Sequencing, Functional Annotation, and Interaction Prediction of mRNAs and Candidate Long Noncoding RNAs Originating from Tea Leaves During Infection by the Fungal Pathogen Causing Tea Leaf Spot, Didymella bellidis.

Tea leaf spot caused by Didymella bellidis can seriously reduce the productivity and quality of tea (Camellia sinensis var. sinensis) leaves in Guizhou Province, southwest China. Analysis of the relationship between messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) of tea could provide insights into the plant-pathogen interaction. In this study, high-throughput sequencing of mRNAs and lncRNAs from tea leaves during infection by D. bellidis was conducted using the Illumina Novaseq 6000 platform. Infection by D. bellidis hyphae resulted in up- or downregulation of 553 and 191 of the differentially expressed mRNAs (DEmRNAs), respectively. As the S gene number (total number of genes with significantly differential expression annotated in the specified Gene Ontology [GO] database), three were enriched with respect to the defense response to the fungus at the biological process level. Expression of the DEmRNAs peroxidase 21 (TEA000222.1) and mcht-2 (TEA013240.1) originating from tea leaves were upregulated during challenge by D. bellidis hyphae, whereas expression of the LRR receptor-like serine/threonine-protein kinase ERECTA (TEA016781.1) gene was downregulated. The infection of D. bellidis hyphae resulted in up- or downregulation of 227 and 958 of the differentially expressed lncRNAs (DElncRNAs). The DEmRNAs associated with uncharacterized LOC101499401 (TEA015626.1), uncharacterized protein (TEA014125.1), structural maintenance of chromosomes protein 1 (TEA001660.1), and uncharacterized protein (TEA017727.1) occurred as a result of cis regulation by DElncRNAs MSTRG.20036, MSTRG.3843, MSTRG.26132, and MSTRG.56701, respectively. The expression profiling and lncRNA/mRNA association prediction in the tea leaves infected by D. bellidis will provide a valuable resource for further research into disease resistance.

Biomimetic Nano-Immunoactivator via Ionic Metabolic Modulation for Strengthened NIR-II Photothermal Immunotherapy.

Reprogramming the immunologically "cold" environment of solid tumors is currently becoming the mainstream strategy to elicit powerful and systemic anticancer immunity. Here, a facile and biomimetic nano-immunnoactivator (CuS/Z@M4T1 ) is detailed by engineering a Zn2+ -bonded zeolitic imidazolate framework-8 (ZIF-8) with CuS nanodots (NDs) and cancer cell membrane for amplified near-infrared-II (NIR-II) photothermal immunotherapy via Zn2+ metabolic modulation. Taking advantage of the NIR-II photothermal effect of CuS NDs and the acidic responsiveness of ZIF-8, CuS/Z@M4T1 rapidly causes intracellular Zn2+ pool overload and disturbs the metabolic flux of 4T1 cells, which effectively hamper the production of heat shock proteins and relieve the resistance of photothermal therapy (PTT). Thus, amplified immunogenic cell death is evoked and initiates the immune cascade both in vivo and in vitro as demonstrated by dendritic cells maturation and T-cell infiltration. Further combination with antiprogrammed death 1 (aPD-1) achieves escalated antitumor efficacy which eliminates the primary, distant tumor and avidly inhibits lung metastasis due to cooperation of enhanced photothermal stimulation and empowerment of cytotoxic T lymphocytes by aPD-1. Collectively, this work provides the first report of using the intrinsic modulation property of meta-organometallic ZIF-8 for enhanced cancer photoimmunotherapy together with aPD-1, thereby inspiring a novel combined paradigm of ion-rich nanomaterials for cancer treatment.

Functional Annotation of circRNAs in Tea Leaves After Infection by the Tea Leaf Spot Pathogen, Lasiodiplodia theobromae.

Tea leaf spot, caused by Lasiodiplodia theobromae, is an important disease that can seriously decrease the production and quality of tea (Camellia sinensis (L.) O. Kuntze) leaves. The analysis of circular RNA (circRNA) in tea leaves after infection by the pathogen could improve understanding about the mechanism of host-pathogen interactions. In this study, high-performance sequencing of circRNA from C. sinensis Fuding-dabaicha leaves that had been infected with L. theobromae was conducted using the Illumina HiSeq 4000 platform. In total, 192 and 153 differentially expressed circRNAs from tea leaves were significantly up- and downregulated, respectively, after infection with L. theobromae. A gene ontology analysis indicated that the differentially expressed circRNA-hosting genes for DNA binding were significantly enriched. The genes with significantly differential expressions that were annotated in the specified database (S genes) were σ factor E isoform 1, triacylglycerol lipase SDP1, DNA-directed RNA polymerase III subunit 2, WRKY transcription factor WRKY24, and regulator of nonsense transcripts 1 homolog. A Kyoto Encyclopedia of Genes and Genomes analysis indicated that the significantly enriched circRNA-hosting genes involved in the plant-pathogen interaction pathway were Calmodulin-domain protein kinase 5 isoform 1, probable WRKY transcription factor 33, U-box domain-containing protein 35, probable inactive receptor-like protein kinase At3g56050, WRKY transcription factor WRKY24, mitogen-activated protein kinase kinase kinase YODA, SGT1, and protein DGS1. Functional annotation of circRNAs in tea leaves infected by L. theobromae will provide a valuable resource for future research on host-pathogen interactions.

Integration of Transcriptomic and Proteomic Data Reveals the Possible Action Mechanism of the Antimicrobial Zhongshengmycin Against Didymella segeticola, the Causal Agent of Tea Leaf Spot.

Tea leaf spot, caused by the fungal phytopathogen Didymella segeticola, is an important foliar disease that can cause huge losses in the production and quality of tea, and there are no effective management measures to control the disease. This study screened a natural antimicrobial chemical for its activity against D. segeticola and studied its mode of action. Antifungal activity of the Streptomyces-derived antimicrobial zhongshengmycin (ZSM) against D. segeticola strain GZSQ-4 was assayed in vitro via the mycelial growth rate method. Optical microscopy and scanning and transmission electron microscopy were used to observe the morphological effects on hyphae treated with ZSM, with these studies complemented by transcriptomic, proteomic, and bioinformatic studies to identify the differentially expressed genes or differentially expressed proteins in hyphae treated with ZSM. Correlation analysis of transcriptomic and proteomic data were used to reveal the mode of action. The results indicated that ZSM could inhibit the growth of hyphae in vitro with a half-maximal effective concentration of 5.9 µg/ml, inducing some morphological changes in organelles, septa, and extracellular polysaccharides, targeting ribosomes to disturb translation, affecting the biosynthesis of some hyphal proteins at the messenger RNA and protein levels, and revealing correlations between findings from transcriptomes and proteomes.

Hexabromocyclododecanes in soils and plants from a plastic waste treatment area in North China: occurrence, diastereomer- and enantiomer-specific profiles, and metabolization.

Plastic waste is a source of organic contaminants such as hexabromocyclododecanes (HBCDs). HBCDs have been found to cause developmental and reproductive toxicity; it is important to investigate the occurrence and metabolization of HBCDs in the soil environments with plastic waste contamination. This work analyzed HBCDs and their metabolites in soil and plant samples collected from Xinle and Dingzhou-the major plastic waste recycling centers in North China. Results showed that total HBCD concentrations in soils followed the order: plastic waste treatment site (11.0-624 ng/g) > roadside (2.96-85.4 ng/g) ≥ farmland (8.69-55.5 ng/g). HBCDs were detected in all the plant samples with total concentrations ranging from 3.47 to 23.4 ng/g. γ-HBCD was the dominant congener in soils, while α-HBCD was preferentially accumulated in plants. Compositions of HBCD isomers in soils and plants were significantly different (P < 0.05) among sampling sites and among plant species. HBCDs in farmland soil and all plant samples exhibited high enantio-selectivity based on the enantiomeric fractions (EFs). Furthermore, metabolites of pentabromocyclododecenes (PBCDEs) were frequently identified in soils, and mono-OH-HBCDs were the most common ones in plants. This study for the first time provides evidences of HBCD contamination in the soil-plant system caused by plastic waste, their stereo-selectivity, and metabolization behavior, improving our understanding of the environmental behavior and fate of HBCDs.

Occurrence and distribution of organophosphorus esters in soils and wheat plants in a plastic waste treatment area in China.

This study for the first time reported the occurrence, distribution and concentrations of organophosphate esters (OPEs) in soils caused by plastic waste treatment, as well as their influence on OPE accumulation in wheat (Triticum aestivum L.). Eight OPEs were detected with the total concentrations of 38-1250 ng/g dry weight in the soils from the treatment sites, and tributoxyethyl phosphate and tri(2-chloroethyl) phosphate present as the dominant OPEs. There were similar distribution patterns of OPEs and significant correlations between the total OPE concentrations in the soils from the plastic waste treatment sites with those in the nearby farmlands (P < 0.005), indicating that plastic waste treatment caused the OPE contamination of farmland soils. The uptake and translocation of OPEs by wheat were determined, with OPEs of high hydrophobicity more easily taken up from soils and OPEs with low hydrophobicity more liable to be translocated acropetally.

Influences of artificial root exudate components on the behaviors of BDE-28 and BDE-47 in soils: desorption, availability, and biodegradation.

Behaviors of BDE-28 and BDE-47 in two distinct soils (Phaeozem and Acrisol) as affected by the separate addition of root exudate components (i.e., oxalic acid, glycine, and fructose) were investigated by a soil microcosm incubation experiment. The results showed that root exudate components promoted the desorption of BDE-28 (57.6-235.0 %) and BDE-47 (56.9-223.7 %) from the soils due to the enhancement of their water solubilities. The addition of root exudate components increased the n-butanol extractability of BDE-28 and BDE-47 by 20.3-72.5 and 48.6-169.2 %, respectively, which had a positive correlation with the concentrations of dissolved organic carbon (DOC) in the soils (p < 0.01), suggesting that the increase of DOC in the soils by root exudate components was the major factor to enhance the extractability. Fructose and oxalic acid promoted the desorption and increased the availability of BDE-28 and BDE-47 in the soils more efficiently than glycine. The addition of different root exudate components resulted in distinct shifts in soil microbial community structure (p < 0.05). Oxalic acid caused the greatest impacts on the soil bacterial communities and increased the degradation rates of BDE-28 and BDE-47 most obviously. The findings of this study clarified the roles of root exudate components in affecting the behaviors of polybrominated diphenyl ethers (PBDEs) in soils.

In vitro biotransformation of PBDEs by root crude enzyme extracts: potential role of nitrate reductase (NaR) and glutathione S-transferase (GST) in their debromination.

In order to investigate the enzyme transformation of PBDEs and to track the key enzymes involved in PBDE degradation in plants, in vivo exposure of plants of ryegrass, pumpkin and maize and in vitro exposure of their root crude enzyme extracts to PBDEs were conducted. Degradation of PBDEs in the root crude enzyme solutions fit well with the first order kinetics (R(2)=0.52-0.97, P<0.05), and higher PBDEs degraded faster than the lower ones. PBDEs could be transformed to lower brominated PBDEs and hydroxylated-PBDEs by the root crude enzyme extracts with debromination as the main pathway which contributed over 90% of PBDE depletion. In vitro and in vivo exposure to PBDEs produced similar responses in root enzyme activities of which the nitroreductase (NaR) and glutathione-transferase (GST) activities decreased significantly, while the peroxidase, catalase and cytochrome P-450 activities had no significant changes. Furthermore, higher enzyme concentrations of NaR and GST led to higher PBDE debromination rates, and the time-dependent activities of NaR and GST in the root crude enzyme extracts were similar to the trends of PBDE depletion. All these results suggest that NaR and GST were the key enzymes responsible for PBDE degradation. This conclusion was further confirmed by the in vitro debromination of PBDEs with the commercial pure NaR and GST.

Accumulation and speciation of selenium in plants as affected by arbuscular mycorrhizal fungus Glomus mosseae.

Effects of arbuscular mycorrhizal fungus (Glomus mosseae) on the accumulation and speciation of selenium (Se) in alfalfa, maize, and soybean were investigated by using Se(IV)-spiked soil. Mycorrhizal inoculation decreased Se accumulation in roots and shoots of all the plants at Se spiked level of 0 or 2 mg kg(-1), while an increased Se accumulation was observed in alfalfa shoots and maize roots and shoots at the spiked level of 20 mg kg(-1). Concentration of inorganic Se (especially Se(VI)) in roots and shoots of the three plants was much higher in mycorrhizal than non-mycorrhizal treatment. Mycorrhizal inoculation decreased the portion of total organic Se in plant tissues with the exception of alfalfa and maize shoots at Se spiked level of 20 mg kg(-1), in which organic Se portion did not reduced greatly (<5%) for mycorrhizal treatment. Mycorrhizal effects on alfalfa and maize were more obvious than on soybean in terms of root colonization rate, biomass, and Se accumulation.

Metabonomics study of urine from Sprague-Dawley rats exposed to Huang-yao-zi using (1)H NMR spectroscopy.

Urinary metabolic perturbations associated with liver toxicity induced by Huang-yao-zi (root of Dioscorea bulifera L.) were studied using nuclear magnetic resonance spectroscopy ((1)H NMR) to determine the correlations between metabonomic profiling and histopathologic/biochemical observations and to discover biomarkers for liver toxicity. Huang-yao-zi with a maximal tolerance dose (MTD) was given to male Sprague-Dawley rats for 72h followed by metabonomic analysis of urine samples collected at 24 and 72h. The results revealed that the levels of taurine, creatine, betaine, dimethylglycine (DMG), acetate, glycine were elevated, whereas, the levels of succinate, 2-oxoglutarate, citrate, hippurate and urea were reduced. Partial least square (PLS)-discrimination analysis (DA) of NMR spectra revealed two apparent clusters between control groups and treatment groups, indicating metabolic changes observed in urine samples in response to Huang-yao-zi treatment. In addition, mechanism associated with oxidative injury of hepatic mitochondria was investigated. These results indicated that (1)H NMR-based metabonomics analysis in urine samples may be useful for predicting hepatotoxicity induced by Huang-yao-zi.

[Study of immunogenicity of chlorogenic acid-BSA with different number of CGA].

OBJECTIVE: To investigate the immunogenicity of chlorogenic acid-BSA(CGA-BSA) and the foundation for the allergization of CGA-BSA. METHOD: The CGA-BSA with different number of CGA was synthesized to allergize the BALB/c mice and SD rats to get antiserum. The level of IgE and histamine was analyzed by ELISA, and the passive cutaneous anaphylaxis (PCA) test was carried to analyze the antibody titer. RESULT: When the coupling rate of CGA-BSA is 20, the highest IgE and histamine level in BALB/c mice and the antibody titer in PCA are the highest. CONCLUSION: It has better immunogenicity when the coupling rate of CGA-BSA is between 10 to 20, while the CGA-BSA 20 with the best immunogenicity.

Phytoestrogen genistein supplementation increases eNOS and decreases caveolin-1 expression in ovariectomized rat hearts.

This study examined whether genistein influences the production of nitric oxide (NO) and expression of endothelial nitric oxide synthase (eNOS) and the modulators of eNOS activity in ovariectomized (OVX) rat hearts. Female mature Sprague-Dawley rats were subjected to bilateral ovariectomy, OVX rats were randomly divided into four groups: 17beta-estradiol (0.1 mg/kg, s.c. daily) was used as the positive control; low dose of genistein (0.5 mg/kg, s.c. daily); high dose of genistein (5.0 mg/kg, s.c. daily) and model. Sham operations as controls, the treatment lasted 6 weeks. Blood pressure, heart rate, plasma estradiol, heart and uterine weights were measured. Nitrite production in the myocardium was determined by nitrate reductase method. Protein level of eNOS, caveolin-1 and calmodulin was determined by Western blot. The results showed that nitrite production and eNOS protein in homogenized ventricular tissue was attenuated by approximately 53% and 67% in OVX rats compared with those in sham rats, respectively. Genistein increased nitrite production in rat heart in a dose-dependent manner, genistein at the dose of 5 mg/kg.d(-1) resumed nitrite production to a level similar to that in sham operated rats. Administration of genistein also increased eNOS protein expression in OVX rats myocardium with a concomitant decrease in the expression of caveolin-1, an endogenous eNOS inhibitory protein. Another eNOS stimulatory protein, calmodulin, was unchanged in these treatments. These effects were also observed in rats treated with 17beta-estradiol. Genistein at the dose of 5.0 mg/kg.d(-1) augmented uterine weight but this side effect in reproductive system was less than that of 17beta-estradiol. These results suggest that genistein supplementation and estrogen replacement therapy directly increase eNOS functional activity and NO production in the hearts of the OVX rats, but genistein has less side effects on the reproductive system than 17beta-estradiol.

Onychin inhibits proliferation of vascular smooth muscle cells by regulating cell cycle.

AIM: To investigate the effects of onychin on the proliferation of cultured rat artery vascular smooth muscle cells (VSMCs) in the presence of 10% new-born calf serum (NCS). METHODS: Rat VSMCs were incubated with onychin 150 micromol/L or genistein 10 micromol/L in the presence of 10% NCS for 24 h. The proliferation of VSMCs was measured by cell counting and MTS/PMS colorimetric assays. Cell cycle progression was evaluated by flow cytometry. Retinoblastoma (Rb) phosphorylation, and expression of cyclin D1 and cyclin E were measured by Western blot assays. The tyrosine phosphorylation of ERK1/2 was examined by immunoprecipitation techniques using anti-phospho-tyrosine antibodies. RESULTS: The proliferation of VSMCs was accelerated significantly in the presence of 10% NCS. Onychin reduced the metabolic rate of MTS and the cell number of VSMCs in the presence of 10% NCS in a dose-dependent manner. Flow cytometry analysis revealed that the G1-phase fraction ratio in the onychin group was higher than that in the 10% NCS group (85.2% vs 70.0%, P<0.01), while the S-phase fraction ratio in the onychin group was lower than that in 10% NCS group (4.3% vs 16.4%, P<0.01). Western blot analysis showed that onychin inhibited Rb phosphorylation and reduced the expression of cyclin D1 and cyclin E. The effects of onychin on proliferation, the cell cycle and the expression of cyclins in VSMCs were similar to those of genistein, an inhibitor of tyrosine kinase. Furthermore immunoprecipitation studies showed that both onychin and genistein markedly inhibited the tyrosine phosphorylation of ERK1/2 induced by 10% NCS. CONCLUSION: Onychin inhibits the proliferation of VSMCs through G1 phase cell cycle arrest by decreasing the tyrosine phosphorylation of ERK1/2, and the expression of cyclin D1 and cyclin E, and sequentially inhibiting Rb phosphorylation.