In vitro pharmacokinetic behavior in lung of harringtonine, an antagonist of SARS-CoV-2 associated proteins: New insights of inhalation therapy for COVID-19.

BACKGROUND: Recent studies have shown that harringtonine (HT) could specifically bind with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein and host cell transmembrane serine protease 2 (TMPRSS2) to block membrane fusion, which is an effective antagonist for SARS-CoV-2. PURPOSE: Our study focused on in-depth exploration of in vitro pharmacokinetic characteristics of HT in lung. METHODS: HPLC-fluorescence detection method was used to detect changes of HT content. Incubation systems of lung microsomes for phase I metabolism and UGT incubation systems for phase II metabolism were performed to elucidate metabolites and metabolic mechanisms of HT, and then the metabolic enzyme phenotypes for HT were clarified by chemical inhibition method and recombinant enzyme method. Through metabolomics, we comprehensively evaluated the physiological dynamic changes in SD rat and human lung microsomes, and revealed the relationship between metabolomics and pharmacological activity of HT. RESULTS: HPLC-fluorescence detection method showed strong specificity, high accuracy, and good stability for rapid quantification of HT. We confirmed that HT mainly underwent phase I metabolism, and the metabolites of HT in different species were all identified as 4'-demethyl HT, with metabolic pathway being hydrolysis reaction. CYP1A2 and CYP2E1 participated in HT metabolism, but as HT metabolism was not NADPH dependent, the esterase HCES1 in lung also played a role. The main KEGG pathways in SD rat and human lung microsomes were cortisol synthesis and secretion, steroid hormone biosynthesis and linoleic acid metabolism, respectively. The downregulated key biomarkers of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME suggested that HT could prevent immunosuppression and interfere with infection and replication of SARS-CoV-2. CONCLUSION: HT was mainly metabolized into 4'-demethyl HT through phase I reactions, which was mediated by CYP1A2, CYP2E1, and HCES1. The downregulation of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME were key ways of HT against SARS-CoV-2. Our study was of great significance for development and clinical application of HT in the treatment of COVID-19.

Structure and histochemistry of the stem of Dracaena cambodiana Pierre ex Gagnep.

Dracaena cambodiana Pierre ex Gagnep is an important plant resource for producing dragon's blood and one of most popular ornamental trees in China. For a better understanding of the physiological function of the stem, the structural characteristics and main substance histological location of the stems of D. cambodiana were studied. The structural characteristics of the different developmental stages of stems of D. cambodiana were observed and described detailly. And then a schematic diagram of the mature stem was created. Histochemical staining showed that two kinds of polysaccharides distributed in parenchymal cells. Saponins distributed mainly in ground tissue and phenolic compounds distributed mainly in the thick cell walls. An abundant of calcium oxalate raphide bundles were identified in cortex and primary tissue. Finally, the role of the above results in the taxonomy of Dracaena species and in their strong adaptability was discussed.

Integrative transcriptomic and metabolomic analyses unveil tanshinone biosynthesis in Salvia miltiorrhiza root under N starvation stress.

Salvia miltiorrhiza is a model plant for Chinese herbal medicine with significant pharmacologic effects due to its tanshinone components. Our previous study indicated that nitrogen starvation stress increased its tanshinone content. However, the molecular mechanism of this low nitrogen-induced tanshinone biosynthesis is still unclear. Thus, this study aimed to elucidate the molecular mechanism of tanshinone biosynthesis in S. miltiorrhiza under different N conditions [N-free (N0), low-N (Nl), and full-N (Nf, as control) conditions] by using transcriptome and metabolome analyses. Our results showed 3,437 and 2,274 differentially expressed unigenes between N0 and Nf as well as Nl and Nf root samples, respectively. N starvation (N0 and Nl) promoted the expression of the genes involved in the MVA and MEP pathway of tanshinone and terpenoid backbone biosynthesis. Gene ontology and KEGG analyses revealed that terpenoid backbone biosynthesis, hormone signal transduction, and phenylpropanoid biosynthesis were promoted under N starvation conditions, whereas starch and sucrose metabolisms, nitrogen and phosphorus metabolisms, as well as membrane development were inhibited. Furthermore, metabolome analysis showed that metabolite compounds and biosynthesis of secondary metabolites were upregulated. This study provided a novel insight into the molecular mechanisms of tanshinone production in S. miltiorrhiza in response to nitrogen stress.

Genetic relationship and source species identification of 58 Qi-Nan germplasms of Aquilaria species in China that easily form agarwood.

Recently, Qi-Nan germplasm, the germplasm of Aquilaria species that easily forms agarwood, has been widely cultivated in Guangdong and Hainan Provinces in China. Since the morphological characteristics of Qi-Nan germplasm are similar to those of Aquilaria species and germplasm is bred by grafting, it is difficult to determine the source species of this germplasm by traditional taxonomic characteristics. In this study, we performed a DNA barcoding analysis of 58 major Qi-Nan germplasms as well as Aquilaria sinensis, A. yunnanensis, A. crassna, A. malaccensis and A. hirta with 5 primers (nuclear gene internal transcribed spacer 2 (ITS2) and the chloroplast genes matK, trnH-psbA, rbcL and trnL-trnF). This field survey in the Qi-Nan germplasm plantations in Guangdong and Hainan Provinces aimed to accurately identify the source species of Qi-Nan germplasm. According to the results, ITS2 and matK showed the most variability and the highest divergence at all genetic distances. This ITS2+matK combination, screened for with TaxonDNA analysis, showed the highest success rate in species identification of the Qi-Nan germplasm. Clustering in the phylogenetic trees constructed with Bayesian inference and maximum likelihood indicated that the Qi-Nan germplasm was most closely related to A. sinensis and more distantly related to A. yunnanensis, A. crassna, A. malaccensis and A. hirta. Therefore, this study determined that the source species of the Qi-Nan germplasm is A. sinensis.

Programmed cell death might involve in progress of wounding induced agarwood formation in stems of Aquilaria sinensis.

Agarwood, a non-timber fragrant wood, is derived from wounded Aquilaria trees (Thymelaeaceae) and is widely used in traditional medicine, incense and perfume. Agarwood-like substances and programmed cell death (PCD) can be induced by wounding signals in the suspension cells and aerial roots of Aquilaria sinensis. In this study, the dynamic process of wound-induced agarwood formation in stems of A. sinensis was observed, and the occurrence of PCD was synchronously detected using techniques such as 4',6-diamidino-2-phenylindole and dUTP nick-end labeling staining. The results showed that the wounding was a induce signal for agarwood resin formation, meanwhile might induce PCD. Interxylary phloem and xylem ray were the main sites of agarwood resin formation and PCD occurrence. There might be a relationship between the spatiotemporal pattern of PCD and agarwood resin formation: more severe PCD corresponded to a higher rate of resin formation but a shorter resin formation time; conversely, slower PCD progression corresponded to a lower rate of resin formation but a longer resin formation time. Our findings are the first to demonstrate that PCD might occur in the process of wound-induced agarwood formation at the tree level, and the spatiotemporal pattern is closely related to the formation of agarwood resin. This study provides valuable insight for further studies on the relationship between PCD and agarwood formation. HIGHLIGHTS: Programmed cell death (PCD) might occur in the process of wound-induced agarwood formation at the tree level. Interxylary phloem and xylem ray were the main sites of agarwood resin formation and PCD occurrence. Spatiotemporal pattern of PCD might have a strong impact on agarwood resin formation.

Transcriptome analysis of Curcuma wenyujin from Haikou and Wenzhou, and a comparison of the main constituents and related genes of Rhizoma Curcumae.

For more than a thousand years, Rhizoma Curcumae (known as E zhu), a Chinese herbal medicine, has been used to eradicate blood stasis and relieve aches. The plant Curcuma wenyujin, which is grown primarily in Wenzhou, China, is considered the best source of Rhizoma Curcumae. In this study, we sought to ascertain differences in transcript profiles of C. wenyujin grown in traditional (Wenzhou) and recently established (Haikou) production areas based on Illumina and RNA (RNA-seq) sequencing. We also examined differences in the main components of the volatile oil terpene; curcumin, polysaccharide, and starch constituents and related genes in the corresponding pathways, in C. wenyujin cultivated in the two production areas. We accordingly found that the essential oil (2.05%), curcumin (1.46%), and polysaccharide (8.90%) content in Wenzhou rhizomes was higher than that in the rhizomes of plants from Haikou (1.60%, 0.91%, and 6.15%, respectively). In contrast, the starch content of Wenzhou rhizomes (17.0%) was lower than that of Haikou rhizomes (23.8%). Furthermore, we detected significant differences in the oil components of Haikou and Wenzhou rhizomes, with curzerene (32.34%), curdione (21.35%), and germacrene B (9.39%) being the primary components of the essential oil derived from Wenzhou rhizomes, and curzerene (20.13%), curdione (14.73%), and cineole (9.76%) being the main constituents in Haikou rhizomes. Transcriptome and qPCR analyses revealed considerable differences in gene expression between Wenzhou and Haikou rhizomes. The expression of terpene, curcumin, and polysaccharide pathway-related genes in Wenzhou rhizomes was significantly up-regulated, whereas the expression of starch-associated genes was significantly down-regulated, compared with those in Haikou rhizomes. Difference in the content of terpene, curcumin, polysaccharides, and starch in rhizomes from the two production areas could be explained in terms of differences in expression of the related genes.

[Characterization of AOX family members from Aquilaria sinensis and their responses to wounding].

Aquilaria sinensis is a typical inducible medicinal plant, that can produce agarwood only after it is wounded by external stimuli. Alternative oxidase(AOX) is one of the terminal oxidases of the plant mitochondrial electron transport, which plays an important role in plants' response to environmental stress. In order to reveal the physiological function of AOX gene in the process of agarwood formation from A.sinensis induced by wounding, AOX gene was cloned based on the transcriptome database and then identified by the bioinformatics analysis, and their expression pattern in different tissues and under wounding stress were detected by qRT-PCR. The results as follows. Three AOX genes were cloned from A.sinensis for the first time. They were named AsAOX1a, AsAOX1d and AsAOX2, respectively. The tissue expression shown that AsAOX1a is mainly expressed in the stem and the seed, and the AsAOX1d and AsAOX2 genes are mainly expressed in the pulp and the stem. AsAOX1a and AsAOX1d genes are highly responsive to wounding stress, and their response time was different. In addition, the expression of AsAOX1a and AsAOX2 induced by wounding are reduced by H_2O_2 treatment, but promoted by AsA treatment. The cloning, bioinformatics analysis and expression characteristics of AOX genes from A.sinensis provided basic information for further study the function of AOX genes in the development of A.sinensis, especially in the process of agarwood formation of A. sinensis induced by wounding.

Distribution Characteristics of Volatile Organic Compounds and Contribution to Ozone Formation in a Coking Wastewater Treatment Plant.

Ozone pollution, which can be caused by photochemical reactions, has become a serious problem. The ozone formation potential (OFP) is used to describe the photochemical reactivity. Volatile organic compounds (VOCs) are main precursors of ozone formation, and wastewater treatment plants (WWTPs) are important sources of VOCs. Therefore, it is necessary to study the concentration level and OFP of VOCs from WWTPs. In this work, a coking WWTP with anaerobic-oxic-oxic (A/O/O) processes in Shaoguan city, Guangdong province, China, was selected to investigate the characteristics of VOCs at wastewater treatment areas and office areas. The OFP of VOCs was estimated by the maximum incremental reactivity (MIR) coefficient method. Results showed that 17 VOCs were detected, and the total concentration of VOCs was the highest at the raw water tank (857.86 µg m-3). The benzene series accounted for 69.0%-86.9% and was the main component of VOCs in the WWTP. Based on OFP data, the top six VOCs contributing most to the OFP were m-xylene, toluene, p-xylene, o-xylene, styrene, and benzene. This study provides field data and information on the environmental risk of VOCs for coking companies and environmental departments. We found that the priority control sources of VOCs were wastewater treatment units because of their larger OFP contributions.

Emission characteristics and associated health risk assessment of volatile organic compounds from a typical coking wastewater treatment plant.

Coking wastewater is a typical industrial wastewater and contains a number of toxic and harmful organic pollutants which threaten human health. However, emission of volatile organic compounds (VOCs) from coking wastewater treatment plants (WWTPs) is rarely studied. Here, the emission characteristics of VOCs were investigated in a full-scale coking WWTP composed of an anaerobic-oxic-oxic (A-O1-O2) treatment system. Furthermore, the potential health risks were assessed in this study. VOC emission rates were estimated at each unit of the coking WWTP and the influencing factors of emissions were discussed. Seventeen VOCs were identified in the air phase by gas chromatography-mass spectrometry combined with Tenax adsorption-thermal desorption method; benzene, toluene, and xylenes were predominant, and the concentration of total VOCs decreased gradually from the raw water tank (857.86 ±â€¯131.30 µg m-3) to the effluent tank (28.56 ±â€¯3.96 µg m-3). The total VOC emission rate from all units was 1773.42 g d-1, corresponding to an annual emission of 0.65 tons year-1. Since the treatment capacity of this coking WWTP was about 1500 m3 d-1, it was estimated that 1.18 g of VOCs are emitted during the treatment of 1 m3 wastewater. Influencing factors of VOC emission mainly include the background concentration of VOCs in wastewater, operational parameters of the treatment processes, and physicochemical properties of VOCs. The carcinogenic risk of VOCs for workers in this coking WWTP ranged from 3.0 × 10-5 to 7.8 × 10-4, which exceeded an acceptable level (1.0 × 10-6). The non-carcinogenic risk hazard ratio of benzene exceeded 1, indicating that benzene has an obvious non-carcinogenic risk. Understanding VOCs emission characteristics and emission rates can help to identify the adverse effects of coking WWTPs on human health and provide relevant information for policy-making.

Molecular cloning and characterization of a Brassica juncea yellow stripe-like gene, BjYSL7, whose overexpression increases heavy metal tolerance of tobacco.

KEY MESSAGE: BjYSL7 encodes a plasma-localized metal-NA transporter and has transport Fe(II)-NA complexes activity. BjYSL7 is involved in the transport of Cd and Ni from roots to shoots. Heavy metal transporters play a key role in regulating metal accumulation and transport in plants. In this study, we isolated a novel member of the yellow stripe-like (YSL) gene family BjYSL7 from the hyperaccumulator Brassica juncea. BjYSL7 is composed of 688 amino acids with 12 putative transmembrane domains and is over 90 % identical to TcYSL7 and AtYSL7. Real-time PCR analysis revealed that BjYSL7 mRNA was mainly expressed in the stem under normal condition. The expression of BjYSL7 was found to be up-regulated by 127.1-, 12.7-, and 3.4-fold in roots and 6.5-, 4.3-, and 2.8-fold in shoots under FeSO4, NiCl2, and CdCl2 stresses, respectively. We have demonstrated that BjYSL7 is a Fe(II)-NA influx transporter by yeast functional complementation. Moreover, a BjYSL7::enhanced green fluorescent protein (EGFP) fusion localized to the plasma membrane of onion epidermal cells. The BjYSL7-overexpressing transgenic tobacco plants exhibited longer root lengths, lower relative inhibition rate of lengths and superior root hair development compared to that of wild-type (WT) plants in the presence of CdCl2 and NiCl2. Furthermore, the concentrations of Cd and Ni in shoots of BjYSL7-overexpressing plants are significantly higher than that of WT plants. Compared with WT plants, BjYSL7-overexpressing plants exhibited Fe concentrations that were higher in the shoots and seeds and lower in the roots. Taken together, these results suggest that BjYSL7 might be involved in the transport of Fe, Cd and Ni to the shoot and improving heavy metal resistance in plants.

[Mechanism of heavy-metal tolerance in Pseudomonas aeruginosa ZGKD2].

The cadmium-resistant bacterium Pseudomonas aeruginosa strain ZGKD2 exhibiting tolerance to various heavy-metals was isolated from gangue pile of coal area in our laboratory. This bacterium could serve as an effective metal sequestering and growth-promoting bioinoculant for plants grown in metal-contaminated soil. However, the mechanism of heavy-metal tolerance is still unclear. When the beef extract-peptone medium was supplemented with 200-3 000 micromol x L(-1) Cd2+, Cu2+, Zn2+, Ni2+, Pb2+ or Mn2+, the maximum biomass of strain ZGKD2 decreased with the increase of heavy-metal concentrations, while different concentrations of heavy-metals had no significant effect on its alkaline production. Stationary-phase cells of strain ZGKD2 were exposed to 0, 200, 600 and 1 000 micromol x L(-1) of Cd2+ , Cu2+, Zn2+ and Ni2+ or 0, 1 000, 2 000 and 3 000 micromol x L(-1) of Pb2+ and Mn2+ for 2 h, respectively. The activity of SOD and CAT increased in a heavy-metal-concentration-dependent manner, especially in the Cd2+ and Cu2+ treatments. The siderophore production of strain ZGKD2 in modified sugar-aspartic acid medium was enhanced by 200- 1 000 micromol x L(-1) of various heavy-metals. Cd2+ and Zn2+ strongly induced the siderophore production of strain ZGKD2, Ni2+ and Mn2+ had little effect, whereas Cu2+ led to significant inhibition. The siderophore production of strain ZGKD2 was positively related with its metal tolerance. These results indicated that alkaline production, siderophore production, and the increase of antioxidant enzyme activities in strain ZGKD2 might be the main mechanisms of heavy-metal tolerance.

The effects of copper, manganese and zinc on plant growth and elemental accumulation in the manganese-hyperaccumulator Phytolacca americana.

Synchrotron radiation X-ray fluorescence (SRXRF) and inductively coupled plasma mass spectrometry were used to estimate major, minor and trace elements in Cu-, Zn- and Mn-treated Phytolacca americana. The effects of the addition of Cu, Zn and Mn on morphological parameters, such as root length, shoot height, and fresh and dry weights of shoots and roots, were also examined. In addition, the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidases (GPX) and catalase (CAT) and the expression of Fe-SOD, Cu/Zn-SOD, metallothionein-2 and glutathione S-transferase (GST) exposed to the highest amounts of Cu, Zn or Mn were detected. Our results confirmed the following: (1) Zn supplementation leads to chlorosis, disturbed elemental homeostasis and decreased concentrations of micro- and macroelements such as Fe, Mg, Mn, Ca and K. Cu competed with Fe, Mn and Zn uptake in plants supplemented with 25 µM Cu. However, no antagonistic interactions took place between Cu, Zn, Mn and Fe uptake in plants supplemented with 100 µM Cu. Mn supplementation at various concentrations had no negative effects on elemental deficits. Mn was co-located with high concentrations of Fe and Zn in mature leaves and the concentrations of macro elements were unchanged. (2) P. americana supplemented with increased concentrations of Zn and Cu exhibited lower biomass production and reduced plant growth. (3) When plants were supplemented with the highest Zn and Cu concentrations, symptoms of toxicity corresponded to decreased SOD or CAT activities and increased APX and GPX activities. However, Mn tolerance corresponded to increased SOD and CAT activities and decreased POD and APX activities. Our study revealed that heavy metals partially exert toxicity by disturbing the nutrient balance and modifying enzyme activities that induce damage in plants. However, P. americana has evolved hyper accumulating mechanisms to maintain elemental balance and redox homeostasis under excess Mn.

Enhancement of Cd tolerance in transgenic tobacco plants overexpressing a Cd-induced catalase cDNA.

Catalase (CAT), an important enzyme of antioxidant system, was investigated the role in preventing the plant from Cd-induced oxidative stress caused by reactive oxygen species. A CAT gene from Brassica juncea was cloned and up-regulated in response to Cd/Zn. The CAT cDNA (BjCAT3) under the control of CaMV35S promoter was introduced into tobacco via Agrobacterium-mediated transformation. Northern blot analysis verified the BjCAT3 was expressed at high level in different transgenic lines. In morphological observation, we found that seedlings from transgenic tobacco plants grew better and showed longer root length in the presence of Cd versus wild-type (WT) seedlings. Under 100 microM Cd stress, WT plants became chlorotic and almost dead while transgenic tobacco plants still remained green and phenotypically normal. The CAT activity of transgenic T(1) generations was approximately two-fold higher than that of WT plants. In WT, endogenous CAT activity is rapidly reduced as a result of 200 microM CdCl2 exposure. Compared with WT plants, lower level of Cd-induced H2O2 accumulation and cell death were detected in roots of transgenic plants with high level of CAT activity. All our findings strongly support that overexpressing BjCAT3 in tobacco could enhance the tolerance under Cd stress.

Cloning and expression analysis of SKn-type dehydrin gene from bean in response to heavy metals.

A heavy metal responsive gene PvSR3 (GenBank accession number U54703) encoding an acid dehydrin was isolated from a mercuric chloride-treated bean (Phaseolus vulgaris L.) leaf cDNA library by differential screening using cDNAs derived from treated and untreated plants. The PvSR3 cDNA is 981-bp long and has a 606-bp open-reading frame with a 202-residue-deduced amino acid sequence. The PvSR3 sequence contains two conserved repeats of the characteristic lysine-rich K segment (EKKGIMDKIKEKLPG) preceded by an 8-serine residue stretch, whereas the Y segment (DEYGNP) conserved motif is absent. The deduced protein has a calculated molecular weight of 23 kDa and an isoelectric point of 5.2. Sequence similarity and comparative analysis showed that PvSR3 shares 70 and 73% similarity with the dehydrin of poplar and pepper, respectively. Southern hybridizations indicated that PvSR3 was a low copy-number gene. Northern blot analysis revealed that PvSR3 mRNA was weakly detected in seedling leaves. However, the gene expression was strongly stimulated by heavy metals, such as mercury, cadmium, arsenic, and copper, whereas virus infection and salt had little effect on it. In contrast, PvSR3 was not responsive to drought or abscisic acid (ABA), and was downregulated by UV radiation. Furthermore, PvSR3 was upregulated by the exogenous signaling molecules, including salicylic acid (SA) and hydrogen peroxide (H2O2). It is suggested that PvSR3 is extremely related to heavy metal stress, and might play an important role in metal detoxification and resistance to the damage caused by heavy metals.