Molecular mechanisms underlying the signal perception and transduction during seed germination.

QuerySeed germination is a vital step in the life cycle of a plant, playing a significant role in seedling establishment and crop yield potential. It is also an important factor in the conservation of plant germplasm resources. This complex process is influenced by a myriad of factors, including environmental conditions, the genetic makeup of the seed, and endogenous hormones. The perception of these environmental signals triggers a cascade of intricate signal transduction events that determine whether a seed germinates or remains dormant. Despite considerable progress in uncovering the molecular mechanisms governing these processes, many questions remain unanswered. In this review, we summarize the current progress in the molecular mechanisms underlying the perception of environmental signals and consequent signal transduction during seed germination, and discuss questions that need to be addressed to better understand the process of seed germination and develop novel strategies for germplasm improvement.

Discovery of a Fungal P450 with an Unusual Two-Step Mechanism for Constructing a Bicyclo[3.2.2]nonane Skeleton.

The successful biomimetic or chemoenzymatic synthesis of target natural products (NPs) and their derivatives relies on enzyme discovery. Herein, we discover a fungal P450 BTG5 that can catalyze the formation of a bicyclo[3.2.2]nonane structure through an unusual two-step mechanism of dimerization and cyclization in the biosynthesis of beticolin 1, whose bicyclo[3.2.2]nonane skeleton connects an anthraquinone moiety and a xanthone moiety. Further investigation reveals that BTG5-T318 not only determines the substrate selectivity but also alters the catalytic reactions, which allows the separation of the reaction to two individual steps, thereby understanding its catalytic mechanism. It reveals that the first heterodimerization undergoes the common oxidation process for P450s, while the second uncommon formal redox-neutral cyclization step is proved as a redox-mediated reaction, which has never been reported. Therefore, this work advances our understanding of P450-catalyzed reactions and paves the way for expansion of the diversity of this class of NPs through synthetic biology.

Synthetic Biology-based Construction of Unnatural Perylenequinones with Improved Photodynamic Anticancer Activities.

The construction of structural complexity and diversity of natural products is crucial for drug discovery and development. To overcome high dark toxicity and poor photostability of natural photosensitizer perylenequinones (PQs) for photodynamic therapy, herein, we aim to introduce the structural complexity and diversity to biosynthesize the desired unnatural PQs in fungus Cercospora through synthetic biology-based strategy. Thus, we first elucidate the intricate biosynthetic pathways of class B PQs and reveal how the branching enzymes create their structural complexity and diversity from a common ancestor. This enables the rational reprogramming of cercosporin biosynthetic pathway in Cercospora to generate diverse unnatural PQs without chemical modification. Among them, unnatural cercosporin A displays remarkably low dark toxicity and high photostability with retention of great photodynamic anticancer and antimicrobial activities. Moreover, it is found that, unlike cercosporin, unnatural cercosporin A could be selectively accumulated in cancer cells, providing potential targets for drug development. Therefore, this work provides a comprehensive foundation for preparing unnatural products with customized functions through synthetic biology-based strategies, thus facilitating drug discovery pipelines from nature.

Designing a cercosporin-bioinspired bifunctional algicide with flocculation and photocatalysis for efficiently controlling harmful cyanobacterial blooms.

Harmful cyanobacterial blooms (HCBs) are spreading in freshwater ecosystems worldwide, adversely affecting drinking water supplies, aquatic production, recreational and tourism activities. Therefore, the efficient and environmentally friendly method is still of interest to be developed to effectively control HCBs. Inspired by the excellent algicidal activity of cercosporin (CP), a novel metal-free algaecide SiO2@EDU@CP (EDU, N-ethyl-N'-(3-dimethylaminopropyl)urea) with flocculation and photoremoval functions, was successfully designed and prepared in one-step to simultaneously introduce CP and EDU on SiO2 nanoparticles. It could rapidly form algae flocs in 20 min with 97.1% flocculation rate, and remove Microcystis aeruginosa within 12 h with 91.0% algicidal rate under 23 W compact fluorescent light irradiation without any leaked CP detected. Additionally, odorant ß-cyclocitral and toxin microcystin-LR were both photodegraded after treatment of SiO2@EDU@CP. Further mechanistic studies showed that the introduction of EDU significantly reversed the zeta potential of SiO2-COOH to achieve the flocculation through neutral charge, and the photophysical characterization of SiO2@EDU@CP revealed the improved charge separation ability to generate reactive oxygen species. More importantly, the utility of SiO2@EDU@CP was well demonstrated by its effectiveness for algae from Taihu Lake under natural sunlight and inability to regrow after treatment. This study not only establishes a bifunctional algicide SiO2@EDU@CP to efficiently control HCBs, but also provides design possibilities to develop more novel and efficient algicides for the better control of practical HCBs.

Enhanced production of aspochalasin D through genetic engineering of Aspergillus flavipes.

Aspochalasin D (AD) belongs to the polyketide-amino acid hybrid natural products with anti-cancer, anti-bacterial, and anti-fouling bioactivities. However, the low production limits its further application. In this study, AD was separated and identified from Aspergillus flavipes 3.17641. Next, besides the optimization of culture conditions using a single-factor experiment and response surface methodology, metabolic engineering was employed to increase the AD production. It shows that the deletion of the shunt gene aspoA and overexpression of the pathway-specific regulator aspoG significantly improve the AD production. Its production reached to 812.1 mg/L under the optimized conditions, with 18.5-fold increase. Therefore, this study not only provides a general method for improving the production of similar natural products in other fungi, but also enables the further biological function development of AD in agriculture and pharmaceutical. KEY POINTS: • The Aspochalasin D (AD) production was improved by optimizing culture conditions. • The deletion of the shunt gene aspoA increased the AD production. • Overexpression of the pathway regulator aspoG further improved the AD production.

The lipoxygenase OsLOX10 affects seed longevity and resistance to saline-alkaline stress during rice seedlings.

Prolonged storage of rice seeds can lead to a decrease in seed vigor and seedling quality. The Lipoxygenase (LOX) gene family is widely distributed in plants, and LOX activity is closely related to seed viability and stress tolerance. In this study, the lipoxygenase OsLOX10 gene from the 9-lipoxygenase metabolic pathway was cloned from rice, and its roles in determining seed longevity and tolerance to saline-alkaline stress caused by Na2CO3 in rice seedlings were mainly investigated. CRISPR/Cas9 knockout of OsLOX10 increased seed longevity compared with the wild-type and OsLOX10 overexpression lines in response to artificial aging. The expression levels of other 9-lipoxygenase metabolic pathway related genes, such as LOX1, LOX2 and LOX3, were increased in the LOX10 overexpression lines. Quantitative real-time PCR and histochemical staining analysis showed that the expression of LOX10 was highest in seed hulls, anthers and the early germinating seeds. KI-I2 staining of starch showed that LOX10 could catalyze the degradation of linoleic acid. Furthermore, we found that the transgenic lines overexpressing LOX10 showed better tolerance to saline-alkaline stress than the wild-type and knockout mutant lines. Overall, our study demonstrated that the knockout LOX10 mutant increased seed longevity, whereas overexpression of LOX10 enhanced tolerance to saline-alkaline stress in rice seedlings.

The natural product dehydrocurvularin induces apoptosis of gastric cancer cells by activating PARP-1 and caspase-3.

The natural product dehydrocurvularin (DSE2) is a fungal-derived macrolide with potent anticancer activity, but the mechanism is still unclear. We found that DSE2 effectively inhibited the growth of gastric cancer cells and induced the apoptosis by activating Poly(ADP-ribose) polymerase 1 (PARP-1) and caspase-3. Pharmacological inhibition and genetic knockdown with PARP-1 or caspase-3 suppressed DSE2-induced apoptosis. PARP-1 was previously reported to be cleaved into fragments during apoptosis. However, PARP-1 was barely cleaved in DSE2-induced apoptosis. DSE2 induced PARP-1 activation as indicated by rapid depletion of NAD+ and the concomitant formation of poly(ADP-ribosylated) proteins (PARs). Interestingly, the PARP-1 inhibitor (Olaparib) attenuated the cytotoxicity of DSE2. Moreover, the combination of Olaparib and Z-DEVD-FMK (caspase-3 inhibitor) further reduced the cytotoxicity. It has been shown that PARP-1 activation triggers cytoplasm-nucleus translocation of apoptosis-inducing factor (AIF). Caspase-3 inhibitors inhibited PARP-1 activation and suppressed PARP-1-induced AIF nuclear translocation. These results indicated that DSE2-induced caspase-3 activation may occur before PARP-1 activation. The ROS inhibitor, N-acetyl-cysteine, significantly inhibited the activation of caspase-3 and PARP-1, indicating that ROS overproduction contributed to DSE2-induced apoptosis. Using an in vivo approach, we further found that DSE2 significantly inhibited gastric tumor growth and promoted translocation of AIF to the nucleus. In conclusion, DSE2 induces gastric cell apoptosis by activating caspase-3 and PARP-1, and shows potent antitumor activity against human gastric carcinoma in vitro and in vivo.

Structural analysis of an anthrol reductase inspires enantioselective synthesis of enantiopure hydroxycycloketones and ß-halohydrins.

Asymmetric reduction of prochiral ketones, particularly, reductive desymmetrization of 2,2-disubstituted prochiral 1,3-cyclodiketones to produce enantiopure chiral alcohols is challenging. Herein, an anthrol reductase CbAR with the ability to accommodate diverse bulky substrates, like emodin, for asymmetric reduction is identified. We firstly solve crystal structures of CbAR and CbAR-Emodin complex. It reveals that Tyr210 is critical for emodin recognition and binding, as it forms a hydrogen-bond interaction with His162 and π-π stacking interactions with emodin. This ensures the correct orientation for the stereoselectivity. Then, through structure-guided engineering, variant CbAR-H162F can convert various 2,2-disubstituted 1,3-cyclodiketones and α-haloacetophenones to optically pure (2S, 3S)-ketols and (R)-ß-halohydrins, respectively. More importantly, their stereoselectivity mechanisms are also well explained by the respective crystal structures of CbAR-H162F-substrate complex. Therefore, this study demonstrates that an in-depth understanding of catalytic mechanism is valuable for exploiting the promiscuity of anthrol reductases to prepare diverse enantiopure chiral alcohols.

Biosynthetic Pathways of Dimeric Natural Products Containing Bisanthraquinone and Related Xanthones.

Many dimeric natural products containing bisanthraquinone and related xanthones with diverse structures and versatile bioactivities have been isolated over the years. However, the complicated biosynthetic pathways of such natural products, which have remained elusive until recently, negatively impact their mass bioproduction and biosynthetic structural modification for drug discovery. In this concept, we summarize the recent progress in gene cluster mining and biosynthetic pathway elucidation of natural products containing bisanthraquinone and related xanthones. These pioneering works may pave the way for further biosynthetic pathway elucidation and structure modification of dimeric natural products through gene and protein engineering.

Stimulating fungal cell wall integrity by exogenous ß-glucanase to improve the production of fungal natural products.

The low production of natural products (NPs) is still the critical restrictive factor in exploiting their potential large-scale applications and a barrier to isolating and identifying other meaningful products. Given that the stimulation of cell wall integrity (CWI) has become a novel strategy to modulate the production of microbial natural products, herein, exogenous ß-glucanase treatment was developed as an external cell wall ß-glucan stress to stimulate the fungal CWI, and then to improve the production of fungal NPs. It was found that the production of fungal NPs cercosporin and sophorolipids, biosynthesized by Cercospora sp. and Starmerella bombicola, respectively, was significantly improved by the treatment of ß-glucanase under a controllable dose. Moreover, it demonstrated that ß-glucanase had an ability to stimulate fungal CWI through slight fungal superficial damage, thus facilitating the secretion of NPs. We expected that this easy-operating method to stimulate fungal CWI could be feasible to improve more fungal NPs production. KEY POINTS: • Exogenous ß-glucanase stimulated the fungal cell wall integrity • Changing fungal cell walls modulated natural product production • ß-glucanase with potential universal effects on more fungal natural products.

Randomised controlled trial: effect of metformin add-on therapy on functional cure in entecavir-treated patients with chronic hepatitis B.

INTRODUCTION AND OBJECTIVES: Hepatitis B surface antigen (HBsAg) clearance, indicating functional cure or resolved chronic hepatitis B (CHB), remains difficult to achieve via nucleos(t)ide analogue monotherapy. We investigated whether metformin add-on therapy could help achieve this goal in entecavir-treated patients with hepatitis B e antigen (HBeAg)-negative CHB. PATIENTS AND METHODS: Patients with HBeAg-negative CHB who met eligibility criteria (entecavir treatment for > 12 months, HBsAg < 1000 IU/mL) were randomly assigned (1:1) to receive 24 weeks of either metformin (1000 mg, oral, once a day) or placebo (oral, once a day) add-on therapy. The group allocation was blinded for both patients and investigators. Efficacy and safety analyses were based on the intention-to-treat set. The primary outcome, serum HBsAg level (IU/mL) at weeks 24 and 36, was analysed using mixed models. RESULTS: Sixty eligible patients were randomly assigned to the metformin (n = 29) and placebo (n = 31) groups. There was no substantial between-group difference in the HBsAg level at week 24 (adjusted mean difference 0.05, 95% confidence interval -0.04 to 0.13, p = 0.278) or week 36 (0.06, -0.03 to 0.15, p = 0.187), and no significant effect of group-by-time interaction on the HBsAg level throughout the trial (p = 0.814). The occurrence of total adverse events between the two groups was comparable (9 [31.0%] of 29 vs. 5 [16.1%] of 31, p = 0.227) and no patient experienced serious adverse events during the study. CONCLUSION: Although it was safe, metformin add-on therapy did not accelerate HBsAg clearance in entecavir-treated patients with HBeAg-negative CHB.

Discovery of the Biosynthetic Pathway of Beticolin 1 Reveals a Novel Non-Heme Iron-Dependent Oxygenase for Anthraquinone Ring Cleavage.

This study used light-mediated comparative transcriptomics to identify the biosynthetic gene cluster of beticolin 1 in Cercospora. It contains an anthraquinone moiety and an unusual halogenated xanthone moiety connected by a bicyclo[3.2.2]nonane. During elucidation of the biosynthetic pathway of beticolin 1, a novel non-heme iron oxygenase BTG13 responsible for anthraquinone ring cleavage was discovered. More importantly, the discovery of non-heme iron oxygenase BTG13 is well supported by experimental evidence: (i) crystal structure and the inductively coupled plasma mass spectrometry revealed that its reactive site is built by an atypical iron ion coordination, where the iron ion is uncommonly coordinated by four histidine residues, an unusual carboxylated-lysine (Kcx377) and water; (ii) Kcx377 is mediated by His58 and Thr299 to modulate the catalytic activity of BTG13. Therefore, we believed this study updates our knowledge of metalloenzymes.

SDR7-6, a short-chain alcohol dehydrogenase/reductase family protein, regulates light-dependent cell death and defence responses in rice.

Lesion mimic mutants resembling the hypersensitive response without pathogen attack are an ideal material to understand programmed cell death, the defence response, and the cross-talk between defence response and development in plants. In this study, mic, a lesion mimic mutant from cultivar Yunyin treated with ethyl methanesulphonate (EMS), was screened. By map-based cloning, a short-chain alcohol dehydrogenase/reductase with an atypical active site HxxxK was isolated and designated as SDR7-6. It functions as a homomultimer in rice and is localized at the endoplasmic reticulum. The lesion mimic phenotype of the mutant is light-dependent. The mutant displayed an increased resistance response to bacterial blight, but reduced resistance to rice blast disease. The mutant and knockout lines showed increased reactive oxygen species, jasmonic acid content, antioxidant enzyme activity, and expression of pathogenicity-related genes, while chlorophyll content was significantly reduced. The knockout lines showed significant reduction in grain size, seed setting rate, 1000-grain weight, grain weight per plant, panicle length, and plant height. SDR7-6 is a new lesion mimic gene that encodes a short-chain alcohol dehydrogenase with atypical catalytic site. Disruption of SDR7-6 led to cell death and had adverse effects on multiple agricultural characters. SDR7-6 may act at the interface of the two defence pathways of bacterial blight and rice blast disease in rice.

PEPC of sugarcane regulated glutathione S-transferase and altered carbon-nitrogen metabolism under different N source concentrations in Oryza sativa.

BACKGROUND: Phosphoenolpyruvate carboxylase (PEPC) plays an important role in the primary metabolism of higher plants. Several studies have revealed the critical importance of PEPC in the interaction of carbon and nitrogen metabolism. However, the function mechanism of PEPC in nitrogen metabolism is unclear and needs further investigation. RESULTS: This study indicates that transgenic rice expressing the sugarcane C4-PEPC gene displayed shorter primary roots and fewer crown roots at the seedling stage. However, total nitrogen content was significantly higher in transgenic rice than in wild type (WT) plants. Proteomic analysis revealed that there were more differentially expressed proteins (DEPs) responding to nitrogen changes in transgenic rice. In particular, the most enriched pathway "glutathione (GSH) metabolism", which mainly contains GSH S-transferase (GST), was identified in transgenic rice. The expression of endogenous PEPC, GST and several genes involved in the TCA cycle, glycolysis and nitrogen assimilation changed in transgenic rice. Correspondingly, the activity of enzymes including GST, citrate synthase, 6-phosphofructokinase, pyruvate kinase and ferredoxin-dependent glutamate synthase significantly changed. In addition, the levels of organic acids in the TCA cycle and carbohydrates including sucrose, starch and soluble sugar altered in transgenic rice under different nitrogen source concentrations. GSH that the substrate of GST and its components including glutamic acid, cysteine and glycine accumulated in transgenic rice. Moreover, the levels of phytohormones including indoleacetic acid (IAA), zeatin (ZT) and isopentenyladenosine (2ip) were lower in the roots of transgenic rice under total nutrients. Taken together, the phenotype, physiological and biochemical characteristics of transgenic rice expressing C4-PEPC were different from WT under different nitrogen levels. CONCLUSIONS: Our results revealed the possibility that PEPC affects nitrogen metabolism through regulating GST, which provide a new direction and concepts for the further study of the PEPC functional mechanism in nitrogen metabolism.

Anti-Tumor Activity and Underlying Mechanism of Phomoxanthone B in MCF7 Cells.

BACKGROUND: Xanthones are a class of heterocyclic natural products, which are promising sources of anti-cancer leads. Phomoxanthone B (PXB) and Phomoxanthone A(PXA)are xanthone dimers. PXA is wellstudied as an anti-cancer agent, but PXB is not. In our study, PXB was isolated from the endophytic fungus Phomopsis sp. By254. OBJECTIVE: The purpose of this study was to identify the underlying anti-tumor mechanisms of PXB in breast cancer MCF7 cell line. METHODS: Apoptosis, cell cycle, proliferation, invasion, and migration assays were used to assess the anti-tumor activity of PXB. RNA sequencing was used to analyze the effect of PXB treatment on gene expression in MCF7 cells. RESULTS: PXB showed cytotoxicity towards a variety of tumor cells, especially MCF7 cells. PXB inhibited the migration and invasion, arrested cell cycle at G2/M phase, and induced apoptosis associated with caspase-3 activation in MCF7 cells. The detailed transcriptome analysis revealed that PXB affected several pathways related to tumorigenesis, metabolisms, and oxidative phosphorylation in MCF7 cells. KEGG transcriptome analysis revealed that PXB upregulated pro-survival signal pathways, such as MAPK, PI3K-AKT, and STAT3 pathways. We found that PXB also significantly upregulated the expression of IL24, DDIT3, and XAF1, which may contribute to PXB-induced apoptosis. We further found that PXB may downregulate oxidative phosphorylation by decreasing the expression of electron transport chain genes, especially MT-ND1, which is a potential unfavorable prognostic marker for ER-positive breast cancer. CONCLUSION: PXB exerts strong cytotoxicity against human tumor cells and has a potential for ER-positive breast cancer treatment.

Identification of Phomoxanthone A and B as Protein Tyrosine Phosphatase Inhibitors.

Phomoxanthone A and B (PXA and PXB) are xanthone dimers and isolated from the endophytic fungus Phomopsis sp. By254. The results demonstrated that PXB and PXA are noncompetitive inhibitors of SHP2 and PTP1B and competitive inhibitors of SHP1. Molecular docking studies showed that PXB and PXA interact with conserved domains of protein tyrosine phosphatases such as the ß5-ß6 loop, WPD loop, P loop, and Q loop. PXA and PXB could significantly inhibit the cell proliferation in MCF7 cells. Our results indicated that these two compounds do not efficiently inhibit PTP1B and SHP2 activity. RNA sequencing showed that PXA and PXB may inhibit SHP1 activity in MCF7 cells leading to the upregulation of inflammatory factors. In addition to PTP inhibition, PXA and PXB are multitarget compounds to inhibit the proliferation of tumor cells. In conclusion, both compounds show inhibition of cancer cells and a certain degree of inflammatory stimulation, which make them promising for tumor immunotherapy.

Brassinosteroid signaling may regulate the germination of axillary buds in ratoon rice.

BACKGROUND: Rice ratooning has traditionally been an important component of the rice cropping system in China. However, compared with the rice of the first harvest, few studies on factors effecting ratoon rice yield have been conducted. Because ratoon rice is a one-season rice cultivated using axillary buds that germinate on rice stakes and generate panicles after the first crop's harvest, its production is mainly affected by the growth of axillary buds. The objectives of this study were to evaluate the sprouting mechanism of axillary buds to improve the ratoon rice yield. RESULTS: First, we observed the differentiation and growth dynamics of axillary buds at different nodes of Shanyou 63, and found that they differentiated from bottom to top before the heading of the mother stem, and that they developed very slowly. After heading they differentiated from top to bottom, and the ones on the top, especially the top 2nd node, developed much faster than those at the other nodes. The average length and dry weight of the axillary buds were significantly greater than those at other nodes by the yellow ripe stage, and they differentiated into pistils and stamens by 6 d after the yellow ripe stage. The morphology of vegetative organs from regenerated tillers of Shanyou 63 also suggested the superior growth of the upper buds, which was regulated by hormones, in ratoon rice. Furthermore, a comprehensive proteome map of the rice axillary buds at the top 2nd node before and after the yellow ripe stage was established, and some proteins involved in steroid biosynthesis were significantly increased. Of these, four took part in brassinosteroid (BR) biosynthesis. Thus, BR signaling may play a role in the germination of axillary buds of ratoon rice. CONCLUSIONS: The data provide insights into the molecular mechanisms underlying BR signaling, and may allow researchers to explore further the biological functions of endogenous BRs in the germination of axillary buds of ratoon rice.

Origin of high thermoelectric performance with a wide range of compositions for BixSb2-xTe3 single quintuple layers.

Composition regulation of semiconductors can engineer the band structures and hence optimize their properties for better applications. Herein, we report a BixSb2-xTe3 (BST) single QL with high ZT values (∼1.2 to ∼1.5) at 300 K across a wide range of compositions 0 < x≤ 1. The improved description of band structures by the unfold method reveals the multi-valley bands near the Fermi energy. The high power factor of a p-type BST single QL originates from the robust multi-valley character of valence bands. The wide composition range is ensured by the valence band maximum dominated by the antibonding states of Sb-Te2 bonds, which would be affected little by the disorder. The optimal composition for the BST single QL is attributed to the different contributions from Sb and Bi to the valence band edge. This work paves the way for the further combination of a large power factor and low thermal conductivity across a wide range of compositions.

Thoracic aggressive vertebral hemangioma with neurologic deficit: A retrospective cohort study.

The aim of this study is to evaluate the safety and effectiveness in the treatment of thoracic aggressive vertebral hemangiomas (AVHs) with neurologic deficit by multiple surgical treatments.The clinical and radiographic data of 5 patients suffering from thoracic AVHs with neurologic deficit and treated by multiple surgical treatments, including percutaneous curved vertebroplasty (PCVP) combined with pedicle screw fixation and decompressive laminectomy, were reviewed and analyzed retrospectively.Five patients (3 women and 2 man, with a mean age of 57.40 ±â€Š11.93) were diagnosed with AVHs from July 2010 to April 2016. All of them had objective neurologic deficit, myelopathy, and back pain. They underwent multiple surgical treatments and were followed-up for 12 to 23 months. At final follow-up, Frankel Grade D was achieved in all 5 patients. Patients were free from pain and neurologic symptoms, and the functional status was improved. No major complication was found.The treatment of AVHs with neurologic deficit is a challenge for surgeons. PCVP combined with pedicle screw fixation and decompressive laminectomy is safe and effective, and can be used for AVHs with neurologic deficit. Further studies with more samples are required to validate the effectiveness and safety of PCVP combined with pedicle screw fixation and decompressive laminectomy.