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.

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.

[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.

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.

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.

Anastomosing hemangioma arising from the left renal vein: A case report.

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.

[Progress on Laboratory Diagnosis of Hereditary Spherocytosis--Review].

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.

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.

Radiological aspects of giant hepatocellular adenoma of the left liver: A case report.

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.

CCN2 Mediates S1P-Induced Upregulation of COX2 Expression in Human Granulosa-Lutein Cells.

CCN1 and CCN2 are members of the CCN family and play essential roles in the regulation of multiple female reproductive functions, including ovulation. Cyclooxygenase-2 (COX2) is a critical mediator of ovulation and can be induced by sphingosine-1-phosphate (S1P) through the S1P1/3-mediated Yes-associated protein (YAP) signaling. However, it is unclear whether CCN1 or CCN2 can mediate S1P-induced upregulation of COX2 expression and increase in prostaglandin E2 (PGE2) production in human granulosa-lutein (hGL) cells. In the present study, we investigated the effects of S1P on the expressions of CCN1 and CCN2 in hGL cells. Additionally, we used a dual inhibition approach (siRNA-mediated silencing and small molecular inhibitors) to investigate the molecular mechanisms of S1P effects. Our results showed that S1P treatment significantly upregulated the expression of CCN1 and CCN2 in a concentration-dependent manner in hGL cells. Additionally, inhibition or silencing of S1P1, but not S1P3, completely abolished the S1P-induced upregulation of CCN2 expression. Furthermore, we demonstrated that S1P-induced nuclear translocation of YAP and inhibition or silencing of YAP completely abolished the S1P-induced upregulation of CCN1 and CCN2 expression. Notably, silencing of CCN2, but not CCN1, completely reversed the S1P-induced upregulation of COX2 expression and the increase in PGE2 production. Thus, CCN2 mediates the S1P-induced upregulation of COX2 expression through the S1P1-mediated signaling pathway in hGL cells. Our findings expand our understanding of the molecular mechanism underlying the S1P-mediated cellular activities in the human ovary.

Bacteria Associated With Shiraia Fruiting Bodies Influence Fungal Production of Hypocrellin A.

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.

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.

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.

Modified unilateral video-assisted thoracoscopic extended thymectomy for myasthenia gravis using 5-mm incisions: A case report.

RATIONALE: Myasthenia gravis (MG) is the most common cause of acquired neuromuscular junction disorder. Thymectomy has been established as an effective therapy for MG, as it attenuates the natural course of the disease and may result in complete remission. PATIENT CONCERNS: We report the case of a 22-year-old female with a 6-year history of MG presented with bilateral ptosis, diplopia, and intermittent dysphagia. She denied shortness of breath, dysarthria, and fatigue. DIAGNOSES: She had been diagnosed with MG 6 years previously at the Neurology Department of our hospital. A computed tomography (CT) scan revealed thymic hyperplasia INTERVENTIONS:: She was treated with modified unilateral VATET that minimized incision size. OUTCOMES: Unilateral VATET was performed using two 5-mm incisions to minimize pressure on intercostal soft tissues/nerves and reduce postoperative pain. LESSONS: The lesson learnt from this case report is that this modified VATET method could be a useful approach to the management of non-thymomatous MG. The ability to achieve complete dissection with good cosmetic results may lead to wider acceptance of this technique by patients with MG and their neurologists for earlier thymectomy and improved outcomes. Additional studies are needed to determine the superiority of this approach to established methods.

[Activation of nuclear factor-κB subunit p50/p65 enhances gefitinib resistance of lung adenocarcinoma H1650 cell line].

OBJECTIVE: To explore the intrinsic connection between activation of classical nuclear factor-κB (NF-κB) pathway and gefitinib resistance in human lung adenocarcinoma H1650 cells. METHODS: Human lung adenocarcinoma H1650 cells were exposed to gefitinib continuously for 60 days to obtain resistant H1650 cells. The expressions of P-IκBα, P-p50 and P-p65 in the cytoplasm or nuclei were detected using Western blotting in human lung adenocarcinoma HCC827 cells, parental H1650 cells and gefitinib-resistant H1650 cells. The effects of gefitinib alone or in combination with PDTC on the survival rate and expressions of NF-κB P-p50 and P-p65 were compared among the 3 cell lines. RESULTS: Gefitinib-resistant H1650 cells showed increased cytoplasmic and nuclear P-IκBα expressions. The expressions of P-p50 and P-p65 differed significantly among the 3 cell line, decreasing in the order of resistant H1650 cells, parental H1650 cells, and gefitinib sensitive HCC827 cell lines (P<0.05 or 0.01). Treatment with gefitinib alone resulted in a significantly lower cell inhibition rate in resistant H1650 cells than in the parental H1650 cells (P<0.05) and HCC827 cells (P<0.01). The resistant H1650 cells had a significantly higher expression of P-p50 and P-p65 than other two cell lines (P<0.05). In both the resistant and parental H1650 cells, gefitinib significantly lowered P-p50 and P-p65 expressions (P<0.05 or 0.01), and the combined treatment with gefitinib and PDTC significantly decreased the cell survival rate and further lowered the cytoplasmic and nuclear expressions of P-p50 and P-p65 (P<0.01 or 0.01). CONCLUSION: The activation of classical NF-κB pathway is a key factor contributing to transformation of the parental H1650 cells into gefitinib-resistant cells. Gefitinib combined with PDTC can inhibit P-IκBα production and NF-κB P-p50 and P-p65 activation to suppress the survival of residual H1650 cells and the generation of gefitinib-resistant cells.

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.

Sonographic Features of Cervical Lymph Nodes in Patients With Hashimoto Thyroiditis and the Impacts From the Levothyroxine With Prednisone Therapy.

OBJECTIVE: 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). METHODS: 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. RESULTS: 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 (P < 0.01). 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 (P < 0.01). After the treatment, the mean thyroid volume, thyroid nodule volume, CLN volume, symptom score, and cosmetic grade of 30 HT patients were remarkably decreased (P < 0.01 or P < 0.001). CONCLUSIONS: 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.

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.

Biosynthesis of silver nanoparticles using Artemisia annua callus for inhibiting stem-end bacteria in cut carnation flowers.

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.