Effect of Curcuma longa extract on reproduction function in mice and testosterone production in Leydig cells.

Curcuma longa, best known for its culinary application as the main constituent of curry powder, has shown potential impact on the reproductive system. This study aimed to investigate the efficacy of Curcuma longa extract (CLE) on Kidney-Yang deficiency mice induced by hydrocortisone and the possible roles in testosterone secretion in Leydig cells. We evaluated male sexual behaviour, reproductive organ weight, testosterone levels, and histological tissue changes in hydrocortisone-induced mice. CLE effectively reversed hydrocortisone-induced Kidney-Yang deficiency syndrome by improving sexual behaviour, testis and epididymis weight, testosterone levels and reducing pathological damage. Our in vitro study further indicated that CLE stimulated testosterone production via upregulating the mRNA and protein expression of steroidogenic enzymes in Leydig cells. It significantly improved H89-inhibited protein expression of StAR and cAMP-response element-binding (CREB), as well as melatonin-suppressed StAR protein expression. The data obtained from this study suggest that CLE could alleviate Kidney-Yang deficiency symptoms and stimulate testosterone production by upregulating the steroidogenic pathway. This research identifies CLE as a potential nutraceutical option for addressing testosterone deficiency diseases.

Chromosome-level reference genome of Tetrastigma hemsleyanum (Vitaceae) provides insights into genomic evolution and the biosynthesis of phenylpropanoids and flavonoids.

Here, we present a high-quality chromosome-scale genome assembly (2.19 Gb) and annotation of Tetrastigma hemsleyanum, a perennial herbaceous liana native to subtropical China with diverse medicinal applications. Approximately 73% of the genome was comprised of transposable elements (TEs), of which long terminal repeat retrotransposons (LTR-RTs) were a predominant group (69% of the genome). The genome size increase of T. hemsleyanum (relative to Vitis species) was mostly due to the proliferation of LTR-RTs. Of the different modes of gene duplication identified, transposed duplication (TRD) and dispersed duplication (DSD) were the predominant ones. Genes, particularly those involved in the phenylpropanoid-flavonoid (PF) pathway and those associated with therapeutic properties and environmental stress resistance, were significantly amplified through recent tandem duplications. We dated the divergence of two intraspecific lineages in Southwest (SW) versus Central-South-East (CSE) China to the late Miocene (approximately 5.2 million years ago). Of those, the former showed more upregulated genes and metabolites. Based on resequencing data of 38 individuals representing both lineages, we identified various candidate genes related to 'response to stimulus' and 'biosynthetic process', including ThFLS11, which is putatively involved in flavonoid accumulation. Overall, this study provides abundant genomic resources for future evolutionary, ecological, and functional genomics studies in T. hemsleyanum and related species.

A transcription factor of SHI family AaSHI1 activates artemisinin biosynthesis genes in Artemisia annua.

BACKGROUND: Transcription factors (TFs) of plant-specific SHORT INTERNODES (SHI) family play a significant role in regulating development and metabolism in plants. In Artemisia annua, various TFs from different families have been discovered to regulate the accumulation of artemisinin. However, specific members of the SHI family in A. annua (AaSHIs) have not been identified to regulate the biosynthesis of artemisinin. RESULTS: We found five AaSHI genes (AaSHI1 to AaSHI5) in the A. annua genome. The expression levels of AaSHI1, AaSHI2, AaSHI3 and AaSHI4 genes were higher in trichomes and young leaves, also induced by light and decreased when the plants were subjected to dark treatment. The expression pattern of these four AaSHI genes was consistent with the expression pattern of four structural genes of artemisinin biosynthesis and their specific regulatory factors. Dual-luciferase reporter assays, yeast one-hybrid assays, and transient transformation in A. annua provided the evidence that AaSHI1 could directly bind to the promoters of structural genes AaADS and AaCYP71AV1, and positively regulate their expressions. This study has presented candidate genes, with AaSHI1 in particular, that can be considered for the metabolic engineering of artemisinin biosynthesis in A. annua. CONCLUSIONS: Overall, a genome-wide analysis of the AaSHI TF family of A. annua was conducted. Five AaSHIs were identified in A. annua genome. Among the identified AaSHIs, AaSHI1 was found to be localized to the nucleus and activate the expression of structural genes of artemisinin biosynthesis including AaADS and AaCYP71AV1. These results indicated that AaSHI1 had positive roles in modulating artemisinin biosynthesis, providing candidate genes for obtaining high-quality new A. annua germplasms.

Manganese dioxide decorated kiwi peel powder for efficient removal of lead from aqueous solutions, blood and Traditional Chinese Medicine extracts.

For human health and environment safety, it is of great significance to develop novel materials with high effectiveness for removal of lead from not only aqueous solutions but also human body and traditional Chinese medicines. Here, functional kiwi peel composite, manganese dioxide decorated kiwi peel powder (MKPP), is proposed for the removal of Pb2+ effectively. The adsorption of Pb2+ in aqueous solution is a highly selective and endothermic process and kinetically follows a pseudo-second-order model, which can reach equilibrium with the capacity of 192.7 mg/g within 10 min. Comprehensive factors of hydration energy, charge-to-radius ratio and softness of Pb2+ make a stronger affinity between MKPP and Pb2+. The possible adsorption mechanism involves covalent bond, electrostatic force and chelation, etc. MKPP can be efficiently regenerated and reused with high adsorption efficiency after five cycles. Besides, MKPP can remove over 97% of Pb2+ from real water samples. MKPP can also alleviate lead poisoning to a certain extent and make the Pb level of TCM extract meet the safety standard. This work highlights that MKPP is a promising adsorbent for the removal of Pb2+ and provides an efficient strategy for reusing kiwi peel as well as dealing with the problem of Pb pollution.

Molecular regulation of the key specialized metabolism pathways in medicinal plants.

The basis of modern pharmacology is the human ability to exploit the production of specialized metabolites from medical plants, for example, terpenoids, alkaloids, and phenolic acids. However, in most cases, the availability of these valuable compounds is limited by cellular or organelle barriers or spatio-temporal accumulation patterns within different plant tissues. Transcription factors (TFs) regulate biosynthesis of these specialized metabolites by tightly controlling the expression of biosynthetic genes. Cutting-edge technologies and/or combining multiple strategies and approaches have been applied to elucidate the role of TFs. In this review, we focus on recent progress in the transcription regulation mechanism of representative high-value products and describe the transcriptional regulatory network, and future perspectives are discussed, which will help develop high-yield plant resources.

Ginsenoside Rh2 augmented anti-PD-L1 immunotherapy by reinvigorating CD8+ T cells via increasing intratumoral CXCL10.

Profiting from the sustained clinical improvement and prolonged patient survival, immune checkpoint blockade of programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis has emerged as a revolutionary cancer therapy approach. However, the anti-PD-1/PD-L1 antibodies only achieve a clinical response rate of approximately 20%. Herein, we identified a novel combination strategy that Chinese medicine ginseng-derived ginsenoside Rh2 (Rh2) markedly improved the anti-cancer efficacy of anti-PD-L1 antibody in mice bearing MC38 tumor. Rh2 combined with anti-PD-L1 antibody (combo treatment) further triggered the infiltration, proliferation and activation of CD8+ T cells in the tumor microenvironment (TME). Depletion of CD8+ T cells by mouse CD8 blocking antibody abolished the anti-cancer effect of combo treatment totally. Mechanistically, combo treatment further increased the expression of CXCL10 through activating TBK1-IRF3 signaling pathway, explaining the increased infiltration of T cells. Employing anti- CXC chemokine receptor 3 (CXCR3) blocking antibody prevented the T cells infiltration and abolished the anti-cancer effect of combo treatment. Meanwhile, combo treatment increased the percentage of M1-like macrophages and raised the ratio of M1/M2 macrophages in TME. By comparing the anti-cancer effect of combo treatment among MC38, CT26 and 4T1 tumors, resident T cells were considered as a prerequisite for the effectiveness of combo treatment. These findings demonstrated that Rh2 potentiated the anti-cancer effect of PD-L1 blockade via promoting the T cells infiltration and activation, which shed a new light on the combination strategy to enhance anti-PD-L1 immunotherapy by using natural product Rh2.

Transcutaneous electrical acupoint stimulation for cancer-related pain management in patients receiving chronic opioid therapy: a randomized clinical trial.

PURPOSE: The opioid crisis resulting from its use disorder and overdose poses additional challenges for cancer pain management. The American Society of Clinical Oncology Practice Guideline recommends acupuncture therapy for the management of adult cancer-related pain (CRP), but the effectiveness of transcutaneous electrical acupoint stimulation (TEAS) on CRP remains uncertain. METHODS: This 5-week prospective randomized clinical trial was conducted at 2 hospitals in China, and participants with CRP receiving chronic opioid therapy were randomized 1:1 into two groups between December 2014 and June 2018. The true TEAS group underwent 15 sessions of TEAS treatments over 3 consecutive weeks, while the control group received sham stimulation. The primary outcome was the numerical rating scale (NRS) score in the past 24h at week 3. The secondary outcomes included morphine equivalent daily dose, quality of life and adverse events. RESULTS: A total of 159 participants were included in the modified intention-to-treat population. The baseline characteristics were similar in both groups. The mean NRS scores were 0.98 points at week 3 in the true TEAS group and 1.41 points in the sham group, with the mean difference between groups of -0.43 points (P < 0.001; OR = 0.68, P < 0.05). The proportion of patients with NRS reduction more than thirty percentage at week 3 was 50.00% in the true TEAS group and 35.44% in the sham group (RD = 0.15, P > 0.05; RR = 1.41, P > 0.05). No significant difference in pain intensity between the two groups was observed during the follow-up period without TEAS intervention (week 4, OR = 0.83, P > 0.05; week 5, OR = 0.83, P > 0.05). The Karnofsky Performance Status value suggested that patients in the true TEAS group experienced an improved quality of life (Between-group differences: week 3, 3.5%, P < 0.05; week 4, 4.6%, P < 0.001; week 5, 5.6%, P < 0.001). CONCLUSIONS: The 3-week application of TEAS in patients with CRP receiving chronic opioid therapy resulted in a statistically significant reduction in pain scores, but the observed reduction was of uncertain clinical significance. The prolonged analgesic effect of TEAS was not confirmed in this trial. CLINICALTRIAL: GOV: ChiCTR-TRC-13003803.

Comparison of Salvianolic Acid A Adsorption by Phenylboronic-Acid-Functionalized Montmorillonites with Different Intercalators.

Due to its success in treating cardio-cerebrovascular illnesses, salvianolic acid A (SAA) from Salvia miltiorrhiza is of major importance for effective acquisition. For the adsorption of salvianolic acid, cationic polyelectrolytes, and amino-terminated silane intercalated with phenylboronic-acid-functionalized montmorillonites, known as phenylboronic-acid-functionalized montmorillonites with PEI (PMP) and phenylboronic-acid-functionalized montmorillonites with KH550 (PMK), respectively, were produced. In this paper, detailed comparisons of the SAA adsorption performance and morphology of two adsorbents were performed. PMP showed a higher adsorption efficiency (>88%) over a wide pH range. PMK showed less pH-dependent SAA adsorption with a faster adsorption kinetic fitting in a pseudo-second-order model. For both PMP and PMK, the SAA adsorption processes were endothermic. Additionally, it was clearer how temperature affected PMP adsorption. PMK has a higher adsorption selectivity. This study demonstrates how the type of intercalator can be seen to have an impact on adsorption behavior through various structural variations and offers an alternative suggestion for establishing a dependable method for the synthesis of functional montmorillonite from the intercalator's perspective.

Antifungal Properties of Biogenic Selenium Nanoparticles Functionalized with Nystatin for the Inhibition of Candida albicans Biofilm Formation.

In the present study, biogenic selenium nanoparticles (SeNPs) have been prepared using Paenibacillus terreus and functionalized with nystatin (SeNP@PVP_Nystatin nanoconjugates) for inhibiting growth, morphogenesis, and a biofilm in Candida albicans. Ultraviolet-visible spectroscopy analysis has shown a characteristic absorption at 289, 303, and 318 nm, and X-ray diffraction analysis has shown characteristic peaks at different 2θ values for SeNPs. Electron microscopy analysis has shown that biogenic SeNPs are spherical in shape with a size in the range of 220-240 nm. Fourier transform infrared spectroscopy has confirmed the functionalization of nystatin on SeNPs (formation of SeNP@PVP_Nystatin nanoconjugates), and the zeta potential has confirmed the negative charge on the nanoconjugates. Biogenic SeNPs are inactive; however, nanoconjugates have shown antifungal activities on C. albicans (inhibited growth, morphogenesis, and a biofilm). The molecular mechanism for the action of nanoconjugates via a real-time polymerase chain reaction has shown that genes involved in the RAS/cAMP/PKA signaling pathway play an important role in antifungal activity. In cytotoxic studies, nanoconjugates have inhibited only 12% growth of the human embryonic kidney cell line 293 cells, indicating that the nanocomposites are not cytotoxic. Thus, the biogenic SeNPs produced by P. terreus can be used as innovative and effective drug carriers to increase the antifungal activity of nystatin.

OpNAC1 transcription factor regulates the biosynthesis of the anticancer drug camptothecin by targeting loganic acid O-methyltransferase in Ophiorrhiza pumila.

Camptothecin (CPT) is an anticancer pentacyclic quinoline alkaloid widely used to treat cancer patients worldwide. However, the biosynthetic pathway and transcriptional regulation of camptothecin are largely unknown. Ophiorrhiza pumila, the herbaceous plant from the Rubiaceae family, has emerged as a model plant for studying camptothecin biosynthesis and regulation. In this study, a high-quality reference genome of O. pumila with estimated size of ~456.90 Mb was reported, and the accumulation level of camptothecin in roots was higher than that in stems and leaves. Based on its spatial distribution in the plant, we examined gene functions and expression by combining genomics with transcriptomic analysis. Two loganic acid O-methyltransferase (OpLAMTs) were identified in strictosidine-producing plant O. pumila, and enzyme catalysis assays showed that OpLAMT1 and not OpLAMT2 could convert loganic acid into loganin. Further knock-out of OpLAMT1 expression led to the elimination of loganin and camptothecin accumulation in O. pumila hairy roots. Four key residues were identified in OpLAMT1 protein crucial for the catalytic activity of loganic acid to loganin. By co-expression network, we identified a NAC transcription factor, OpNAC1, as a candidate gene for regulating camptothecin biosynthesis. Transgenic hairy roots and biochemical assays demonstrated that OpNAC1 suppressed OpLAMT1 expression. Here, we reported on two camptothecin metabolic engineering strategies paving the road for industrial-scale production of camptothecin in CPT-producing plants.

Genome-wide survey of the GATA gene family in camptothecin-producing plant Ophiorrhiza pumila.

BACKGROUND: Ophiorrhiza pumila (Rubiaceae) is capable of producing camptothecin (CPT), one monoterpene indole alkaloid extensively employed in the treatment of multiple cancers. Transcription factors (TFs) GATA are a group of transcription regulators involved in plant development and metabolism, and show the feature of binding to the GATA motif within the promoters of target genes. However, GATA TFs have not been characterized in O. pumila. RESULT: In this study, a total of 18 GATA genes classified into four subfamilies were identified, which randomly distributed on 11 chromosomes of O. pumila. Synteny analysis of GATA genes between O. pumila and other plant species such as Arabidopsis thaliana, Oryza sativa, Glycine max, Solanum lycopersicum, Vitis vinifera, and Catharanthus roseus genomes were analyzed. Tissue expression pattern revealed that OpGATA1 and OpGATA18 were found to be correlated with ASA, MK, CPR and GPPS, which were highly expressed in leaves. OpGATA7, showed high expression in roots as most of the CPT biosynthetic pathway genes did, suggesting that these OpGATAs may be potential candidates regulating CPT biosynthesis in O. pumila. CONCLUSIONS: In this study, we systematically analyzed the OpGATA TFs, and provided insights into the involvement of OpGATA TFs from O. pumila in CPT biosynthesis.

Endophytic fungus Pseudodidymocyrtis lobariellae KL27 promotes taxol biosynthesis and accumulation in Taxus chinensis.

BACKGROUND: Taxol from Taxus species is a precious drug used for the treatment of cancer and can effectively inhibit the proliferation of cancer cells. However, the growth of Taxus plants is very slow and the content of taxol is quite low. Therefore, it is of great significance to improve the yield of taxol by modern biotechnology without destroying the wild forest resources. Endophytic fungus which symbiosis with their host plants can promote the growth and secondary metabolism of medicinal plants. RESULTS: Here, an endophytic fungus KL27 was isolated from T. chinensis, and identified as Pseudodidymocyrtis lobariellae. The fermentation broth of KL27 (KL27-FB) could significantly promote the accumulation of taxol in needles of T. chinensis, reaching 0.361 ± 0.082 mg/g·DW (dry weight) at 7 days after KL27-FB treatment, which is 3.26-fold increase as compared to the control. The RNA-seq and qRT-PCR showed that KL27-FB could significantly increase the expression of key genes involved in the upstream pathway of terpene synthesis (such as DXS and DXR) and those in the taxol biosynthesis pathway (such as GGPPS, TS, T5OH, TAT, T10OH, T14OH, T2OH, TBT, DBAT and PAM), especially at the early stage of the stimulation. Moreover, the activation of jasmonic acid (JA) biosynthesis and JA signal transduction, and its crosstalk with other hormones, such as gibberellin acid (GA), ethylene (ET) and salicylic acid (SA), explained the elevation of most of the differential expressed genes related to taxol biosynthesis pathway. Moreover, TF (transcriptional factor)-encoding genes, including MYBs, ethylene-responsive transcription factors (ERFs) and basic/helix-loop-helix (bHLH), were detected as differential expressed genes after KL27-FB treatment, further suggested that the regulation of hormone signaling on genes of taxol biosynthesis was mediated by TFs. CONCLUSIONS: Our results indicated that fermentation broth of endophytic fungus KL27-FB could effectively enhance the accumulation of taxol in T. chinensis needles by regulating the phytohormone metabolism and signal transduction and further up-regulating the expression of multiple key genes involved in taxol biosynthesis. This study provides new insight into the regulatory mechanism of how endophytic fungus promotes the production and accumulation of taxol in Taxus sp.

A novel WRKY34-bZIP3 module regulates phenolic acid and tanshinone biosynthesis in Salvia miltiorrhiza.

Phenolic acids and tanshinones are main bioactive compounds produced in Salvia miltiorrhiza widely used in treatment of cardiovascular diseases, which could be promoted by abscisic acid elicitation. However, the regulation mechanism remained to be elucidated. An ABA-inducible IIa WRKY transcription factor (TF) named SmWRKY34 exhibiting high homology with AtWRKY40 was isolated. SmWRKY34 exhibited a negative role on phenolic acids and tanshinones by directly regulating SmRAS and SmGGPPS. Moreover, ABA-responsive bZIP TF member named SmbZIP3 expressing significantly in SmWRKY34 transcriptome was screened. SmWRKY34 showed a negative regulatory role on SmbZIP3. SmbZIP3 acted as a positive regulator in the biosynthesis of phenolic acids and tanshinones by targeting SmTAT and two tanshinone-promoting TFs SmERF128 and SmMYB9b. Taken together, we identify a new module WRKY34-bZIP3 involved in ABA signaling that manipulates phenolic acid and tanshinone accumulation, shedding new insights in metabolic engineering application in S. miltiorrhiza.

Nano-priming as emerging seed priming technology for sustainable agriculture-recent developments and future perspectives.

Nano-priming is an innovative seed priming technology that helps to improve seed germination, seed growth, and yield by providing resistance to various stresses in plants. Nano-priming is a considerably more effective method compared to all other seed priming methods. The salient features of nanoparticles (NPs) in seed priming are to develop electron exchange and enhanced surface reaction capabilities associated with various components of plant cells and tissues. Nano-priming induces the formation of nanopores in shoot and helps in the uptake of water absorption, activates reactive oxygen species (ROS)/antioxidant mechanisms in seeds, and forms hydroxyl radicals to loosen the walls of the cells and acts as an inducer for rapid hydrolysis of starch. It also induces the expression of aquaporin genes that are involved in the intake of water and also mediates H2O2, or ROS, dispersed over biological membranes. Nano-priming induces starch degradation via the stimulation of amylase, which results in the stimulation of seed germination. Nano-priming induces a mild ROS that acts as a primary signaling cue for various signaling cascade events that participate in secondary metabolite production and stress tolerance. This review provides details on the possible mechanisms by which nano-priming induces breaking seed dormancy, promotion of seed germination, and their impact on primary and secondary metabolite production. In addition, the use of nano-based fertilizer and pesticides as effective materials in nano-priming and plant growth development were also discussed, considering their recent status and future perspectives.

Promising natural products against SARS-CoV-2: Structure, function, and clinical trials.

The corona virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus type 2 (SARS-COV-2) poses a severe threat to human health and still spreads globally. Due to the high mutation ratio and breakthrough infection rate of the virus, vaccines and anti-COVID-19 drugs require continual improvements. Drug screening research has shown that some natural active products can target the critical proteins of SARS-CoV-2, including 3CLpro, ACE2, FURIN, and RdRp, which could produce great inhibitory effects on SARS-COV-2. In addition, some natural products have displayed activities of immunomodulation, antiinflammatory, and antihepatic failure in COVID-19 clinical trials, which may relate to their non-monomeric structures. However, further evaluation and high-quality assessments, including safety verification tests, drug interaction tests, and clinical trials, are needed to substantiate natural products' multi-target and multi-pathway effects on COVID-19. Here, we review the literature on several promising active natural products that may act as vaccine immune enhancers or provide targeted anti-COVID-19 drugs. The structures, mechanisms of action, and research progress of these natural products are analyzed, to hopefully provide effective ideas for the development of targeted drugs that possess better structure, potency, and safety.

Total flavonoids from the dried root of Tetrastigma hemsleyanum Diels et Gilg inhibit colorectal cancer growth through PI3K/AKT/mTOR signaling pathway.

The dried root of Tetrastigma hemsleyanum Diels et Gilg is used as a traditional Chinese medicine in southern China, as a folk remedy for carcinomas and gastrointestinal diseases. The total flavonoids of T. hemsleyanum (THTF) provide its main bioactive constituents. However, the mechanisms underlying its potential activity on colorectal cancer are still unknown. Here, we investigated the antitumor effect of THTF on colorectal cancer in vitro and in vivo. It was found that THTF inhibited HCT-116 and HT-29 cell growth, with an IC50 of 105.60 and 140.80 µg/mL, respectively. THTF suppressed clonogenicity and promoted apoptosis in HCT-116. In vivo, THTF (120 mg/kg) delayed tumor growth in HCT-116 xenografts without influencing on body weight, organ pathology and indexes, and blood routine level. Mechanistically, THTF inhibited the expression of PI3K, AKT, and mTOR at the protein level and transcriptional levels. Molecular docking indicated eight compounds in THTF (kaempferol 3-rutinoside, rutinum, isoquercitrin, L-epicatechin, quercetin, astragalin, kaempferol 3-sambubioside, and catechin) strongly bound with amino acid sites of PI3K and mTOR proteins, indicating a high affinity. The results suggest that THTF delayed colorectal tumor growth by inhibiting the PI3K/AKT/mTOR pathway and might be a potential candidate for colorectal cancer prevention.

Divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila.

BACKGROUND: The anticancer drug camptothecin (CPT), first isolated from Camptotheca acuminata, was subsequently discovered in unrelated plants, including Ophiorrhiza pumila. Unlike known monoterpene indole alkaloids, CPT in C. acuminata is biosynthesized via the key intermediate strictosidinic acid, but how O. pumila synthesizes CPT has not been determined. RESULTS: In this study, we used nontargeted metabolite profiling to show that 3α-(S)-strictosidine and 3-(S), 21-(S)-strictosidinic acid coexist in O. pumila. After identifying the enzymes OpLAMT, OpSLS, and OpSTR as participants in CPT biosynthesis, we compared these enzymes to their homologues from two other representative CPT-producing plants, C. acuminata and Nothapodytes nimmoniana, to elucidate their phylogenetic relationship. Finally, using labelled intermediates to resolve the CPT biosynthesis pathway in O. pumila, we showed that 3α-(S)-strictosidine, not 3-(S), 21-(S)-strictosidinic acid, is the exclusive intermediate in CPT biosynthesis. CONCLUSIONS: In our study, we found that O. pumila, another representative CPT-producing plant, exhibits metabolite diversity in its central intermediates consisting of both 3-(S), 21-(S)-strictosidinic acid and 3α-(S)-strictosidine and utilizes 3α-(S)-strictosidine as the exclusive intermediate in the CPT biosynthetic pathway, which differs from C. acuminata. Our results show that enzymes likely to be involved in CPT biosynthesis in O. pumila, C. acuminata, and N. nimmoniana have evolved divergently. Overall, our new data regarding CPT biosynthesis in O. pumila suggest evolutionary divergence in CPT-producing plants. These results shed new light on CPT biosynthesis and pave the way towards its industrial production through enzymatic or metabolic engineering approaches.

First Report of Leaf Spot of Atractylodes macrocephala Caused by Fusarium commune in Zhejiang province, China.

Atractylodes macrocephala Koidz is a very common herbs in China, also famous for its high medicinal value (Lee et al., 2007). In summer of 2019, in Fuyang county of Zhejiang province, the main production area of China, 74 plants of A. macrocephala from a total of about 300 plants, showed black leaf spots . The incidence of the disease was 25% and increased under high temperature and humidity conditions. Initial leaf symptoms appeared as black or tan spots surrounded by brown margins and expanded irregularly. Finally, large blackish brown spots appeared on the leaves, elliptical or irregular, 1.0 to 1.5 cm in diameter, and then lesions turned necrotic. To isolate the pathogen, small pieces (5×5 mm) from the margin of symptomatic leaves were surface-sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite for 2 min, rinsed five times with sterile water, and incubated on potato dextrose agar (PDA) at 28°C in darkness. Purified colonies were white to pink with densely floccose to fluffy aerial mycelium and peach-orange pigmentation. Macroconidia, usually three-septate, were 26.7 to 43.3×3.1 to 5.3µm (n=50), thin-walled, slightly curved, with apical and basal cells curved when cultured in continuous darkness. Microconidia were mostly aseptate, ovate-oblong, straight to slightly curved, and measuring 5.9 to 14.3×2.3 to 3.9µm in size (n=50). Spherical chlamydospores were produced singly or in pairs from mycelium and spores. These characteristics were consistent with the description of Fusarium spp. (Leslie and Summerell, 2006). To identify the species, the translation elongation factor-1 alpha regions (TEF-1α) and the mitochondrial small subunit (mtSSU) were amplified using primers EF-3/ EF-22 (Palmore et al.,2010 and O'Donnell et al., 1998) and MS3F/ MS3R (Stenglein et al., 2010), respectively. Sequences were deposited in GenBank (MT263720, OM203177, OM203178, OM203179, OM203180, OM203181 and MN853662, MZ028170.1, MZ028171.1, MZ028172.1, MZ028173.1, MZ028174.1). These six isolates clustered in the Fusarium commune clade with 100% and 98% similarity, respectively. To test pathogenicity of every isolate, five 8-week-old potted A. macrocephalae plants were wound-inoculated and mycelial discs of 5-mm diameter were used to inoculate. As a control, five plants were inoculated with 5-mm PDA plugs. All plants were individually covered with a plastic bag and kept in a greenhouse at 25 ± 2°C with a 12-h photoperiod at 70 to 80% relative humidity. Typical symptoms similar to those of the field appeared only in inoculated plants after five days. In addition, a conidial suspension (1×105 spores/ml) was sprayed onto young leaves of three potted healthy plants. Three plants sprayed with sterile distilled water served as controls. After 7 days, typical symptoms were observed on all inoculated leaves. Experiments were replicated three times. F. commune was successfully re-isolated from diseased plants. Based on morphological and molecular identification, the pathogen was identified as F. commune. In China, Alternaria alternata (Zhuang, 2005), A. longipes (Tan et al., 2012), Phyllosticta commonsii (Sang et al., 2006) and Phoma exigua (Zhang et al., 2018) were reported as causal agents of the leaf spot disease of A. macrocephalae. To our knowledge, this is the first report of leaf spot disease on A. macrocephalae caused by F. commune in China. Effective control strategies need to be established to reduce the losses.

Dihydrotanshinone I Inhibits the Lung Metastasis of Breast Cancer by Suppressing Neutrophil Extracellular Traps Formation.

Breast cancer (BC) is a common female malignancy, worldwide. BC death is predominantly caused by lung metastasis. According to previous studies, Dihydrotanshinone I (DHT), a bioactive compound in Salvia miltiorrhiza Bunge (S. miltiorrhiza), has inhibitory effects on numerous cancers. Here, we investigated the anti-metastatic effect of DHT on BC, where DHT more strongly inhibited the growth of BC cells (MDA-MB-231, 4T1, MCF-7, and SKBR-3) than breast epithelial cells (MCF-10a). Additionally, DHT repressed the wound healing, invasion, and migration activities of 4T1 cells. In the 4T1 spontaneous metastasis model, DHT (20 mg/kg) blocked metastasis progression and distribution in the lung tissue by 74.9%. DHT reversed the formation of neutrophil extracellular traps (NETs) induced by phorbol 12-myristate 13-acetate, as well as ameliorated NETs-induced metastasis. Furthermore, it inhibited Ly6G+Mpo+ neutrophils infiltration and H3Cit expression in the lung tissues. RNA sequencing, western blot, and bioinformatical analysis indicated that TIMP1 could modulate DHT acting on lung metastasis inhibition. The study demonstrated a novel suppression mechanism of DHT on NETs formation to inhibit BC metastasis.

Medioresinol as a novel PGC-1α activator prevents pyroptosis of endothelial cells in ischemic stroke through PPARα-GOT1 axis.

AIM: Brain microvascular endothelial cells (BMVECs), as the important structure of blood-brain barrier (BBB), play a vital role in ischemic stroke. Pyroptosis of different cells in the brain may aggravate cerebral ischemic injury, and PGC-1α plays a major role in pyroptosis. However, it is not known whether BMVECs undergo pyroptosis after ischemic stroke and whether PGC-1α activator Medioresinol (MDN) we discovered may be useful against pyroptosis of endothelial cells and ischemic brain injury. METHODS: For in vitro experiments, the bEnd.3 cells and BMVECs under oxygen and glucose-deprivation (OGD) were treated with or without MDN, and the LDH release, tight junction protein degradation, GSDMD-NT membrane location and pyroptosis-associated proteins were evaluated. For in vivo experiments, mice underwent transient middle cerebral artery occlusion (tMCAO) for ischemia model, and the neuroprotective effects of MDN were measured by infarct volume, the permeability of BBB and pyroptosis of BMVECs. For mechanistic study, effects of MDN on the accumulation of phenylalanine, mitochondrial reactive oxygen species (mtROS) were tested by untargeted metabolomics and MitoSOX Red probe, respectively. RESULTS: BMVECs underwent pyroptosis after ischemia. MDN dose-dependently activated PGC-1α, significantly reduced pyroptosis, mtROS and the expressions of pyroptosis-associated proteins (NLRP3, ASC, cleaved caspase-1, IL-1ß, GSDMD-NT), and increased ZO-1 and Occludin protein expressions in BMVECs. In tMCAO mice, MDN remarkably reduced brain infarct volume and the permeability of BBB, inhibited pyroptosis of BMVECs, and promoted long-term neurobehavioral functional recovery. Mechanistically, MDN promoted the interaction of PGC-1α with PPARα to increase PPARα nuclear translocation and transcription activity, further increased the expression of GOT1 and PAH, resulting in enhanced phenylalanine metabolism to reduce the ischemia-caused phenylalanine accumulation and mtROS and further ameliorate pyroptosis of BMVECs. CONCLUSION: In this study, we for the first time discovered that pyroptosis of BMVECs was involved in the pathogenesis of ischemic stroke and MDN as a novel PGC-1α activator could ameliorate the pyroptosis of endothelial cells and ischemic brain injury, which might attribute to reduction of mtROS through PPARα/GOT1 axis in BMVECs. Taken together, targeting endothelial pyroptosis by MDN may provide alternative therapeutics for brain ischemic stroke.