Polystyrene nanoplastics induce apoptosis, autophagy, and steroidogenesis disruption in granulosa cells to reduce oocyte quality and fertility by inhibiting the PI3K/AKT pathway in female mice.

BACKGROUND: Nanoplastics (NPs) are emerging pollutants that pose risks to living organisms. Recent findings have unveiled the reproductive harm caused by polystyrene nanoparticles (PS-NPs) in female animals, yet the intricate mechanism remains incompletely understood. Under this research, we investigated whether sustained exposure to PS-NPs at certain concentrations in vivo can enter oocytes through the zona pellucida or through other routes that affect female reproduction. RESULTS: We show that PS-NPs disrupted ovarian functions and decreased oocyte quality, which may be a contributing factor to lower female fertility in mice. RNA sequencing of mouse ovaries illustrated that the PI3K-AKT signaling pathway emerged as the predominant environmental information processing pathway responding to PS-NPs. Western blotting results of ovaries in vivo and cells in vitro showed that PS-NPs deactivated PI3K-AKT signaling pathway by down-regulating the expression of PI3K and reducing AKT phosphorylation at the protein level, PI3K-AKT signaling pathway which was accompanied by the activation of autophagy and apoptosis and the disruption of steroidogenesis in granulosa cells. Since PS-NPs penetrate granulosa cells but not oocytes, we examined whether PS-NPs indirectly affect oocyte quality through granulosa cells using a granulosa cell-oocyte coculture system. Preincubation of granulosa cells with PS-NPs causes granulosa cell dysfunction, resulting in a decrease in the quality of the cocultured oocytes that can be reversed by the addition of 17ß-estradiol. CONCLUSIONS: This study provides findings on how PS-NPs impact ovarian function and include transcriptome sequencing analysis of ovarian tissue. The study demonstrates that PS-NPs impair oocyte quality by altering the functioning of ovarian granulosa cells. Therefore, it is necessary to focus on the research on the effects of PS-NPs on female reproduction and the related methods that may mitigate their toxicity.

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.

[Role of histone posttranslational modifications in the regulation of ovarian function].

The ovary is the reproductive organ of female mammals, which is responsible for producing mature eggs and secreting sex hormones. The regulation of ovarian function involves the ordered activation and repression of genes related to cell growth and differentiation. In recent years, it has been found that histone posttranslational modification can affect DNA replication, damage repair and gene transcriptional activity. Some regulatory enzymes mediating histone modification are co-activators or co-inhibitors associated with transcription factors, which play important roles in the regulation of ovarian function and the development of ovary-related diseases. Therefore, this review outlines the dynamic patterns of common histone modifications (mainly acetylation and methylation) during the reproductive cycle and their regulation of gene expression for important molecular events, focusing on the mechanisms of follicle development and sex hormone secretion and function. For example, the specific dynamics of histone acetylation are important for the arrest and resumption of meiosis in oocytes, while histone (especially H3K4) methylation affects the maturation of oocytes by regulating their chromatin transcriptional activity and meiotic progression. Besides, histone acetylation or methylation can also promote the synthesis and secretion of steroid hormones before ovulation. Finally, the abnormal histone posttranslational modifications in the development of two common ovarian diseases (premature ovarian insufficiency and polycystic ovary syndrome) are briefly described. It will provide a reference basis for understanding the complex regulation mechanism of ovarian function and further exploring the potential therapeutic targets of related diseases.

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.

Nitric Oxide and Hydrogen Peroxide Signaling in Extractive Shiraia Fermentation by Triton X-100 for Hypocrellin A Production.

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.

Hippo pathway functions as a downstream effector of AKT signaling to regulate the activation of primordial follicles in mice.

Clarifying the molecular mechanisms by which primordial follicles are initiated is crucial for the prevention and treatment of female infertility and ovarian dysfunction. The Hippo pathway has been proven to have a spatiotemporal correlation with the size of the primordial follicle pool in mice in our previous work. But the role and underlying mechanisms of the Hippo pathway in primordial follicle activation remain unclear. Here, the localization and expression of the core components were examined in primordial follicles before and after activation. And the effects of the Hippo pathway on primordial follicle activation were determined by genetically manipulating yes-associated protein 1 (Yap1), the key transcriptional effector. Furthermore, an AKT specific inhibitor (MK2206) was added to determine the interaction between the Hippo pathway and AKT, an important signaling regulator of ovarian function. Results showed that the core components of the Hippo pathway were localized in both primordial and primary follicles and the expression levels of them changed significantly during the initiation of primordial follicles. Yap1 knockdown suppressed primordial follicle activation, while its overexpression led to the opposite trend. MK2206 downregulated the ratio of P-MST/MST1 and upregulated the ratio of P-YAP1/YAP1 significantly, whereas Yap1-treatment had no influence on AKT. In addition, YAP1 upregulation partially rescued the suppression of the primordial follicle activation induced by MK2206. Our findings revealed that the Hippo-YAP1 regulates primordial follicular activation, which is mediated by AKT signaling in mice, thus providing direct and new evidence to highlight the role of Hippo signaling in regulating ovarian follicles development.

Inducing perylenequinone production from a bambusicolous fungus Shiraia sp. S9 through co-culture with a fruiting body-associated bacterium Pseudomonas fulva SB1.

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.

Comparative Transcriptome Analysis Identifies Genes Putatively Involved in 20-Hydroxyecdysone Biosynthesis in Cyanotis arachnoidea.

Cyanotis arachnoidea contains a rich array of phytoecdysteroids, including 20-hydroxyecdysone (20E), which displays important agrochemical, medicinal, and pharmacological effects. To date, the biosynthetic pathway of 20E, especially the downstream pathway, remains largely unknown. To identify candidate genes involved in 20E biosynthesis, the comparative transcriptome of C. arachnoidea leaf and root was constructed. In total, 86.5 million clean reads were obtained and assembled into 79,835 unigenes, of which 39,425 unigenes were successfully annotated. The expression levels of 2427 unigenes were up-regualted in roots with a higher accumulation of 20E. Further assignments with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways identified 49 unigenes referring to the phytoecdysteroid backbone biosynthesis (including 15 mevalonate pathway genes, 15 non-mevalonate pathway genes, and 19 genes for the biosynthesis from farnesyl pyrophosphate to cholesterol). Moreover, higher expression levels of mevalonate pathway genes in roots of C. arachniodea were confirmed by real-time quantitative PCR. Twenty unigenes encoding CYP450s were identified to be new candidate genes for the bioreaction from cholesterol to 20E. In addition, 90 transcription factors highly expressed in the roots and 15,315 unigenes containing 19,158 simple sequence repeats (SSRs) were identified. The transcriptome data of our study provides a valuable resource for the understanding of 20E biosynthesis in C. arachnoidea.

Exposure to Cadmium Impairs Sperm Functions by Reducing CatSper in Mice.

BACKGROUND: Cadmium (Cd), a common environmental heavy metal and endocrine disruptor, is known to exert toxic effects on the testes. However, the mechanisms accounting for its toxicity in mature spermatozoa remain unclear. METHODS: Adult male C57BL/6 mice were orally administered with CdCl2 for 5 weeks at 3 mg·kg-1·day-1. Additionally, mouse spermatozoa were incubated in vitro with different doses of CdCl2 (0, 10, 50, 250 µM). Several sperm functions including the sperm motility, viability and acrosome reaction (AR) ratio were then examined. Furthermore, the current and expression levels of both the sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper) were evaluated by patch-clamping and western blotting, respectively. RESULTS: Our data showed that the motility, viability and AR of sperm exposed to cadmium significantly decreased in vivo and in vitro. Interestingly, these changes were correlated with changes in CatSper but not KSper. CONCLUSION: The findings indicate sperm dysfunction during both chronic and acute cadmium exposure as well as a specific role for CatSper in the reproductive toxicity of cadmium.

Diethylstilbestrol activates CatSper and disturbs progesterone actions in human spermatozoa.

STUDY QUESTION: Is diethylstilbestrol (DES), a prototypical endocrine-disrupting chemical (EDC), able to induce physiological changes in human spermatozoa and affect progesterone actions? SUMMARY ANSWER: DES promoted Ca2+ flux into human spermatozoa by activating the cation channel of sperm (CatSper) and suppressed progesterone-induced Ca2+ signaling, tyrosine phosphorylation and sperm functions. WHAT IS KNOWN ALREADY: DES significantly impairs the male reproductive system both in fetal and postnatal exposure. Although various EDCs affect human spermatozoa in a non-genomic manner, the effect of DES on human spermatozoa remains unknown. STUDY DESIGN, SIZE, DURATION: Sperm samples from normozoospermic donors were exposed in vitro to a range of DES concentrations with or without progesterone at 37°C in a 5% CO2 incubator to mimic the putative exposure to this toxicant in seminal plasma and the female reproductive tract fluids. The incubation time varied according to the experimental protocols. All experiments were repeated at least five times using different individual sperm samples. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human sperm intracellular calcium concentrations ([Ca2+]i) were monitored with a multimode plate reader following sperm loading with Ca2+ indicator Fluo-4 AM, and the whole-cell patch-clamp technique was performed to record CatSper and alkalinization-activated sperm K+ channel (KSper) currents. Sperm viability and motility parameters were assessed by an eosin-nigrosin staining kit and a computer-assisted semen analysis system, respectively. The ability of sperm to penetrate into viscous media was examined by penetration into 1% methylcellulose. The sperm acrosome reaction was measured using chlortetracycline staining. The level of tyrosine phosphorylation was determined by western blot assay. MAIN RESULTS AND THE ROLE OF CHANCE: DES exposure rapidly increased human sperm [Ca2+]i dose dependently and even at an environmentally relevant concentration (100 pM). The elevation of [Ca2+]i was derived from extracellular Ca2+ influx and mainly mediated by CatSper. Although DES did not affect sperm viability, motility, penetration into viscous media, tyrosine phosphorylation or the acrosome reaction, it suppressed progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation. Consequently, DES (1-100 µM) significantly inhibited progesterone-induced human sperm penetration into viscous media and acrosome reaction. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although DES has been shown to disturb progesterone actions on human spermatozoa, this study was performed in vitro, and caution must be taken when extrapolating the results in practical applications. WIDER IMPLICATIONS OF THE FINDINGS: The present study revealed that DES interfered with progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation, ultimately inhibited progesterone-induced human sperm functions and, thereby, might impair sperm fertility. The non-genomic manner in which DES disturbs progesterone actions may be a potential mechanism for some estrogenic endocrine disruptors to affect human sperm function. STUDY FUNDING/COMPETING INTERESTS: National Natural Science Foundation of China (No. 31400996); Natural Science Foundation of Jiangxi, China (No. 20161BAB204167 and No. 20142BAB215050); open project of National Population and Family Planning Key Laboratory of Contraceptives and Devices Research (No. 2016KF07) to T. Luo; National Natural Science Foundation of China (No. 81300539) to L.P. Zheng. The authors have no conflicts of interest to declare.

Antioxidant and DNA Damage Protecting Activity of Exopolysaccharides from the Endophytic Bacterium Bacillus cereus SZ1.

An endophytic bacterium was isolated from the Chinese medicinal plant Artemisia annua L. The phylogenetic and physiological characterization indicated that the isolate, strain SZ-1, was Bacillus cereus. The endophyte could produce an exopolysaccharide (EPS) at 46 mg/L. The 1,1-diphenyl-2-picrylhydracyl (DPPH) radical scavenging activity of the EPS reached more than 50% at 3-5 mg/mL. The EPS was also effective in scavenging superoxide radical in a concentration dependent fashion with an EC50 value of 2.6 mg/mL. The corresponding EC50 for scavenging hydroxyl radical was 3.1 mg/mL. Moreover, phenanthroline-copper complex-mediated chemiluminescent emission of DNA damage was both inhibited and delayed by EPS. The EPS at 0.7-1.7 mg/mL also protected supercoiled DNA strands in plasmid pBR322 against scission induced by Fenton-mediated hydroxyl radical. The preincubation of PC12 cells with the EPS prior to H2O2 exposure increased the cell survival and glutathione (GSH) level and catalase (CAT) activities, and decreased the level of malondialdehyde (MDA) and lactate dehydrogenase (LDH) activity in a dose-dependent manner, suggesting a pronounced protective effect against H2O2-induced cytotoxicity. Our study indicated that the EPS could be useful for preventing oxidative DNA damage and cellular oxidation in pharmaceutical and food industries.

[Signaling molecules and pathways involved in maintaining the quiescence of primordial follicles].

Reproductive lifespan in female mammals is related to the size of primordial follicles pool, which relies on the balance between activated and quiescent primordial follicles. Therefore, the molecular mechanisms of recruiting and maintaining quiescence of primordial follicles have become hot research topics recently. Multiple studies have shown that genetic mutations, local ovarian autocrine and paracrine factors, proto-oncogene and tumor-suppressor genes are involved in the maintenance of balance between quiescent and activated primordial follicles. In the present review, we summarize recent research progress of the important signaling molecules and pathways that maintain the quiescence of primordial follicles.

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.

Undifferentiated high-grade pleomorphic sarcoma of the common bile duct: A case report and review of literature.

BACKGROUND: Undifferentiated pleomorphic sarcoma (UPS) is a rare malignant mesenchymal tumor with a poor prognosis. It mainly occurs in the extremities, trunk, head and neck, and retroperitoneum regions. Owing to the lack of specific clinical manifestations and imaging features, UPS diagnosis mainly depends on pathological and immunohistochemical examinations for exclusive diagnosis. Here we report an extremely rare case of high-grade UPS in the common bile duct (CBD). There are limited available data on such cases. CASE SUMMARY: A 70-year-old woman was admitted to our department with yellow eyes and urine accompanied by upper abdominal distending pain for 2 wk. Her laboratory data suggested significantly elevated hepatorenal function levels. The imaging data revealed calculous cholecystitis, intrahepatic and extrahepatic bile duct dilation with extrahepatic bile duct calculi, and a space-occupying lesion at the distal CBD. After endoscopic biliary stenting and symptomatic support therapy, CBD exploration and biopsy were performed. The frozen section indicated malignant spindle cell tumor of the CBD mass, and further radical pancreaticoduodenectomy was performed. Finally, the neoplasm was diagnosed as a high-grade UPS combined with the light-microscopic morphology and immunohistochemical results. CONCLUSION: This extremely rare case highlighted the need for increasing physicians' vigilance, reducing the odds of misdiagnosis, and providing appropriate treatment strategies.

RNA interference mediated pten knock-down inhibit the formation of polycystic ovary.

Pten (phosphatase and tensin homolog deleted on chromosome 10), a kind of tumor suppressor gene, plays important roles in female reproductive system. But its expression and roles in the formation of polycystic ovaries are yet to be known. In this study, we constructed a rat model of PCOS using norethindrone and HCG injections and found the expressions of pten mRNA and PTEN protein increased significantly in the polycystic ovary tissue by immunohistochemistry, RT-PCR, and western blot. Furthermore, the results showed that in vivo ovaries could be effectively transfected by lentiviral vectors through the ovarian microinjection method and indicated that pten shRNA may inhibit the formation of polycystic ovaries by pten down-regulation. Our study provides new information regarding the role of PTEN in female reproductive disorders, such as polycystic ovary syndrome.

[Jiedu sangen decoction intervened carcinoma-associated fibroblasts and inhibited migration and invasion of colon cancer: an experimental research].

OBJECTIVE: To observe the effect of Jiedu Sangen Decoction (JSD, consisting of Polygonum cuspidatum, Geum Japonicum Thumnb, Radix Actinidiae Chinensis) on the migration capability of colon cancer CT-26 cells were observed, and on expressions of carcinoma-associated fibroblasts (CAFs) such as transforming growth factor-beta1 (TGF-beta1), matrix metalloproteinase 9 (MMP-9), and alpha-smooth muscle actin (alpha-SMA). METHODS: The BALB/C mice were subcutaneously inoculated with colon cancer CT-26 cells (1.2 x 10(6)mL) and then randomly divided into 3 groups, i.e., the normal control group, the model group, the JSD treated group. The effects of three different serums on the migration ability of colon cancer CT-26 cells were observed using Transwell. The expression quantities of TGF-beta1 and MMP-9 in the supernatant of CAFs were detected using ELISA. The mRNA expression quantities of TGF-beta1 and alpha-SMA in CAFs were detected by real-time fluorescence quantitative PCR. RESULTS: The number of semi-permeable film cells in the JSD treated group significantly decreased, when compared with the model group, showing statistical significance (P < 0.01). Compared with the model group, the expressions of TGF-beta1 and MMP-9 in the supernatant of CAFs decreased in the JSD treated group at 24 and 48 h, showing statistical difference (P < 0.05, P < 0.01). Compared with the model group, the mRNA expressions of TGF-beta1 and alpha-SMA in the JSD treated group obviously decreased, showing statistical difference (P < 0.01). CONCLUSION: JSD could decrease expressions of TGF-beta1 and MMP-9 in the supernatant of CAFs, lower mRNA expressions of alpha-SMA and TGF-beta1, which might be possible mechanisms for inhibiting the migration and invasion of tumor cells.

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.

The proto-oncogene c-src is involved in primordial follicle activation through the PI3K, PKC and MAPK signaling pathways.

BACKGROUND: C-src is an evolutionarily conserved proto-oncogene that regulates cell proliferation, differentiation and apoptosis. In our previous studies, we have reported that another proto-oncogene, c-erbB2, plays an important role in primordial follicle activation and development. We also found that c-src was expressed in mammalian ovaries, but its functions in primordial follicle activation remain unclear. The objective of this study is to investigate the role and mechanism of c-src during the growth of primordial follicles. METHODS: Ovaries from 2-day-old rats were cultured in vitro for 8 days. Three c-src-targeting and one negative control siRNA were designed and used in the present study. PCR, Western blotting and primordial follicle development were assessed for the silencing efficiency of the lentivirus c-src siRNA and its effect on primordial follicle onset. The expression of c-src mRNA and protein in primordial follicle growth were examined using the PCR method and immunohistochemical staining. Furthermore, the MAPK inhibitor PD98059, the PKC inhibitor Calphostin and the PI3K inhibitor LY294002 were used to explore the possible signaling pathways of c-src in primordial folliculogenesis. RESULTS: The results showed that Src protein was distributed in the ooplasmic membrane and the granulosa cell membrane in the primordial follicles, and c-src expression level increased with the growth of primordial follicle. The c-src -targeting lentivirus siRNAs had a silencing effect on c-src mRNA and protein expression. Eight days after transfection of rat ovaries with c-src siRNA, the GFP fluorescence in frozen ovarian sections was clearly discernible under a fluorescence microscope, and its relative expression level was 5-fold higher than that in the control group. Furthermore, the c-src-targeting lentivirus siRNAs lowered its relative expression level 1.96 times. We also found that the development of cultured primordial follicles was completely arrested after c-src siRNA knockdown of c-src expression. Furthermore, our studies demonstrated that folliculogenesis onset was inhibited by Calphostin, PD98059 or LY294002 treatment,but none of them down-regulated c-src expression. In contrast, the expression levels of p-PKC, p-ERK1/2 and p-PI3K in the follicles were clearly decreased by c-src siRNA transfection. Correspondingly, both Calphostin and LY294002 treatment resulted in a decrease in the p-PKC level in follicles, but no change was observed in the PD98059 group. Finally, LY294002 treatment decreased the p-PI3K expression level in the follicles, but no changes were observed in the PD98059 and Calphostin groups. CONCLUSIONS: C-src plays an important role in regulating primordial follicle activation and growth via the PI3K-PKC- ERK1/2 pathway.

Nitric oxide elicitation for secondary metabolite production in cultured plant cells.

Nitric oxide (NO) is an important signal molecule in stress responses. Accumulation of secondary metabolites often occurs in plants subjected to stresses including various elicitors or signal molecules. NO has been reported to play important roles in elicitor-induced secondary metabolite production in tissue and cell cultures of medicinal plants. Better understanding of NO role in the biosynthesis of such metabolites is very important for optimizing the commercial production of those pharmaceutically significant secondary metabolites. This paper summarizes progress made on several aspects of NO signal leading to the production of plant secondary metabolites, including various abiotic and biotic elicitors that induce NO production, elicitor-triggered NO generation cascades, the impact of NO on growth development and programmed cell death in medicinal plants, and NO-mediated regulation of the biosynthetic pathways of such metabolites. Cross-talks among NO signaling and reactive oxygen species, salicylic acid, and jasmonic acid are discussed. Some perspectives on the application of NO donors for induction of the secondary metabolite accumulation in plant cultures are also presented.

Design of fast-onset antidepressant by dissociating SERT from nNOS in the DRN.

Major depressive disorder (MDD) is one of the most common mental disorders. We designed a fast-onset antidepressant that works by disrupting the interaction between the serotonin transporter (SERT) and neuronal nitric oxide synthase (nNOS) in the dorsal raphe nucleus (DRN). Chronic unpredictable mild stress (CMS) selectively increased the SERT-nNOS complex in the DRN in mice. Augmentation of SERT-nNOS interactions in the DRN caused a depression-like phenotype and accounted for the CMS-induced depressive behaviors. Disrupting the SERT-nNOS interaction produced a fast-onset antidepressant effect by enhancing serotonin signaling in forebrain circuits. We discovered a small-molecule compound, ZZL-7, that elicited an antidepressant effect 2 hours after treatment without undesirable side effects. This compound, or analogous reagents, may serve as a new, rapidly acting treatment for MDD.