pure compounds, the volatiles produced by the bacterium were characterized. The eliciting roles of bacterial volatile organic compounds (VOCs) on HA production via transcriptional regulation of host Shiraia fungus were confirmed. In the established submerged bacterial volatile co-culture, bacterial volatiles could not only promote HA production in the mycelium culture, but also facilitate the release of HA into the medium. The total production of HA was reached to 225.9 mg/L, about 1.87 times that of the fungal mono-culture. In contrast, the live bacterium suppressed markedly fungal PQ production in both confrontation plates and mycelium cultures by direct contact. The live bacterium not only down-regulated the transcript levels of HA biosynthetic genes, but also degraded extracellular HA quickly to its reductive product. CONCLUSION: Our results indicated that bacterial volatile release could be a long-distance signal to elicit fungal PQ production. Biodegradation and inhibition by direct contact on fungal PQs were induced by the dominate Bacillus to protect themselves in the fruiting bodies. This is the first report on the regulation of Bacillus volatiles on fungal PQ production. These findings could be helpful for both understanding the intimate fungal-bacterial interactions in a fruiting body and establishing novel cultures for the enhanced production of bioactive PQs.

Two Novel Flavonoids with Lipid-Lowering Activity from Yi Medicine Shekaqi.

Yi medicine Shekaqi is the most attractive traditional ethnic medicine due to its significant and diverse pharmacological activities. Two novel flavonoids, including 5,2'-dihydroxy-6-methoxy-7-decyloxyflavone and tenaxin II-7-O-ß-D-glucuronopyranosyl acid butyl ester, along with six known flavonoids, were isolated from Yi medicine Shekaqi. Their structures were elucidated based on the analysis of their comprehensive spectral data. The in vitro lipid-lowering activities of the eight compounds showed that all the compounds significantly inhibited the lipopolysaccharide (LPS)-induced increase in the total cholesterol (TC) level, while compounds 1, 4, 6, 7, and 8 significantly inhibited the LPS-induced increase in the triglyceride (TG) level.

Effects of Blue Light on Hypocrellin A Production in Shiraia Mycelium Cultures.

Blue light is a crucial environmental cue for fungi. Hypocrellin A (HA) is a photoactive perylenequinone from Shiraia with strong antimicrobial and anticancer properties. In this study, effects of the illumination of blue-light-emitting diode (LED) at 470 nm on Shiraia sp. S8 were investigated. Blue light at 50-200 lx and 4-6 h day-1 could enhance HA content in the mycelia, but suppress it at 300-400 lx or with longer exposure (8-24 h day-1 ). The intermittent blue light (6 h day-1 ) at 200 lx not only enhanced the fungal conidiation but also stimulated HA production without any growth retardation. The generation of fungal reactive oxygen species was induced to upregulate HA biosynthetic gene expressions. When the culture was maintained under the intermittent blue light for 8 days, HA production reached 242.76 mg L-1 , 2.27-fold of the dark control. On the other hand, both the degradation of HA and downregulation of HA biosynthetic genes occurred under long exposure time (8-24 h day-1 ), leading to the suppression of HA production. These results provide a basis for understanding the regulation of blue light on the biosynthesis of fungal photoactivated perylenequinones, and the application of a novel light elicitation to Shiraia mycelium cultures for enhanced HA production.

All-cause mortality risk in aged femoral intertrochanteric fracture patients.

INTRODUCTION: The 1-year mortality rate after femoral intertrochanteric fracture is higher than that of femoral neck fracture, which also belongs to hip fracture (Cui et al. in Arch Osteoporos 14(1):55, 2019). With the application of the concept of co-management model of orthopedics and geriatrics, the short-term and long-term mortality of all types of hip fractures has decreased (Van Heghe et al. in Calcif Tissue Int, 2021, https://doi.org/10.1007/s00223-021-00913-5 ). However, the mortality of Chinese femoral intertrochanteric fracture patients under this model has not been reported in the literatures. AIM: This paper aims to study the risk factors of postoperative all-cause mortality in aged patients with femoral intertrochanteric fracture under the co-management model of orthopedics and geriatrics. MATERIALS AND METHODS: This is a single-center prospective cohort study based on the real world, under the co-management of orthopedics and geriatrics, 363 patients aged ≥ 65 years with femoral intertrochanteric fracture were enrolled and followed up for 2-3 years; 52 patients were lost to follow up. Age, gender, body mass index (BMI), history of comorbidities, hip Bone Mineral Density (BMD), fracture history, 25(OH)D level, hemoglobin level, anti-osteoporosis treatment were risk factors to be tested. Kaplan-Meier survival curves and multivariate Cox proportional hazards models were constructed to analyze the impact of factors on all-cause mortality. RESULTS: (1) Most of the dead patients were older (the mean age was 83.4 years, compared with 79.8 years for surviving patients), with more complications and without anti-osteoporosis medication; gender, pre-fracture history, BMI, total hip BMD, hemoglobin, 25(OH)D had no difference between the dead and the living patients. (2) Elderly patients with Intertrochanteric fracture can benefit from the early treatment of Zoledronic Acid (within 3 days after the operation). CONCLUSION: Under the co-management of orthopedics and geriatrics, to Chinese patients with Femoral Intertrochanteric fracture, Doctors should pay more attention to their age and chronic disease, and give anti-osteoporosis treatment if allowed.

The signaling role of extracellular ATP in co-culture of Shiraia sp. S9 and Pseudomonas fulva SB1 for enhancing hypocrellin A production.

BACKGROUND: Adenosine 5'-triphosphate (ATP) plays both a central role as an intracellular energy source, and a crucial extracellular signaling role in diverse physiological processes of animals and plants. However, there are less reports concerning the signaling role of microbial extracellular ATP (eATP). Hypocrellins are effective anticancer photodynamic therapy (PDT) agents from bambusicolous Shiraia fungi. The co-culture of Shiraia sp. S9 and a bacterium Pseudomonas fulva SB1 isolated from Shiraia fruiting bodies was established for enhanced hypocrellin A (HA) production. The signaling roles of eATP to mediate hypocrellin biosynthesis were investigated in the co-culture. RESULTS: The co-culture induced release of eATP at 378 nM to the medium around 4 h. The eATP release was interdependent on cytosolic Ca2+ concentration and reactive oxygen species (ROS) production, respectively. The eATP production could be suppressed by the Ca2+ chelator EGTA or abolished by the channel blocker La3+, ROS scavenger vitamin C and NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). The bacterium-induced H2O2 production was strongly inhibited by reactive blue (RB), a specific inhibitor of membrane purinoceptors, but dependent on the induced Ca2+ influx in the co-culture. On the other hand, the application of exogenous ATP (exATP) at 10-300 µM to Shiraia cultures also promoted fungal conidiation and HA production, both of which were blocked effectively by the purinoceptor inhibitors pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS) and RB, and ATP hydrolase apyrase. Both the induced expression of HA biosynthetic genes and HA accumulation were inhibited significantly under the blocking of the eATP or Ca2+ signaling, and the scavenge of ROS in the co-culture. CONCLUSIONS: Our results indicate that eATP release is an early event during the intimate bacterial-fungal interaction and eATP plays a signaling role in the bacterial elicitation on fungal metabolites. Ca2+ and ROS are closely linked for activation of the induced ATP release and its signal transduction. This is the first report on eATP production in the fungal-bacterial co-culture and its involvement in the induced biosynthesis of fungal metabolites.

Identification of the prognostic value of elevated ANGPTL4 expression in gallbladder cancer-associated fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) with different gene profiles from normal fibroblasts (NFs) have been implicated in tumor progression. Angiopoietin-like protein 4 (ANGPTL4) has been shown to regulate tumor angiogenesis and metastasis, and predict poor prognosis. However, the ANGPTL4 expression in CAFs, especially in gallbladder CAFs (GCAFs) and its relationship with patient prognosis is unclear. METHODS: Affymetrix gene profile chip analysis in vitro was performed to detect the different gene expression profiles between GCAFs and NFs. RT-qPCR, immunohistochemistry, and western blotting were performed to investigate the different expression levels of ANGPTL4 in GCAFs/NFs in vitro and in an in vivo nude mouse model of xenograft tumors. Finally, the ANGPTL4 expression was investigated in the stroma of different lesion tissues of the human gallbladder by immunohistochemistry, especially the expression in GCAFs in vivo by co-immunofluorescence, and their prognostic significance in patients with gallbladder cancer (GBC) was assessed. RESULTS: ANGPTL4 was upregulated in both GCAFs in vitro and in the xenograft stroma of nude mice in vivo, and its expression was also significantly upregulated in human GBC stroma co-localized with the interstitial markers fibroblast secreted protein-1 and α-smooth muscle actin. In addition, the elevated ANGPTL4 expression in GCAFs was correlated with tumor differentiation, liver metastasis, venous invasion and Nevin staging, and GBC patients with an elevated ANGPTL4 expression in GACFs were found to have a lower survival rate. CONCLUSIONS: Increased ANGPTL4 expression in GCAFs correlates with poor patient prognosis, which indicates a potential therapeutic target for human GBCs.

Nitric oxide donor sodium nitroprusside-induced transcriptional changes and hypocrellin biosynthesis of Shiraia sp. S9.

BACKGROUND: Nitric oxide (NO) is a ubiquitous signaling mediator in various physiological processes. However, there are less reports concerning the effects of NO on fungal secondary metabolites. Hypocrellins are effective anticancer photodynamic therapy (PDT) agents from fungal perylenequinone pigments of Shiraia. NO donor sodium nitroprusside (SNP) was used as a chemical elicitor to promote hypocrellin biosynthesis in Shiraia mycelium cultures. RESULTS: SNP application at 0.01-0.20 mM was found to stimulate significantly fungal production of perylenequinones including hypocrellin A (HA) and elsinochrome A (EA). SNP application could not only enhance HA content by 178.96% in mycelia, but also stimulate its efflux to the medium. After 4 days of SNP application at 0.02 mM, the highest total production (110.34 mg/L) of HA was achieved without any growth suppression. SNP released NO in mycelia and acted as a pro-oxidant, thereby up-regulating the gene expression and activity of reactive oxygen species (ROS) generating NADPH oxidase (NOX) and antioxidant enzymes, leading to the increased levels of superoxide anion (O2-) and hydrogen peroxide (H2O2). Gene ontology (GO) analysis revealed that SNP treatment could up-regulate biosynthetic genes for hypocrellins and activate the transporter protein major facilitator superfamily (MFS) for the exudation. Moreover, SNP treatment increased the proportion of total unsaturated fatty acids in the hypha membranes and enhanced membrane permeability. Our results indicated both cellular biosynthesis of HA and its secretion could contribute to HA production induced by SNP. CONCLUSIONS: The results of this study provide a valuable strategy for large-scale hypocrellin production and can facilitate further understanding and exploration of NO signaling in the biosynthesis of the important fungal metabolites.

Biotransformation of artemisinic acid to bioactive derivatives by endophytic Penicillium oxalicum B4 from Artemisia annua L.

As a biosynthetic precursor of the antimalarial drug artemisinin, artemisinic acid (AA) is abundant in Artemisia annua L. with a content of 8-10-fold higher than artemisinin, but less effective. In this study, the biotransformation of AA was carried out with an endophytic fungus Penicillium oxalicum B4 to extend its utility. After 10-day-culture of the endophyte with AA at 2 mg/mL, eight biotransformation metabolites were isolated from the culture broth, including five undescribed metabolites, namely 3α,14-dihydroxyartemisinic acid, 14-hydroxy-3-oxo-artemisinic acid, 15-hydroxy-3-oxo-artemisinic acid, 12, 15-artemisindioic acid and 1,2,3,6-tetradehydro-12, 15-artemisindioic acid. The fungal enzymes possess the selective capacity to hydroxylate, carbonylate and ketonize the allyl group of AA. The major biotransformation metabolite was the hydroxylated product 3-α-hydroxyartemisinic acid (33.3%) in the cultures of early stage (day 1-6), whereas most of the other biotransformation products were synthesized in the later stage (day 8-10). Compared with AA, some metabolites (3α,14-dihydroxyartemisinic acid, 15-hydroxy-3-oxo-artemisinic acid and 1,2,3,6-tetradehydro-12, 15-artemisindioic acid) possessed stronger cytotoxic activity to the human colon carcinoma cell line (LS174T) and promyelocytic leukemia cell line (HL-60). The metabolites 12, 15-artemisindioic acid and 3-α-hydroxyartemisinic acid exhibited significant inhibitory activity to the lipopolysaccharide-induced nitrite production of RAW 264.7 cells at 10.00 µM and 2.50 µM, respectively. The results demonstrated the potential of fungal endophytes on biotransforming AA to its bioactive derivatives.

Changes of bone turnover markers after elderly hip fracture surgery.

INTRODUCTION: Bone turnover markers (BTMs) can be used to monitor bone metabolism, while the actual clinical changing in hip fracture had not been certified to evaluate the changes of BTMs during the healing process after surgery of elderly hip fractures; and to get the effects of operation type, gender, serum 25(OH)D level, and age on bone turnover markers. MATERIALS AND METHODS: A total of 100 elderly cases with hip fracture were selected, including 74 females and 26 males, and the patients were followed to 180-230 days after surgery. Serum levels of N-propeptide of type 1 collagen (P1NP), C-terminal crosslinking telopeptides of type 1 collagen (CTX), Osteocalcin (OC), and 25 hydroxy vitamin D (25OHD) were investigated. Bone mineral density (BMD) was measured with dual-energy X-ray absorptiometry (DXA). RESULTS: (1) P1NP and CTX showed peak time at 30-60 days after operation, while OC keep going even at 180-230 days; P1NP showed less than 4 times elevation during healing, CTX and OC only had less than 2 times rise. (2) Female had higher serum CTX and OC than male, intramedullary nailing for intertrochanteric fracture patients had higher P1NP than hip replacement for femoral neck fracture patients, and both the degrees of increase were less than 50%. (3) Serum average 25(OH)D level had no effect on BTMs during the fracture healing; different from the young old (65-84 years), serum OC level of eldest older patients(≥ 85 years) decreased early in the process of fracture healing. CONCLUSIONS: BTMs reached the peak level in 30-60 days after surgery, P1NP showed less than 4 times elevation, and CTX and OC had less than 2 times rise. It was not necessary to take gender into account when observing P1NP, and it was not necessary to take fracture and operation type into account when observing CTX and OC.

Overexpression of ThMYB8 mediates salt stress tolerance by directly activating stress-responsive gene expression.

MYB transcription factors are important in abiotic stress responses; however, the detailed mechanisms are unclear. Tamarix hispida contains multiple MYB genes. The present study characterized T. hispida MYB8 (ThMYB8) during salt stress using transgenic T. hispida and Arabidopsis assays. ThMYB8 overexpression and ThMYB8 RNAi analysis demonstrated that ThMYB8 enhanced the salt stress tolerance. Transgenic Arabidopsis ectopic expression of ThMYB8 significantly increased root growth, fresh weight, and seed germination rate compared with that of the wild-type under salt stress. Physiological parameters analysis in T. hispida and Arabidopsis showed that ThMYB8 overexpressing plants had the lowest levels of O2, H2O2, cell death, malondialdehyde, and electrolyte leakage. Overexpression of ThMYB8 regulated Na+ and K+ concentrations in plant tissues while maintaining K+/Na+ homeostasis. Analysis using qRT-PCR and ChIP-PCR identified possible downstream ThMYB8-regulated genes. ThMYB8 regulated the expression of ThCYP450-2 (cytochrome p450-2), Thltk (leucine-rich repeat transmembrane protein kinase), and ThTIP (aquaporin TIP) by binding to the MBSI motif ('CAACTG') in their promoters. The results indicated that ThMYB8 enhanced salt stress tolerance in T. hispida by regulating gene expression related to the activation of stress-associated physiological changes, such as enhanced reactive oxygen species scavenging capability, maintaining K+/Na+ homeostasis, and decreasing the malondialdehyde content and lipid peroxidation cell membranes.

Gallbladder cancer-associated fibroblasts promote vasculogenic mimicry formation and tumor growth in gallbladder cancer via upregulating the expression of NOX4, a poor prognosis factor, through IL-6-JAK-STAT3 signal pathway.

BACKGROUND: Cancer-associated fibroblasts (CAFs) and vasculogenic mimicry (VM) play important roles in the occurrence and development of tumors. However, the relationship between CAFs and VM formation, especially in gallbladder cancer (GBC) has not been clarified. In this study, we investigated whether gallbladder CAFs (GCAFs) can promote VM formation and tumor growth and explored the underlying molecular mechanism. METHODS: A co-culture system of human GBC cells and fibroblasts or HUVECs was established. VM formation, proliferation, invasion, migration, tube formation assays, CD31-PAS double staining, optic/electron microscopy and tumor xenograft assay were used to detect VM formation and malignant phenotypes of 3-D co-culture matrices in vitro, as well as the VM formation and tumor growth of xenografts in vivo, respectively. Microarray analysis was used to analyze gene expression profile in GCAFs/NFs and VM (+)/VM (-) in vitro. QRT-PCR, western blotting, IHC and CIF were used to detected NOX4 expression in GCAFs/NFs, 3-D culture/co-culture matrices in vitro, the xenografts in vivo and human gallbladder tissue/stroma samples. The correlation between NOX4 expression and clinicopathological and prognostic factors of GBC patients was analyzed. And, the underlying molecular mechanism of GCAFs promoting VM formation and tumor growth in GBC was explored. RESULTS: GCAFs promote VM formation and tumor growth in GBC; and the finding was confirmed by facts that GCAFs induced proliferation, invasion, migration and tube formation of GBC cells in vitro, and promoted VM formation and tumor growth of xenografts in vivo. NOX4 is highly expressed in GBC and its stroma, which is the key gene for VM formation, and is correlated with tumor aggression and survival of GBC patients. The GBC patients with high NOX4 expression in tumor cells and stroma have a poor prognosis. The underlying molecular mechanism may be related to the upregulation of NOX4 expression through paracrine IL-6 mediated IL-6/JAK/STAT3 signaling pathway. CONCLUSIONS: GCAFs promote VM formation and tumor growth in GBC via upregulating NOX4 expression through the activation of IL-6-JAK-STAT3 signal pathway. NOX4, as a VM-related gene in GBC, is overexpressed in GBC cells and GCAFs, which is related to aggression and unfavorable prognosis of GBC patients.