Transcriptomics integrated with metabolomics to characterize key pigment compounds and genes related to anthocyanin biosynthesis in Zanthoxylum bungeanum peel.

Zanthoxylum bungeanum is an important condiment with high economic value and its peel color is one of the main quality indexes. However, the key pigment compounds and related genes are still unclear affecting the quality control of the plants. In this study, the contents of four types of pigments were measured in Z. bungeanum and flavonoids were identified as the most important pigments. Based on the targeted flavonoid metabolomics of Z. bungeanum peels, 14 key pigment compounds were screened out from 152 flavonoids, among which cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside were the most critical compounds for peel color. They were further verified to be present in nine varieties of Z. bungeanum by HPLC fingerprints. The 14 compounds were all associated with flavonoid and anthocyanin biosynthesis pathways and the 39 differentially expressed genes related to these pathways were annotated and screened based on transcriptomics. The genes ZbDFR, ZbANS, and ZbUFGT were identified as three key genes for anthocyanin synthesis in Z. bungeanum peels. Further qRT-PCR results confirmed the reliability of transcriptomics and the accuracy of gene screening. Subsequent protein induced expression demonstrated that ZbANS and ZbUFGT were expressed after 12 h induced by IPTG while ZbDFR was expressed after 15 h. Further transient and stable transformation analysis confirmed that both anthocyanin content and the expression of ZbDFR were significantly increased in overexpression Z. bungeanum leaves and Nicotiana benthamiana. The functional effect of stable transformation of ZbDFR was more significant than that of transient transformation with a 7.67-fold/1.49-fold difference in total anthocyanin content and a 42.37-fold/12.32-fold difference in the expression of ZbDFR. This study provides new insights into the chemical composition and the molecular mechanisms of Z. bungeanum peel color and lays an effective foundation for the color quality control, multi-purpose utilization of Z. bungeanum and the creation of new germplasm.

Broadband Waterborne Multiphase Pentamode Metastructure with Simultaneous Wavefront Manipulation and Energy Absorption Capabilities.

Acoustic metastructures are artificial structures which can manipulate the wavefront in sub-wavelength dimensions, and previously proposed acoustic metastructures have been mostly realized with single materials. An acoustic metastructure with composite structure is proposed for underwater acoustic stealth considering both wavefront manipulation and sound absorption. The unit cells of the metastructure are composed of a metallic supporting lattice, interconnecting polymer materials and mass balancing columns. With the gradual modulations of equivalent physical properties along the horizontal direction of metastructure, the incident acoustic wave is reflected to other directions. Meanwhile, the polymer material inside the unit cells will dissipate the acoustic wave energy due to inherent damping properties. With the simultaneous modulations of reflected wave direction and scattering acoustic amplitude, significant improvement of the underwater stealth effect is achieved. Compared with single-phase metastructure, the Far-Field Sound Pressure Level (FFSPL) of multiphase metastructure decreases by 4.82 dB within the frequency range of 3 kHz~30 kHz. The linearized mean stress for multiphase metastructure is only 1/3 of that of single-phase metastructure due to it having much thicker struts and much more uniform stress distribution under the same hydrostatic pressure. The proposed composite structure possesses potential applications due to its acceptable thickness (80 mm) and low equivalent density (1100 kg/m3).

Relative quantification of phenolic compounds in exocarp-mesocarp and endocarp of sumac (Toxicodendron vernicifluum) combined with transcriptome analysis provides insights into glycosylation of flavonoids and biflavonoid biosynthesis.

The pericarp of fruit can be differentiated into endocarp, mesocarp, and exocarp. To explore the differences in gene expression and metabolites in different tissues of the pericarp, the fruits of sumac (Toxicodendron vernicifluum) were separated into endocarp and mesocarp-exocarp. The metabolites and transcriptome of exocarp-mesocarp and endocarp of Toxicodendron vernicifluum were analyzed by HPLC-QTOF-MS/MS and RNA sequencing, respectively. A total of 52 phenolic compounds were identified, including 3 phenylpropane derivatives, 10 urushiol compounds and 39 flavonoids. The exocarp-mesocarp contained more urushiol compounds and flavonoid glycosides while the endocarp contained more biflavonoids, such as rhusflavone and dihydromorelloflavone. The characteristic component of endocarp was rhusflavone and the characteristic component of exocarp-mesocarp was urushiol (triene). Most of the genes involved in flavonoid synthesis pathway were upregulated in endocarp compared with exocarp-mesocarp and positively correlated with the content of flavonoids. The candidate genes related to the synthesis of components of flavonoid glycosides and biflavonoids were screened. Metabolomic and transcriptomic analyses provide new insights into the synthesis and distribution of flavonoid glycosides and biflavonoids in the fruits of Toxicodendron vernicifluum.

Efficacy and Mechanisms of Oleuropein in Postmenopausal Osteoporosis.

Background: Postmenopausal osteoporosis (PMOP) has a supernal morbidity rate in elderly females. Objective: To appraise the effects of oleuropein on bone densitometry, bone metabolic index, oxidative stress, and inflammatory index in PMOP. In addition, the mechanism of olive bittersweet preventing bone loss was explored. Methods: We grouped 80 salubrious female Sprague-Dawley rats into four teams: (1) sham operation team (sham, N = 20), (2) ovariectomy (OVX, N = 20), (3) castrated mice fed with oleuropein (OVX+ole, N = 20), and (4) castrated mice fed with estrogen (OVX+E2, N = 20). The ovariectomized SD rats were continuously raised with 200 µg/kg/dose of oleuropein. Bone mineral density and bone metabolism indexes were recorded. In order to assess the effectiveness of oleuropein on osteopenia, an enzyme-linked immunosorbent assay (ELISA) was devoted to examining the bone marrow indexes. The bone metabolism standards of PMOP rats were appraised by assessing serum levels of calcium, alkaline phosphatase (ALP), phosphorus, malondialdehyde (MDA), and nitrate content by experimental detection methods and levels of osteoclastogenesis inhibitory factor (OPG) and receptor activator for nuclear factor-κB ligand (RANKL) by ELISA. The OPG-RANK-RANKL signal passage was examined by Western blot (WB). We measured bone mineral density using dual-energy X-rays. Results: Our animal experimental results indicated that oleuropein could significantly improve the bone mineral density of ovariectomized SD rats. In the meantime, it could reduce ending interleukin-6 (IL-6), malondialdehyde (MDA), nitrate, alkaline phosphatase (ALP), and phosphorus (P) serum concentration and would not affect Ca2+ concentration. In cell experiments, oleuropein also can promote the proliferation of osteoblasts. Furthermore, it can promote the expression of OPG protein and mRNA. In reverse, it inhibits the expression of RANKL protein and mRNA. Conclusion: Oleuropein can not only improve the inflammatory and oxidative indexes of castrated rats but also prevent osteoporosis. Oleuropein avoids bone resorption by regulating OPG/RANKL expression.

Risk Factors for Pulmonary Metastasis in Differentiated Thyroid Carcinoma Patients and the Significance of Changes in Matrix Metalloproteinase 13 and microRNA-142 Levels.

This work aims to explore the risk factors of lung metastasis (LM) in differentiated thyroid cancer (DTC) (LM-DTC) and the effect of treatment and to detect the relationship between LM-DTC and the levels of matrix metalloproteinase-13 (MMP-13) and micro ribonucleic acid (RNA)-142 (miR-142) in peripheral blood. The data of 420 patients with DTC who are admitted from March 2020 to December 2021 are collected and divided into a non-metastasis group (non-LM group) of 400 cases and metastasis group (LM group) of 20 cases according whether the mung metastasis is found. In addition, risk factors of LM-DTC are analysed and compared. The results of multivariate logistic analysis show that age, disease course, and imaging timing are independent influencing factors of the radionuclide treatment effect. Follicular carcinoma, abnormal expressions of MMP-13, and miR-142 can increase the risk of LM-DTC. MMP-13 and miR-142 can be undertaken as auxiliary diagnostic biological indicators.

Time-series based metabolomics reveals the characteristics of the color-related metabolites during the different coloration stages of Zanthoxylum bungeanum peel.

As the most visualized evaluation indicator of the commercial quality, the color of Zanthoxylum bungeanum peels is an important external economic characteristic and largely impacts the purchase behavior of consumers. To explore the potential coloration mechanism, the popular cultivars of Z. bungeanum (Fengxian Dahongpao, FXDHP) were selected to investigate its pigment composition and intracellular metabolic characteristics. The results indicated that the red-color formation of FXDHP peels was along with the degradation of chlorophyll and carotenoid, and the accumulation of the total anthocyanin and flavonoids. Furthermore, a widely targeted metabolomics analysis was performed to analyze the changes of intracellular metabolism during five different coloration stages by ultra-performance liquid chromatography-tandem mass spectrometry. Among the identified 803 intracellular metabolites, the largest number of differential metabolites were flavonoids in each comparasion group. In particular, dihydrokaempferol and dihydroquercetin might be the important metabolic nodes during the red-color formation. Cyanidin-3-O-galactoside, keracyanin and kuromanin were the key pigments in the red-color formation of FXDHP peels. Except for red group, four distinct metabolic modules were identified to be specifically associated with the rest four coloration stages of FXDHP peels through the weighted correlation network analysis. Combined with characteristic metabolites and the enzymatic activities related to anthocyanin biosynthesis, the dynamic metabolic changes of anthocyanin accumulation were further clarified during the color transformation of FXDHP peels. The above information would provide novel insights into the red-color formation of FXDHP peels, and it will certainly assist in efforts to improve the outward appeal and quality of Z. bungeanum.

Structure and Mechanism-Guided Design of Dual Serine/Metallo-Carbapenemase Inhibitors.

Serine/metallo-carbapenemase-coproducing pathogens, often referred to as "superbugs", are a significant clinical problem. They hydrolyze nearly all available ß-lactam antibiotics, especially carbapenems considered as last-resort antibiotics, seriously endangering efficacious antibacterial treatment. Despite the continuous global spread of carbapenem resistance, no dual-action inhibitors are available in therapy. This Perspective is the first systematic investigation of all chemotypes, modes of inhibition, and crystal structures of dual serine/metallo-carbapenemase inhibitors. An overview of the key strategy for designing dual serine/metallo-carbapenemase inhibitors and their mechanism of action is provided, as guiding rules for the development of clinically available dual inhibitors, coadministrated with carbapenems, to overcome the carbapenem resistance issue.

Crystal structure of Arabidopsis thaliana glutamyl-tRNAGlu reductase in complex with NADPH and glutamyl-tRNAGlu reductase binding protein.

In higher plants, the tetrapyrrole biosynthesis pathway starts from the reaction catalyzed by the rate-limiting enzyme, glutamyl-tRNAGlu reductase (GTR). In Arabidopsis thaliana, GTR is controlled by post-transcriptional regulators such as GTR binding protein (GBP), which stimulates AtGTR activity. The NADPH-binding domain of AtGTR undergoes a substantial movement upon GBP binding. Here, we report the crystal structure of AtGTR-NADPH-GBP ternary complex. NADPH binding causes slight structural changes compared with the AtGTR-GBP binary complex, and possibly take a part of the space needed by the substrate glutamyl-tRNAGlu. The highly reactive sulfhydryl group of the active-site residue Cys144 shows an obvious rotation, which may facilitate the hydride transfer from NADPH to the thioester intermediate to form glutamate-1-semialdehyde. Furthermore, Lys271, Lys274, Ser275, Asn278, and Gln282 of GBP participate in the interaction between AtGTR and GBP, and the stimulating effect of GBP decreased when all of these residues were mutated to Ala. When the Cys144 of AtGTR was mutated to Ser, AtGTR activity could not be detected even in the presence of GBP.

The Arabidopsis glutamyl-tRNA reductase (GluTR) forms a ternary complex with FLU and GluTR-binding protein.

Tetrapyrrole biosynthesis is an essential and tightly regulated process, and glutamyl-tRNA reductase (GluTR) is a key target for multiple regulatory factors at the post-translational level. By binding to the thylakoid membrane protein FLUORESCENT (FLU) or the soluble stromal GluTR-binding protein (GBP), the activity of GluTR is down- or up-regulated. Here, we reconstructed a ternary complex composed of the C-terminal tetratricopepetide-repeat domain of FLU, GBP, and GluTR, crystallized and solved the structure of the complex at 3.2 Å. The overall structure resembles the shape of merged two binary complexes as previously reported, and shows a large conformational change within GluTR. We also demonstrated that GluTR binds tightly with GBP but does not bind to GSAM under the same condition. These findings allow us to suggest a biological role of the ternary complex for the regulation of plant GluTR.

Structural insights into the catalytic mechanism of Synechocystis magnesium protoporphyrin IX O-methyltransferase (ChlM).

Magnesium protoporphyrin IX O-methyltransferase (ChlM) catalyzes transfer of the methyl group from S-adenosylmethionine to the carboxyl group of the C13 propionate side chain of magnesium protoporphyrin IX. This reaction is the second committed step in chlorophyll biosynthesis from protoporphyrin IX. Here we report the crystal structures of ChlM from the cyanobacterium Synechocystis sp. PCC 6803 in complex with S-adenosylmethionine and S-adenosylhomocysteine at resolutions of 1.6 and 1.7 Å, respectively. The structures illustrate the molecular basis for cofactor and substrate binding and suggest that conformational changes of the two "arm" regions may modulate binding and release of substrates/products to and from the active site. Tyr-28 and His-139 were identified to play essential roles for methyl transfer reaction but are not indispensable for cofactor/substrate binding. Based on these structural and functional findings, a catalytic model is proposed.

LcWRKY5: an unknown function gene from sheepgrass improves drought tolerance in transgenic Arabidopsis.

KEY MESSAGE: The expression of LcWRKY5 was induced significantly by salinity, mannitol and cutting treatments. Arabidopsis- overexpressing LcWRKY5 greatly increased dehydration tolerance by regulating the expression of multiple stress-responsive genes. Based on the data of sheepgrass 454 high-throughout sequencing and expression analysis results, a drought-induced gene LcWRKY5 was isolated and cloned, and the biological role of the gene has not been reported until now. Bioinformatics analysis showed that LcWRKY5 contains one conserved WD domain and belongs to the group II WRKY protein family. LcWRKY5 shows high sequence identity with predicted or putative protein products of Hordeum vulgare, Aegilops tauschii, Triticum aestivum, Brachypodium distachyon, Oryza sativa, but it has low homology with WRKYs from dicotyledonous plants. Several drought-inducibility, fungal elicitor, MeJA-responsiveness, endosperm, light, anoxic specific inducibility, and circadian control elements were found in the promoter region of LcWRKY5. Tissue-specific expression patterns showed that LcWRKY5 is expressed in roots and leaves, without expression in other tissues. The expression of LcWRKY5 was induced significantly under salinity and mannitol stresses but was not notably changed under cold and Abscisic acid stress. The LcWRKY5 protein exhibits transcription activation activity in the yeast one-hybrid system. Overexpressing LcWRKY5 exhibited increased rates of cotyledon greening and plant survival in transgenic Arabidopsis compared with wild-type plants under drought stress, and the expression levels of DREB2A and RD29A in transgenic plants were enhanced under drought stress. These results indicated that LcWRKY5 may play an important role in drought-response networks through regulation of the DREB2A pathway. LcWRKY5 can be a candidate gene for engineering drought tolerance in other crops.

Crystal structure of Arabidopsis glutamyl-tRNA reductase in complex with its stimulator protein.

Tetrapyrrole biosynthesis in plants, algae, and most bacteria starts from the NADPH-dependent reduction of glutamyl-tRNA by glutamyl-tRNA reductase (GluTR). The GluTR-catalyzed reaction is the rate-limiting step, and GluTR is the target of multiple posttranslational regulations, such as heme feedback inhibition, for the tetrapyrrole biosynthetic pathway. A recently identified GluTR regulator, GluTR binding protein (GluBP), has been shown to spatially organize tetrapyrrole synthesis by distributing GluTR into different suborganellar locations. Here we report the complex structure of GluTR-GluBP from Arabidopsis thaliana. The dimeric GluBP binds symmetrically to the catalytic domains of the V-shaped GluTR dimer via its C-terminal domain. A substantial conformational change of the GluTR NADPH-binding domain is observed, confirming the postulated rotation of the NADPH-binding domain for hydride transfer from NADPH to the substrate. Arg146, "guarding the door" for metabolic channeling, adopts alternative conformations, which may represent steps involved in substrate recognition and product release. A coupled enzyme assay shows that GluBP stimulates GluTR catalytic efficiency with an approximate threefold increase of the 5-aminolevulinic acid formation rate. In addition, the GluTR activity can be inhibited by heme in a concentration-dependent way regardless of the presence of GluBP. A structural alignment indicates that GluBP belongs to a heme-binding family involved in heme metabolism. We propose a catalytic mechanism model for GluTR, through which photosynthetic organisms can achieve precise regulation of tetrapyrrole biosynthesis.

Cloning two P5CS genes from bioenergy sorghum and their expression profiles under abiotic stresses and MeJA treatment.

Sweet sorghum (Sorghum bicolor (Linn.) Moench) has promise as a bioenergy feedstock in China and other countries for its use in the production of ethanol as the result of its high fermentable sugar accumulation in stems. To boost biofuel production and extend its range, we seek to improve its stress tolerance. Proline acts as an osmolyte that accumulates when plants are subjected to abiotic stress. P5CS (Δ1-pyrroline-5-carboxylate synthetase) is a key regulatory enzyme that plays a crucial role in proline biosynthesis. We isolated two closely related P5CS genes from sweet sorghum, designated SbP5CS1 (GenBank accession number: GQ377719) and SbP5CS2 (GenBank accession number: GQ377720), which are located on chromosome 3 and 9 and encode 729 and 716 amino acid polypeptides, respectively. The homology between the two sweet sorghum P5CS genes was 76%. Promoter analysis of the two P5CS genes revealed that both sequences not only contained the expected cis regulatory regions such as TATA and CAAT boxes, but also had many stress response elements. Expression analysis revealed that SbP5CS1 and SbP5CS2 transcripts were up-regulated after treatment of 10-day-old seedlings of sweet sorghum with drought, salt (250mM NaCl) and MeJA (10µM). The expression levels of the both SbP5CS genes were significantly increased after 3-day drought stress. Under high salt treatment, peak SbP5CS1 expression was detected at 4h and 8h for SbP5CS2 in roots, while the trends of expression were nearly identical in leaves. In contrast, under drought and high salt stress, the up-regulated expression of SbP5CS1 was higher than that of SbP5CS2. When the seedlings were exposed to MeJA, rapid transcript induction of SbP5CS1 was detected at 2h in leaves, and the SbP5CS2 expression level increase was detected at 4h post-treatment. SbP5CS1 and SbP5CS2 also show different temporal and spatial expression patterns. SbP5CS2 gene was ubiquitously expressed whereas SbP5CS1 was mainly expressed in mature vegetative and reproductive organs. Proline concentration increased after stress application and was correlated with SbP5CS expression. Our results suggest that the SbP5CS1 and SbP5CS2 are stress inducible genes but might play non-redundant roles in plant development. The two genes could have the potential to be used in improving stress tolerance of sweet sorghum and other bioenergy feedstocks.

Genes and pathways induced in early response to defoliation in rice seedlings.

How plant gene expression respond to grazing defoliation is critical for plant re-growth, survival, and composition in the natural and dairy farming grassland environments. Rice, with genome sequence available, was used as a model plant to study grazing-induced pathway selections. When seedlings were 18 cm in height, the top 12 cm was removed by simulated grazing. The gene expression activities within 2 to 24 hours in the remaining aboveground tissues were profiled using the Affymetrix Rice GeneChips and RT-qPCR. The seedlings responded to grazing by immediately adjusting their global gene expression, e.g., enhancing anaerobic respiration, starch-to-sugar conversion, sucrose synthesis, and sugar transport. The results suggest that (1) remaining aboveground tissues used anaerobic respiration as an emergency measure for energy/substrates supply; (2) Sink tissues reduced its demand after 2 h; (3) Sucrose synthesis enhancement around the 24th hour is likely driven by shoot re-growth. In total, the expression activity of 466 genes, involved in signal transduction, miRNA regulation, cell wall modification, metabolism, hormone synthesis, and molecule transporters, had been significantly changed at least twofold. These genes and their biochemical pathways identified provide insights into how plants respond to grazing at the molecular physiology level.

[Effect of CDK7 gene silencing against hepatoblastoma HepG2 cell proliferation].

OBJECTIVE: To screen the antisense oligodeoxynucleotides (ASODN) that inhibit cultured hepatoblastoma HepG2 cell proliferation, and evaluate the antiproliferative potency of modified ASODN. METHODS: ASODN sequences were selected based on the secondary structure of human CDK7 mRNA predicted with RNAStructure 3.71 software. The binding thermodynamics of CDK7 mRNA to ASODN was analyzed by OligoWalk program. The sequences with the strongest effect against cultured HepG2 cell proliferation in vitro were selected, and the fragments complementary to 1-5 bases upstream or downstream to the complementary region were structurally modified and screened. RESULTS: The partial phosphorothioate ASODN complementary to 284-303 region of human CDK7 mRNA was the most powerful inhibitor, and the antiproliferative activity reached 40.4+/-12.6%; in the second round of screening, the antiproliferative activity of the full phosphorothioate ASODN complementary to the 287-306 region of the mRNA on HepG2 cells was 68.3+/-2.6%, with IC50 of 51.9+/-8.6 nmol/L. CONCLUSION: Proliferation of HepG2 cells can be significantly inhibited by the screened ASODN, which might be used as a lead compound in the development of specific CDK7 inhibitors.

[Pharmacokinetics of Nolaterex in tumor cells].

OBJECTIVE: To investigate the relationship of the transportation characteristics of Nolaterex, a new anti-cancer drug, with its sensitivity in tumor cells. METHODS: The sensitivities of 3 tumor cell lines (C6, SRS-82 and LoVo) to nolaterex were determined by growth inhibition test. After Nolaterex exposure, the intracellular drug concentration was measured by high-performance liquid chromatography. RESULTS: C6 was the most sensitive among the 3 cell lines, and the IC50 values of SRS-82 and LoVo cells were 6.8-fold and 13.8-fold higher, respectively, than that of C6 cells. In all the 3 cell lines, linear relationship between intracellular and extracellular drug concentrations was noted. The intracellular steady-state level achieved in C6 was significantly higher than the levels in the other two cell lines. CONCLUSIONS: The results indicate that nolaterex quickly enters the cells, and different cell lines may have different nolaterex-transporting capacities, thus partially accounting for different states of sensitivity of the tumor cells to nolaterex.