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The medicinal plant Artemisia annua L. is presently the sole plant source of artemisinin, the key ingredient of first-line antimalarial drug. As the content of artemisinin in the native plant is very low, the growth and artemisinin biosynthesis of A. annua attract extensive attentions. Endophytic microbes, which spend their entire life spans within plant tissue are widespread in healthy A. annua plants. Although endophytes have emerged in recent years as the novel resources for secondary metabolites of antioxidant, antimicrobial and anticancer activities, the roles of endophytes in plant growth and metabolism have not been fully understood. This review presents the current development of researches into endophytes in model medicinal herb A. annua and focuses on the regulatory roles of endophytes. We introduced the isolation, cultivation and identification method for endophytes in A. annua. Distribution, biodiversity and novel bioactive metabolites of the endophytes were presented. Endophytes promoted the growth of A. annua through the enhanced photosynthesis, nutrient acquisition and phytohormones. The defense resistance against microbial pathogens of A. annua was partially involved with endophyte metabolites. The development of glandular secretory trichomes and the oxidative stress induced by endophytes could be key factors for enhanced artemisinin production. Understanding the endophyte-host partnerships has the potential to improve agricultural practices for A. annua growth and artemisinin production. The aim of this review is to shed light on the importance of endophytes in plant growth and the production of medicinally important secondary metabolites.

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.

Obesity is a metabolic disease and its relation with male subfertility has aroused a growing concern. However, it is unclear whether gene expression and post-translational modifications (PTMs), two vital molecular mechanisms regulating cellular functions, are associated with obesity-induced male reproductive dysfunction. In this study, male obesity with compromised sperm motility was induced by a high-fat diet (HFD) using a mouse model. The expression of motility related-genes, the level of histone modifications, and the global profiles of post-translational modifications (PTMs), were examined in testes of HFD and control mice by quantitative real-time PCR and western blot, respectively. Outer dense fiber protein 2, a major component of outer dense fibers in the sperm tail, is the most obviously down-regulated gene out of 11 evaluated genes, showing a reduction of about 50% RNA level in testes of obese male mice compared with that in control mice. Semi-quantitative analysis of the western blot demonstrated that ∼56% enrichment of di-methylated histone (H)3 lysine (K)36, ∼59% enrichment of 2-hydroxyisobutyrylated H4K8, ∼32% decrease of propionylated H3K23, ∼33% decrease of crotonylated H4K8, and ∼45% decrease of acetylated H3K122 and H4K8 were detected in testes of male HFD mice compared with that in control mice. In addition, male obesity up-regulated the testicular levels of ubiquitination by ∼18%, tyrosine nitration by ∼20%, lysine succinylation by ∼25%, lysine benzoylation by ∼28%, lysine malonylation by ∼32%, lysine glutarylation by ∼36%, lysine propionylation by ∼42%, lysine 2-hydroxyisobutyrylation by ∼45%, and SUMO1 modification by ∼59%, and down-regulated the testicular levels of O-GlcNAcylation by ∼12%, lysine crotonylation by ∼22%, and lysine acetylation by 35%. These findings indicate that altered gene expression and PTMs are associated with the obesity-induced male reproductive dysfunction.

Background Anastomosing hemangioma (AH) is a rare subtype of benign hemangioma that is most commonly found in the genitourinary tract. Due to the lack of specific clinical and radiologic manifestations, it is easily misdiagnosed preoperatively. Here, we report a case of AH arising from the left renal vein that was discovered incidentally and confirmed pathologically, and then describe its imaging characteristics from a radiologic point of view and review its clinicopathologic features and treatment. Case summary A 74-year-old woman was admitted to our department for a left retroperitoneal neoplasm measuring 2.6 cm × 2.0 cm. Her laboratory data showed no significant abnormalities. A non-contrast-enhanced computed tomography (CT) scan showed a heterogeneous density in the neoplasm. Non-contrast-enhanced magnetic resonance imaging (MRI) revealed a heterogeneous hypointensity on T1-weighed images and a heterogeneous hyperintensity on T2-weighed images. On contrast-enhanced CT and MRI scans, the neoplasm presented marked septal enhancement in the arterial phase and persistent enhancement in the portal phase, and its boundary with the left renal vein was ill-defined. Based on these clinical and radiological manifestations, the neoplasm was initially considered to be a neurogenic neoplasm in the left retroperitoneum. Finally, the neoplasm was completely resected and pathologically diagnosed as AH. Conclusion AH is an uncommon benign hemangioma. Preoperative misdiagnoses are common not only because of a lack of specific clinical and radiologic manifestations but also because clinicians lack vigilance and diagnostic experience in identifying AH. AH is not exclusive to the urogenital parenchyma. We report the first case of this neoplasm in the left renal vein. Recognition of this entity in the left renal vein can be helpful in its diagnosis and distinction from other neoplasms.

Background Hepatocellular adenoma (HCA) is very rare and has a high misdiagnosis rate through clinical and imaging examinations. We report a case of giant HCA of the left liver in a young woman that was diagnosed by medical imaging and pathology. Case summary A 21-year-old woman was admitted to our department for a giant hepatic tumor measuring 22 cm × 20 cm × 10 cm that completely replaced the left hepatic lobe. Her laboratory data only suggested mildly elevated liver function parameters and C-reactive protein levels. A computed tomography (CT) scan showed mixed density in the tumor. Magnetic resonance imaging (MRI) of the tumor revealed a heterogeneous hypointensity on T1-weighed MR images and heterogeneous hyperintensity on T2-weighed MR images. On dynamic contrast CT and MRI scans, the tumor presented marked enhancement and the subcapsular feeding arteries were clearly visible in the arterial phase, with persistent enhancement in the portal and delayed phases. Moreover, the tumor capsule was especially prominent on T1-weighted MR images and showed marked enhancement in the delayed phase. Based on these imaging manifestations, the tumor was initially considered to be an HCA. Subsequently, the tumor was completely resected and pathologically diagnosed as an HCA. Conclusion HCA is an extremely rare hepatic tumor. Preoperative misdiagnoses were common not only due to the absence of special clinical manifestations and laboratory examination findings, but also due to the clinicians' lack of practical diagnostic experience and vigilance in identifying HCA on medical images. Our case highlights the importance of the combination of contrast-enhanced CT and MRI in the preoperative diagnosis of HCA.

This study was designed to investigate the protective effects of puerarin (PUE), which work via the Wnt/β-catenin signaling pathway, and oxidative stress in the premature ovarian failure (POF) model. Two-month-old female mice were randomly divided into four groups. One group was used as the control, and the other three groups were injected with cyclophosphamide and busulfan to create POF models. Two POF treatment groups were gavaged with 100 or 200 mg/kg PUE for 28 days. Next, the ovaries were fixed, and the numbers of different stage follicles were measured, and the ovarian surface epithelium (OSE) was collected. Oct4 and Mvh expression, Wnt/β-catenin signaling pathway activity, the oxidative stress factors SOD2 and Nrf2, and the apoptosis-related proteins Bcl-2 and Bax were detected by IHC, RT-QPCR, and western blotting. We found that the number of follicles, Oct4 and Mvh expression, and Wnt/β-catenin-signaling activity were reduced in the POF groups (p

As a plasticizer widely used in society, tri-ortho-cresyl phosphate (TOCP) is reported to inhibit spermatogenesis and growth of spermatogonial stem cells. However, its effects on female reproductive system are virtually unknown. The present study investigated the effects of TOCP on ovarian follicle development by using mouse model of chronic TOCP exposure, and examined the expression of the core components of the Hippo pathway, which had been proven to be crucial for ovarian follicle development. Furthermore, through up-regulation of Hippo-yes-associated protein 1 (Yap1) in ovaries, the potential protective effects of Yap1 over-expression on TOCP-induced ovarian dysfunction were observed. The results showed that TOCP impaired ovarian function in a dose-dependent manner, and the expression of the Hippo pathway changed significantly in TOCP-exposed ovaries. Further, YAP1 over-expression partially reversed the TOCP-induced ovarian impairment. Collectively, these data indicate that the Hippo pathway is involved in the mechanism by which TOCP elicits ovarian function impairment.

In recent years, the diagnostic methods of hereditary spherocytosis (HS) have been developed rapidly, including eosin-5'-maleimide (EMA) binding test, flow cytometric osmotic fragility test, osmotic gradient ektacytometry and next-generation sequencing. EMA binding test and flow cytometric osmotic fragility test are recommended as HS screening tests due to their high sensitivity and easy operation. Osmotic gradient ektacytometry has high sensitivity and specificity, thus which can be used to distinguish HS from other hereditary membrane disease, but can not differentiate between HS and auto-immune hemolytic anemia (AIHA) and it is difficult operation, which is used as an intermediate step between screening and diagnostic tests. Next-generation sequencing can detect the molecular defects, identifying the gene encoding defective protein, thus achieving accurate diagnosis. This diagnostic test of HS has become an important diagnostic tool. The development of laboratory diagnosis has reduced misdiagnosis, and significantly improved the level of HS diagnosis.遗传性球形红细胞增多症实验室诊断进展.近年来，遗传性球形红细胞增多症（hereditary spherocytosis，HS）的诊断方法得到了快速发展，主要包括EMA结合试验、流式细胞渗透脆性试验、激光衍射法及二代测序技术。EMA结合试验、流式细胞渗透脆性试验由于其敏感性较高且容易操作，被推荐为HS的筛查试验。激光衍射法由于敏感性及特异性均高，可以区分HS和其他遗传性红细胞膜疾病，但不能区分HS和自身免疫性溶血性贫血（auto-immune hemolytic anemia，AIHA），且操作较复杂，被认为是筛查试验和诊断试验的中间步骤。二代测序可以检出患者的分子缺陷，明确编码缺陷蛋白的基因，实现精准诊断，被认为是HS的诊断试验，其快速发展已成为诊断HS的重要工具。实验室诊断技术的发展使得HS患者误诊和漏诊数量减少，显著提高了HS的诊断水平。.

The activation of primordial follicles is critical to ovarian follicle development, which directly influences female fertility and reproductive life span. Several studies have suggested a role for long noncoding RNAs (lncRNAs) in ovarian function. However, the precise involvement of lncRNAs in the initiation of primordial follicles is still unknown. Here, an in vitro culture model was used to investigate the roles of lncRNAs in primordial follicle activation. We found that primordial follicles in day 3 mouse ovaries were activated after culturing for 8 days in vitro, as indicated by ovarian morphology changes, increases in primary follicle number, and downregulation of mammalian Sterile 20-like kinase messenger RNA (mRNA) and upregulation of growth differentiation factor 9 mRNA. We next examined lncRNA expression profiles by RNA sequencing at the transcriptome level and found that among 60 078 lncRNAs, 6541 lncRNA were upregulated and 2135 lncRNA were downregulated in 3-day ovaries cultured for 8 days in vitro compared with ovaries from day 3 mice. We also found that 4171 mRNAs were upregulated and 1795 were downregulated in the cultured ovaries. Gene ontology and pathway analyses showed that the functions of differentially expressed lncRNA targets and mRNAs were closely linked with many processes and pathways related to ovary development, including cell proliferation and differentiation, developmental processes, and other signaling transduction pathways. Additionally, many novel identified lncRNAs showed inducible expression, suggesting that these lncRNAs may be good candidates for investigating mouse primordial follicle activation. This study provides a foundation for further exploring lncRNA-related mechanisms in the initiation of mouse primordial follicles.

Progestin and adipoQ receptor 7 (PAQR7) as an indispensable member of membrane progestin receptors in the Progestin and adipoQ receptor (PAQR) family that mediates nongenomic progesterone actions, initiated rapidly at the cell surface. Previous research demonstrated the distribution of PAQR7, which was mainly expressed in reproductive tissues, including ovary and testis. In the male reproductive system, PAQR7 is involved in progestin-induced sperm hypermotility. However, reports studying PAQR7 in female reproductive tissue mainly concentrate on oocyte maturation in fish, its expression in the ovary and gestational tissue, and regulation of uterine functions in mammals. Despite recent advances, many aspects of progestin signaling through PAQR7 are still unclear, especially in female reproductive tissue. Therefore, we reveal the structure and characteristics of PAQR7 and conclude the putative progestin-induced action mediated by PAQR7 in female reproductive tissue, such as the development of ovarian follicles, apoptosis of granulosa cells, oocyte maturation, and development of certain diseases, among others, to review the function of PAQR7 in the female reproductive system in detail.

Artemisinin, a natural sesquiterpenoid isolated from Artemisia annua L., is regarded as the most efficient drug against malaria in the world. Artemsinin production in NaCl-treated A. annua seedlings and its relationships with the glucose-6-phosphate dehydrogenase (G6PDH) activity and generation of H2O2 and nitric oxide (NO) were investigated. Results revealed that artemisinin content in the seedlings was increased by 79.3 % over the control after 1-month treatment with 68 mM NaCl. The G6PDH activity was enhanced in the presence of NaCl together with stimulated generation of H2O2 and NO. Application of 1.0 mM glucosamine (GlcN), an inhibitor of G6PDH, blocked the increase of NADPH oxidase and nitrate reductase (NR) activities, as well as H2O2 and NO production in A. annua seedlings under the salt stress. The induced H2O2 was found to be involved in the upgrading gene expression of two key enzymes in the later stage of artemisinin biosynthetic pathway: amorphadiene synthase (ADS) and amorpha-4,11-diene monooxygenase (CYP71AV1). The released NO being attributed mainly to the increase of NR activity, negatively interacted with H2O2 production and enhanced gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Inhibition of NO generation partly blocked NaCl-induced artemisinin accumulation, and NO donor strongly rescued the decreased content of artemisinin caused by GlcN. These results suggest that G6PDH could play a critical role in NaCl-induced responses and artemisinin biosynthesis in A. annua.

Achyranthes bidentata, a traditional perennial medicine herb in China, contains a rich source of important pharmaceutically active phytoecdysteroids including 20-hydroxyecdysone (20E). The growth of A. bidentata plants and 20E production are suppressed under abiotic stress condition. Expansins are plant cell wall-loosening proteins to play important roles in different environment stresses. In this study, we tried to clone an expansin gene (designed as AbEXP) from A. bidentata and characterize its transcriptional response to abiotic stresses (salt stress and nutrient deficiency). The cDNA of AbEXP contained 1584 nucleotides with a complete opening reading frame of 786 nucleotides to encode a peptide of 261 amino acids. The expression of the gene was detected in stems, roots or leaves, and was relatively abundant in stems. The expression levels of AbEXP under the salinity stress (25 mM NaCl) were increased 51.7-folds in roots but decreased in stems significantly. P and K deficiency enhanced the expression of AbEXP in roots and stems, but down-regulated the levels in leaves compared with control conditions. Under N deficiency AbEXP expression levels in roots and leaves were down-regulated by 9.95-fold and 4.58-fold, respectively. The expression level in roots was 43.9-fold of the control level under Fe deficiency while no significant changes of the expression were detected in stems and leaves. Although the growth of A. bidentata seedlings was inhibited under both the salinity stress and nutrient deficiency, 20E content in roots was enhanced significantly under N and P deficiency. These results demonstrate that AbEXP could be involved in the responses to abiotic stresses and manipulation of the gene expression of AbEXP could promote growth and 20E production of A. bidentata.

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.

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.

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. Electronic supplementary material The online version of this article (10.1186/s12934-019-1170-5) contains supplementary material, which is available to authorized users.

The fixed primordial follicles pool theory, which monopolized reproductive medicine for more than one hundred years, has been broken by the discovery, successful isolation and establishment of ovarian stem cells. It has brought more hope than ever of increasing the size of primordial follicle pool, improving ovarian function and delaying ovarian consenescence. Traditional view holds that stem cell aging contributes to the senility of body and organs. However, in the process of ovarian aging, the main factor leading to the decline of the reproductive function is the aging and degradation of ovarian stem cell nests, rather than the senescence of ovarian germ cells themselves. Recent studies have found that the immune system and circulatory system are involved in the formation of ovarian germline stem cell niches, as well as regulating the proliferation and differentiation of ovarian germline stem cells through cellular and hormonal signals. Therefore, we can improve ovarian function and delay ovarian aging by improving the immune system and circulatory system, which will provide an updated program for the treatment of premature ovarian failure (POF) and infertility.

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.

Achyranthes bidentata contains a rich source of important pharmaceutically active triterpene acids including oleanolic acid (OA) as a major one. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a key enzyme to provide mevalonate for biosynthesis of triterpene acids. In this study, in order to develop a sustainable source of OA, cell suspension cultures were established from shoot cultures of A. bidentata, and a full length cDNA encoding HMGR (designated as AbHMGR) was cloned and characterized. The cDNA contained 2078 nucleotides with a complete open reading frame of 1593 nucleotides, which was predicted to encode a peptide of 530 amino acids. Expression analysis by real-time PCR revealed that AbHMGR mRNA was abundant in A. bidentata roots, stems and leaves. When cultivated in Murashige and Skoog medium supplemented with 1.5 mg/L 1-naphthlcetic acid (NAA) and 1.5 mg/L 6-benzyladenine (6-BA), cells in suspension culture grew rapidly, yielding OA (100.9 mg/L) after 15 days. OA content in cell cultures was increased under the elicitation of methyl jasmonate (MeJA), yeast elicitor (YE) or cadmium chloride (CdCl2). The ultrahigh production of OA was achieved to 371.8 mg/L, a 5.4-fold of the control after 2-day treatment of 0.2 mM MeJA in the cell cultures. Quantitative real-time PCR analysis showed that AbHMGR was expressed at a higher level under the elicitation of MeJA or YE. Our results suggested that OA production may be the result of the up-regulated expression of AbHMGR under the treatment of various elicitors.

A biological method for synthesising silver nanoparticles (AgNPs) was developed using the callus extracts from Artemisia annua L. under sunlight at 25,000 lx. The AgNPs were characterised using transmission electron microscopy, atomic force microscope, X-ray diffraction and Fourier transform infrared spectroscopy. The AgNPs were mostly spherical with the size of 2.1 to 45.2 nm (average 10.9 nm). Pulse treatments of AgNPs at 125, 250 and 500 mg/l for 1 h extended vase life of cut carnation (Dianthus caryophyllus cv. Green Land) flowers. Four dominant bacteria strains Arthrobacter arilaitensis, Kocuria sp., Staphylococcus equorum and Microbacterium oxydans were isolated from the stem-ends of cut D. caryophyllus flowers. AgNP pulse inhibited significantly bacterial growth in vase solution and cut stem ends during all of the vase period. The bacteria related blockage in the stem-ends was significantly alleviated by AgNP pulse because of its higher antibacterial efficacy against the dominant bacteria. In addition, ethylene release of cut carnation flowers was inhibited in response to AgNP pulse. This is the first time that the biologically synthesised AgNPs could be applied as a promising preservative agent for cut carnation flowers.

Tanshinones are major bioactive diterpenoids of Salvia miltiorrhiza roots used for the treatment of cardiocerebral diseases. To develop effective elicitation and bioprocess strategies for the enhanced production of tanshinones, ultraviolet-B (UV-B) irradiation and methyl jasmonate (MeJA) elicitation were applied alone or in combination respectively in S. miltiorrhiza hairy root cultures. Our results showed 40-min UV-B irradiation at 40μW/cm(2) stimulated tanshinone production without any suppression of root growth, suggesting a new effective elicitor to S. miltiorrhiza hairy root cultures for tanshinone production. Moreover, the combined treatment of UV-B irradiation and MeJA exhibited synergistic effects on the expression levels of 3-hydroxy-3-methylglutaryl-CoA reductase (SmHMGR) and geranylgeranyl diphosphate synthase (SmGGPPS) genes in the tanshinone biosynthetic pathway. When hairy roots of 18-day-old cultures were exposed to the combined elicitation for 9days, the maximum production of tanshinone reached to 28.21mg/L, a 4.9-fold increase over the control. The combined elicitation of UV-B and MeJA was firstly used to stimulate the production of biologically important secondary metabolites in hairy root cultures.

Bisphenol A (BPA), an endocrine-disrupting chemical, is widely used in the manufacture of daily necessities. Previous studies showed that BPA could impair spermatogenesis. However, its effects on mature spermatozoa are not well known. We aimed to investigate the in vivo and in vitro toxicity of BPA on mature mouse spermatozoa. Different doses of BPA (0, 10, 50, and 250 μg·kg(-1)·d(-1)) were administrated orally to C57BL/6 mice for 8 weeks. Subsequently, the sperm viability, motility, acrosome reaction (AR) ratio together with the expression/current levels of the sperm-specific Ca(2+ )channel (CatSper) and K(+ )channel (KSper) were examined. These parameters were also evaluated after applying BPA directly to normal mouse sperm to appraise the toxicity of BPA to mature sperm in vitro Significant decreases in sperm motility and AR were found in BPA administrated mice, possibly resulting from a BPA caused CatSper down-regulation which was supported by both western blot and patch clamping results. Moreover, direct application of BPA to spermatozoa inhibited CatSper transiently and also caused significant reductions in sperm total motility and AR ratio. In conclusion, both in vivo administration and in vitro application of BPA impair mature sperm functions by a CatSper-relevant mechanism.

Artemisia annua L. is presently the sole natural source of antimalarial drug artemisinin. We established dual cultures of A. annua callus or regenerated plantlets with endophytic Penicillium oxalicum B4 to explore endophyte-mediated effects on artemisinin biosynthesis. Although A. annua callus could not produce artemisinin with or without the endophyte, simultaneous growth stimulation of the endophyte and inhibition of A. annua callus were observed in dual cultures. In an in vitro dual culture of endophyte-regenerated plantlets, the endophyte enhanced growth and artemisinin content of host plant. The endophyte could simultaneously induce oxidative stress in regenerated plantlets through the generation of reactive oxygen species (ROS) including O 2 •− and H2O2, which was then accompanied by the activation of antioxidant enzymes such as peroxidase, catalase and superoxide dismutase during the later stages. There was a significant increase in amorphadiene synthase (ADS) and amorpha-4,11-diene monooxygenase (CYP71AV1) transcripts in dual culture of endophyte-plantlets. The induced ROS could modulate the expression of those key genes for artemisinin biosynthesis and might be responsible for conversion of artemisinin acid into artemisinin production. Our results demonstrated that endophytic P. oxalicum B4 could be applied as a promising means to enhance artemisinin production in plants.

One new Zn(Ⅱ) metal–organic framework, [Zn3(tib)2(BPT)2]·7H2O (ZZIT-1) [ZZIT-1 = Zhengzhou Institute of technology, tib = 1,3,5-tris(1-imidazolyl)benzene, H3BPT = biphenyl-3,4′,5-tricarboxylic acid], has been synthesized and characterized. ZZIT-1 is a two-fold interpenetrating 3D framework structure. The gas/vapor adsorption behavior and luminescence of the complex have been studied. Notably, ZZIT-1 can selectively adsorb CO2 over N2, and MeOH over EtOH, and these adsorption results have been corroborated by theoretical calculations. Most interestingly, ZZIT-1 shows unique selectivity for the detection of acetone through a fluorescence quenching mechanism in the solid state at room temperature.

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.

Zn metal-organic framework material [Zn2(tib)(HBTB)2(H2O)]·2H2O (1) [tib = 1,3,5-tris(1- imidazolyl)benzene, H3BTB = 4,4′,4″-benzene-1,3,5-triyl-tribenzoic acid] was synthesized and characterized by Thermogravimetric analyses (TGA), Powder X-ray diffraction (PXRD) analyses and Bruker D8 Advance X-ray diffractometer. The results showed that 1 was a 2D network to be joined together by hydrogen bonds to generate a 3D supramolecular structure. Gas, vapor adsorption behavior of the material was studied. It is meaningful that 1 can selective sorption of CO2 and MeOH.

Background: The Hippo signaling pathway plays fundamental roles in stem cell maintenance in a variety of tissues and has thus implications for stem cell biology. Key components of this recently discovered pathway have been shown to be associated with primordial follicle activation. However, whether the Hippo signaling pathway plays a role in the development of Ovarian Germline Stem Cells (OGSCs) during physiological and pathological ovarian aging in mice is unknown. Methods: Mice at the age of 7 days (7D), or of 2, 10, or 20 months (2M, 10M, 20M) and mice at 2M treated with TPT and CY/BUS drugs were selected as physiological and pathological ovarian aging models, respectively. Immunohistochemistry was used to assess the development of follicles, and the co-localization of genes characteristic of OGSCs with MST1, LATS2 and YAP1 was assessed by immunofluorescence, western blotting and real-time PCR methods. Results: The Hippo signal pathway and MVH/OCT4 genes were co-expressed in the mouse ovarian cortex. The level and co-localization of LATS2, MST1, MVH, and OCT4 were significantly decreased with increased age, but YAP1 was more prevalent in the mouse ovarian cortex of 2M mice than 7D mice and was not observed in 20M mice. Furthermore, YAP1, MVH, and OCT4 were gradually decreased after TPT and CY/BUS treatment, and LATS2 mRNA and protein up-regulation persisted in TPT- and CY/BUS-treated mice. However, the expression of MST1 was lower in the TPT and CY/BUS groups compared with the control group. In addition, pYAP1 protein showed the highest expression in the ovarian cortexes of 7D mice compared with 20M mice, and the value of pYAP1/YAP1 decreased from 7D to 20M. Moreover, pYAP1 decreased in the TPT- and CY/BUS-treated groups, but the value of pYAP1/YAP1 increased in these groups. Conclusion: Taken together, our results show that the Hippo signaling pathway is associated with the changes that take place in OGSCs during physiological and pathological ovarian aging in mice. Thus, the Hippo signaling pathway may be involved in the development schedule of OGSCs.

Background/Aims: Ovarian germline stem cells (OGSCs) have been shown to mainly exist in the ovarian surface epithelium (OSE), but the activity changes of germline stem cells during different reproductive stages and the potential regulatory signaling pathway are still unknown. The Notch signaling pathway plays a key role in cell development, primordial follicles and stem cell proliferation. However, whether it plays a role in the proliferation of OGSCs is unknown. Here, we analyzed the activity changes of germline stem cells and the correlation between germline stem cells and the Notch signaling pathway. Methods: The expression of germline stem cell markers Mvh, Ooc4 and the Notch molecules Notch1, Hes1, and Hes5 were detected during 3 days (3d), and 2, 12, 20 months (2m, 12m, 20m) mouse ovarian surface epithelium samples. DAPT, a specific inhibitor of the Notch pathway, was used to observe the influence of Notch signaling in the germline stem cells. Results: The results showed that the levels of MVH and OCT4 decreased substantially with reproductive age in ovarian surface epithelium, and the same tendency was detected in the Notch signaling molecules Notch1, Hes1 and Hes5. Dual-IF results showed that the germline stem cell markers were co-expressed with Notch molecules in the ovarian surface epithelium. While, the expression of MVH and OCT4 were reduced when the ovaries were treated with DAPT and the levels were attenuated with increasing dose of DAPT. Conclusion: Taken together, our results indicate that the viability of OGSCs decreased with the age of the mouse ovaries, and the activity of OGSCs in the ovarian surface epithelium may be related to the Notch signaling pathway.

Background: The Hippo signaling pathway, a highly conserved cell signaling system, exists in most multicellular organisms and regulates cell proliferation, differentiation, and apoptosis. It has been reported that the members of Hippo signaling are expressed in mammalian ovaries, but the exact functions of this pathway in primordial follicle development remains unclear. Methods: To analyze the spatio-temporal correlation between the core component of Hippo pathway and the size of primordial follicle pool, Western blot, Real-time PCR and immunohistochemistry were used, and the expression and localization of MST1, LATS2 and YAP1 mRNA and protein were examined in 3 d, 1 m, 5 m, 16 m postnatal mice ovary and the culture model of mice primordial follicle in vitro. Results: Both the protein and mRNA expression of the MST1 and LATS2 were decreased significantly as mouse age increased (p < 0.05), however, the mRNA expression of them increased significantly in 16 m compared with 5 m as well as the protein expression of LATS2.The expression of YAP showed the opposite trend, and the significant protein expression of pYAP was increased before 1 m, after which no significant change was observed. Moreover, the ratio of pYAP/YAP decreased significantly. Culturing ovaries for 8 d in vitro resulted in the activation of primordial follicles in 3 d postnatal mice ovaries, and these developed into primary follicles with the expression of PCNA increasing significantly (p < 0.05). The mRNA and protein expression of MST and LATS decreased significantly (p < 0.05), and the expression of YAP increased significantly (p < 0.05, p < 0.01), whereas the ratio of pYAP/YAP decreased significantly (p < 0.05). Conclusion: The above results reveal that the expression of the core components of Hippo pathway changed during mouse follicular development, especially before and after primordial follicle activation in vitro. The primordial follicle activation may be related to the significant decrease of the ratio of pYAP1/YAP1. In conclusion, Hippo signaling pathway expressed in mice ovaries and have spatio-temporal correlation with the size of primordial follicle pool.

This study aimed to evaluate the ultrasonographic pattern of cervical lymph nodes (CLNs) and whether levothyroxine with prednisone therapy is effective for lymphadenopathy in patients with Hashimoto thyroiditis (HT).This retrospective study was looking at patients with confirmed diagnosis of HT who underwent comprehensive neck ultrasound examination. We reviewed sonographic findings in 127 patients with HT, 234 euthyroid patients with goiter, and 122 healthy subjects. In addition, 30 untreated HT patients with cervical lymphadenopathy were recruited for the levothyroxine with prednisone therapy. We rescanned the patients 9 months after treatment with levothyroxine and prednisone.Patients with HT had a higher rate of CLN detection on ultrasound than euthyroid patients with goiter and healthy subjects at cervical levels III, IV, and VI (P0.01). In addition, patients with HT had a higher rate of detection of CLNs with abnormal sonographic features than the other 2 groups, most notably at cervical levels III, IV, and VI (P0.01). After the treatment, the mean thyroid volume, thyroid nodule volume, CLN volume, symptom score, and cosmetic grade of 30 HT patients were remarkably decreased (P0.01 or P0.001).Hashimoto thyroiditis seems to be associated with an increased rate of detection of CLNs with abnormal sonographic features, particularly at cervical levels III, IV, and VI. Therapy with levothyroxine with prednisone is effective for cervical lymphadenopathy in patients with HT.

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.

A biological method for synthesising silver nanoparticles (AgNPs) was developed using the callus extracts from Artemisia annua L. under sunlight at 25,000 lx. The AgNPs were characterised using transmission electron microscopy, atomic force microscope, X‐ray diffraction and Fourier transform infrared spectroscopy. The AgNPs were mostly spherical with the size of 2.1 to 45.2 nm (average 10.9 nm). Pulse treatments of AgNPs at 125, 250 and 500 mg/l for 1 h extended vase life of cut carnation (Dianthus caryophyllus cv. Green Land) flowers. Four dominant bacteria strains Arthrobacter arilaitensis, Kocuria sp., Staphylococcus equorum and Microbacterium oxydans were isolated from the stem‐ends of cut D. caryophyllus flowers. AgNP pulse inhibited significantly bacterial growth in vase solution and cut stem ends during all of the vase period. The bacteria related blockage in the stem‐ends was significantly alleviated by AgNP pulse because of its higher antibacterial efficacy against the dominant bacteria. In addition, ethylene release of cut carnation flowers was inhibited in response to AgNP pulse. This is the first time that the biologically synthesised AgNPs could be applied as a promising preservative agent for cut carnation flowers.

Cyanotis arachnoidea contains a rich source of bioactive phytoecdysteroids (i.e. analogues of insect steroid hormones). 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) supplies mevalonate for the synthesis of many secondary metabolites including 20-hydroxyecdysone (20E), one of metabolism-enhancing phytoecdysteroids. In this study, in order to develop a sustainable source of 20E, cell suspension cultures were established from shoot cultures of C . arachnoidea , and a full length cDNA encoding HMGR (designated as CaHMGR ) was cloned and characterized. The cDNA contained 2037 nucleotides with a complete open reading frame (ORF) of 1800 nucleotides, which was predicted to encode a peptide of 599 amino acids. Expression analysis by real-time PCR revealed that CaHMGR mRNA was abundant in C . arachnoidea stems, roots and leaves. When cultivated in Murashige & Skoog medium supplemented with 0.2 mg L −1 1-naphthlcetic acid (NAA) and 3.0 mg L −1 6-benzyladenine (6-BA), C . arachnoidea cells in suspension culture grew rapidly, yielding 20E (124.14 μg L −1 ) after 12 days. The content of 20E in cell cultures elicited by 0.2 mM methyl jasmonate (MeJA), 100 mg L −1 yeast elicitor (YE) or 25 μM AgNO 3 was increased 8-, 2-, and 6-fold over the control, respectively. Quantitative real-time PCR analysis showed that CaHMGR was expressed at a higher level under the treatment of MeJA or Ag + elicitor. Our results suggested that 20E accumulation may be the result of the expression up-regulation of CaHMGR involved in the biosynthesis under the treatment of various elicitors.

Artemisinin, an endoperoxide sesquiterpene lactone, is an effective antimalarial drug isolated from Artemisia annua L. In this study, a low dose (1.44 kJm(-2)d(-1)) of UV-B radiation (280-320 nm) for short-term (1h per day for 10 days) was applied to A. annua seedlings to stimulate artemisinin production. UV-B treatment not only induced the generation of reactive oxygen species (ROS), enhanced peroxidase activity and endogenous content of abscisic acid (ABA), but stimulated the biosynthesis of artemisinin in the seedlings. Here, transcriptomic changes during UV-B radiation in A. annua were detected using an Agilent GeneChip with 43,692 probe sets. In total, 358 transcripts were identified as differentially expressed under UV-B stress, of which 172 transcripts increased and 186 transcripts decreased in abundance. In terms of biological processes, gene ontology (GO) terms including primary carbohydrate and nitrogen compound metabolic processes were enriched in UV-B-repressed genes. The up-regulated genes were enriched in response to stress, ROS generation, hormone (ethylene, ABA) stimulus and cell cycle control. The expression of key enzymes such as amorpha-4,11-diene synthase (ADS) and cytochrome P450 dependent monooxygenase/hydroxylase (CYP71AV1), and related WRKY transcription factors was up-regulated significantly for artemisinin biosynthesis. This profile of global gene expression patterns during UV-B stress will be valuable for further identification of the enzymes involved in artemisinin biosynthesis.

Glaucocalyxin (Gla) A–C are major ent-kaurane diterpenoids isolated from Isodon japonicus var. glaucocalyx (Maxim.) H. W. Li. This study investigated the possible interference of these diterpenoids with root growth and its mechanism of action in lettuce (Lactuca sativa L.) seedlings. Results indicated the dual stimulatory and inhibitory effects of Gla A and B on root growth and their phytotoxic effects on root hair development. The promotion of root growth by lower levels of Gla A and B (20–40 μM) resulted from enhanced cell length and increased mitotic activity. However, higher concentrations (80–200 μM) of Gla A and B had inhibitory effects. In addition, Gla A and B inhibited root hair development of lettuce seedlings in a dose-dependent manner at concentrations between 20 and 200 μM. Exposure of lettuce roots to Gla A and B at 200 μM increased levels of malondialdehyde and the generation of O 2 ·− , indicating lipid peroxidation and induction of oxidative stress. Activities of the antioxidant enzymes superoxide dismutase, catalase, and peroxidase were significantly elevated. Reactive oxygen species (ROS) scavengers dihydroxybenzene disulfonic acid (Tiron) and dimethylthiourea at 100 μM could efficiently alleviate the phytotoxicity induced by Gla A and B at 200 μM. These results demonstrated that the deleterious effect of Gla A and B at higher concentrations (80–200 μM) on roots may occur through the imposition of oxidative stress on cell growth and cell division. Due to the lack of an α,β-unsaturated ketone in α-methylenecyclopentanone moiety, Gla C could not induce ROS generation and exhibited no effect on the roots, even at the highest concentration (200 μM). Therefore, the α-methylenecyclopentanone moiety in the ent-kaurene diterpenoids was presented as an essential possible active center for the phytotoxicity.

There is a global need for an ideal method of male contraception. However, the development of male contraceptives has not been well successful. Research on sperm-specific ion channels, especially the recent advance obtained from electrophysiological studies, has emphasized the conception that those channels are targets with the most potential to develop non-hormonal male contraceptives. While summarizing the general options for male contraception, this review focuses on the properties and functions of sperm ion channels together with the attempts of utilizing these channels to develop male contraceptives. We believe that a deeper insight into the signaling and molecular mechanisms by which ion channels regulate sperm functions will pave the way for developing novel male-based contraceptives.