Oxidative damage induced by combined exposure of titanium dioxide nanoparticles and cypermethrin in rats for 90 days.

Titanium dioxide nanoparticles (TiO2NPs) and cypermethrin (CPM) are widely used in various fields, and they can enter the environment in different ways. Combined exposure of TiO2NPs and CPM may increase the accumulation of pollutants in organisms and affect human health. This study was undertaken to evaluate the oxidative and inflammatory parameters associated with the combined exposure of TiO2NPs and CPM in rats. Twenty-four healthy male adult SD rats were randomly divided into four groups. The first group served as the control, while groups 2, 3, and 4 were treated with TiO2NPs (450 mg/m3); CPM (6.67 mg/m3) or combined exposure of TiO2NPs and CPM by inhalation for 90 days. We investigated the oxidative damage induced through combined exposure of TiO2NPs and CPM in rats by evaluating hematology of the rats and determining the blood biochemical index. Our results demonstrated that inhalation of TiO2NPs and CPM increased the levels of oxidative stress markers such as malondialdehyde and alkaline phosphatase in the serum of rats. These were accompanied by a decreased glutathione peroxidase and total superoxide dismutase levels. Furthermore, the level of glutathione peroxidase was further decreased while malondialdehyde was increased in the combined exposure of TiO2NPs and CPM. Interestingly, pathological sections showed that different degrees of tissue injury could be seen in the liver and lung tissues of each exposure group. In summary, the combined exposure of TiO2NPs and CPM can cause increased oxidative damage in rats and damage the tissue structure of the liver and lung.

Temperature Prediction of Seasonal Frozen Subgrades Based on CEEMDAN-LSTM Hybrid Model.

Improving the temperature prediction accuracy for subgrades in seasonally frozen regions will greatly help improve the understanding of subgrades' thermal states. Due to the nonlinearity and non-stationarity of the temperature time series of subgrades, it is difficult for a single general neural network to accurately capture these two characteristics. Many hybrid models have been proposed to more accurately forecast the temperature time series. Among these hybrid models, the CEEMDAN-LSTM model is promising, thanks to the advantages of the long short-term memory (LSTM) artificial neural network, which is good at handling complex time series data, and its combination with the broad applicability of the complete ensemble empirical mode decomposition with adaptive noise (CEEMDAN) in the field of signal decomposition. In this study, by performing empirical mode decomposition (EMD), ensemble empirical mode decomposition (EEMD), and CEEMDAN on temperature time series, respectively, a hybrid dataset is formed with the corresponding time series of volumetric water content and frost heave, and finally, the CEEMDAN-LSTM model is created for prediction purposes. The results of the performance comparisons between multiple models show that the CEEMDAN-LSTM model has the best prediction performance compared to other decomposed LSTM models because the composition of the hybrid dataset improves predictive ability, and thus, it can better handle the nonlinearity and non-stationarity of the temperature time series data.

Optimal ablation index parameters for radiofrequency ablation therapy of atrial fibrillation.

Objectives: To explore the optimal ablation index (AI) parameters for radiofrequency catheter ablation (RA) for treating atrial fibrillation (AF). Methods: Patients with AF (186) who underwent bilateral PVAI in the Department of Cardiology, Zhuhai People's Hospital, Guangdong Province, from March 2018 to October 2019 and received catheter ablation as first-round treatment, were grouped according to the received AI. Control group included patients (95) who received the recommended AI ablation (350-400 for posterior wall, 400-450 for non-posterior wall). Patients in optimal AI group were ablated with optimal AI (300-330 for posterior wall, 350-380 for non-posterior wall). Recurrence was defined as any AF, atrial tachycardia, or atrial flutter lasting more than 30 seconds without anti-arrhythmic drugs after the 3-month blank period. Results: Of 186 patients, 66 patients had paroxysmal atrial fibrillation and a mean CHA2DS2-VASc score of 2.83±1.64. Isolation rates of bilateral PVI in both groups were 91.4% and 93.6%, for patients with paroxysmal atrial fibrillation, and 81.7% and 80% for patients with persistent atrial fibrillation (P > 0.05). Left atrial function index (LAFI) decreased under the condition of sinus rhythm at the 3rd and 6th months (P < 0.05). LAFI improvement was significantly better in the optimal AI group than in the control group (P < 0.05). Rates of pain and cough during the ablation, and postoperative gastrointestinal discomfort and use of PPIs were higher in the control group (P < 0.05). The recurrence rate was 14.7% and 14.3% after 12 months of follow-up, respectively, and the difference was not statistically significant (P > 0.05). Conclusion: Radiofrequency ablation of AF, guided by optimal AI combined with impedance, can minimize atrial injury, prevent atrial failure, promote the recovery of atrial function, reduces intraoperative cough, pain, and postoperative gastrointestinal discomfort and use of PPIs.

A Freezing-Thawing Damage Characterization Method for Highway Subgrade in Seasonally Frozen Regions Based on Thermal-Hydraulic-Mechanical Coupling Model.

Seasonally frozen soil where uneven freeze-thaw damage is a major cause of highway deterioration has attracted increased attention in China with the rapid development of infrastructure projects. Based on Darcy's law of unsaturated soil seepage and heat conduction, the thermal-hydraulic-mechanical (THM) coupling model is established considering a variety of effects (i.e., ice-water phase transition, convective heat transfer, and ice blocking effect), and then the numerical solution of thermal-hydraulic fields of subgrade can be obtained. Then, a new concept, namely degree of freeze-thaw damage, is proposed by using the standard deviation of the ice content of subgrade during the annual freeze-thaw cycle. To analyze the freeze-thaw characteristics of highway subgrade, the model is applied in the monitored section of the Golmud to Nagqu portion of China National Highway G109. The results show that: (1) The hydrothermal field of subgrade has an obvious sunny-shady slopes effect, and its transverse distribution is not symmetrical; (2) the freeze-thaw damage area of subgrade obviously decreased under the insulation board measure; (3) under the combined anti-frost measures, the maximum frost heave amount of subgrade is significantly reduced. This study will provide references for the design of highway subgrades in seasonally frozen soil areas.

Metabolic engineering of anthocyanins in dark tobacco varieties.

In this study, we investigate the metabolic engineering of anthocyanins in two dark tobacco crops (Narrow Leaf Madole and KY171) and evaluate the effects on physiological features of plant photosynthesis. Arabidopsis PAP1 (production of anthocyanin pigment 1) gene (AtPAP1) encodes a R2R3-type MYB transcript factor that is a master component of regulatory complexes controlling anthocyanin biosynthesis. AtPAP1 was introduced to Narrow Leaf Madole and KY171 plants. Multiple transgenic plants developed red/purple pigmentation in different tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that the expression levels of six pathway genes were increased two- to eight-fold in AtPAP1 transgenic plants compared with vector control plants. Dihydroflavonol reductase and anthocyanidin synthase genes that were not expressed in wild-type plants were activated. Spectrophotometric measurement showed that the amount of anthocyanins in AtPAP1 transgenic plants were 400-800 µg g-1 fresh weight (FW). High-performance liquid chromatography (HPLC) analysis showed that one main anthocyanin molecule accounted for approximately 98% of the total anthocyanins. Tandem MS/MS analysis using HPLC coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry identified the main anthocyanin as cyanidin 3-O-rutinoside, an important medicinal anthocyanin. Analysis of photosynthesis rate, chlorophylls and carotenoids contents showed no differences between red/purple transgenic and control plants, indicating that this metabolic engineering did not alter photosynthetic physiological traits. This study shows that AtPAP1 is of significance for metabolic engineering of anthocyanins in crop plants for value-added traits.

Metabolic engineering of proanthocyanidin production by repressing the isoflavone pathways and redirecting anthocyanidin precursor flux in legume.

MtPAR is a proanthocyanidin (PA) biosynthesis regulator; the mechanism underlying its promotion of PA biosynthesis is not fully understood. Here, we showed that MtPAR promotes PA production by a direct repression of biosynthesis of isoflavones, the major flavonoids in legume, and by redirecting immediate precursors, such as anthocyanidins, flux into PA pathway. Ectopic expression of MtPAR repressed isoflavonoid production by directly binding and suppressing isoflavone biosynthetic genes such as isoflavone synthase (IFS). Meanwhile, MtPAR up-regulated PA-specific genes and decreased the anthocyanin levels without altering the expression of anthocyanin biosynthetic genes. MtPAR may shift the anthocyanidin precursor flux from anthocyanin pathway to PA biosynthesis. MtPAR complemented PA-deficient phenotype of Arabidopsis tt2 mutant seeds, demonstrating their similar action on PA production. We showed the direct interactions between MtPAR, MtTT8 and MtWD40-1 proteins from Medicago truncatula and Glycine max, to form a ternary complex to trans-activate PA-specific ANR gene. Finally, MtPAR expression in alfalfa (Medicago sativa) hairy roots and whole plants only promoted the production of small amount of PAs, which was significantly enhanced by co-expression of MtPAR and MtLAP1. Transcriptomic and metabolite profiling showed an additive effect between MtPAR and MtLAP1 on the production of PAs, supporting that efficient PA production requires more anthocyanidin precursors. This study provides new insights into the role and mechanism of MtPAR in partitioning precursors from isoflavone and anthocyanin pathways into PA pathways for a specific promotion of PA production. Based on this, a strategy by combining MtPAR and MtLAP1 co-expression to effectively improve metabolic engineering performance of PA production in legume forage was developed.

MtPAR MYB transcription factor acts as an on switch for proanthocyanidin biosynthesis in Medicago truncatula.

MtPAR (Medicago truncatula proanthocyanidin regulator) is an MYB family transcription factor that functions as a key regulator of proanthocyanidin (PA) biosynthesis in the model legume Medicago truncatula. MtPAR expression is confined to the seed coat, the site of PA accumulation. Loss-of-function par mutants contained substantially less PA in the seed coat than the wild type, whereas levels of anthocyanin and other specialized metabolites were normal in the mutants. In contrast, massive accumulation of PAs occurred when MtPAR was expressed ectopically in transformed hairy roots of Medicago. Transcriptome analysis of par mutants and MtPAR-expressing hairy roots, coupled with yeast one-hybrid analysis, revealed that MtPAR positively regulates genes encoding enzymes of the flavonoid-PA pathway via a probable activation of WD40-1. Expression of MtPAR in the forage legume alfalfa (Medicago sativa) resulted in detectable levels of PA in shoots, highlighting the potential of this gene for biotechnological strategies to increase PAs in forage legumes for reduction of pasture bloat in ruminant animals.

Functional characterization of proanthocyanidin pathway enzymes from tea and their application for metabolic engineering.

Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blight-resistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (-)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (-)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols.

MATE2 mediates vacuolar sequestration of flavonoid glycosides and glycoside malonates in Medicago truncatula.

The majority of flavonoids, such as anthocyanins, proanthocyanidins, and isoflavones, are stored in the central vacuole, but the molecular basis of flavonoid transport is still poorly understood. Here, we report the functional characterization of a multidrug and toxin extrusion transporter (MATE2), from Medicago truncatula. MATE 2 is expressed primarily in leaves and flowers. Despite its high similarity to the epicatechin 3'-O-glucoside transporter MATE1, MATE2 cannot efficiently transport proanthocyanidin precursors. In contrast, MATE2 shows higher transport capacity for anthocyanins and lower efficiency for other flavonoid glycosides. Three malonyltransferases that are coexpressed with MATE2 were identified. The malonylated flavonoid glucosides generated by these malonyltransferases are more efficiently taken up into MATE2-containing membrane vesicles than are the parent glycosides. Malonylation increases both the affinity and transport efficiency of flavonoid glucosides for uptake by MATE2. Genetic loss of MATE2 function leads to the disappearance of leaf anthocyanin pigmentation and pale flower color as a result of drastic decreases in the levels of various flavonoids. However, some flavonoid glycoside malonates accumulate to higher levels in MATE2 knockouts than in wild-type controls. Deletion of MATE2 increases seed proanthocyanidin biosynthesis, presumably via redirection of metabolic flux from anthocyanin storage.

Combined exposure of beta-cypermethrin and emamectin benzoate interferes with the HPO axis through oxidative stress, causing an imbalance of hormone homeostasis in female rats.

The impact of pesticides on reproductive health has been increasingly recognized. ß-cypermethrin (ß-CYP) and emamectin benzoate (EMB) are commonly used with agricultural workers. There are few published studies on the effects of combined poisoning of these two pesticides on the reproductive system. This study investigated the toxic effects and mechanism of ß-CYP and EMB on the reproductive system of female rats based on the hypothalamic-pituitary-ovarian (HPO) axis. The hypothalamic GnRH content tended to decrease, and Kiss-1 and GPR-54 mRNA and protein expression tended to increase in exposed rats. FSH content was elevated for the pituitary gland, and Kiss-1 and GPR-54 mRNA and protein expression were enhanced in all experimental groups compared with the control group. E2 content in rat ovaries and ERα mRNA and protein expression were reduced by ß-CYP and EMB. Furthermore, there were interactive effects of ß-CYP and EMB on FSH and E2 release, pituitary GPR-54 mRNA and protein, and ovarian ERα mRNA expression. To investigate causes of damage, oxidative damage indicators were tested and showed that exposure to ß-CYP and EMB decreased GSH-Px and SOD activities in the HPO axis, increased MDA levels in the hypothalamus and ovary together with LDH activities in the HPO axis, with an interaction effect on GSH-Px and SOD activities in the hypothalamus and pituitary gland as well as on MDA in the ovary. The above results support the screening of sensitive molecular biomarkers and evaluation of the adverse effects of pesticide exposure in greenhouse operations on reproductive health.

Experimental study on new structure teeth in a high-temperature environment.

In high-temperature drilling, especially in high-temperature geothermal drilling, cone bits often experience common and severe tooth loss. This issue significantly reduces the cone bit's service life and has a detrimental impact on drilling efficiency. The quality of the fixed teeth plays a crucial role in the performance of the cone bit. In high-temperature environments, conventional methods fail to meet the requirements for securing the cone bit's teeth. Therefore, to address the tooth loss problem in high-temperature drilling, a new tapered tooth structure is proposed. Laboratory experiments were conducted to secure teeth with varying tapers at both normal and high temperatures. The results revealed that the maximum fastening force increased progressively with the degree of taper, reaching its peak at C50. Compared to conventional cylindrical teeth, the maximum fastening force increased by approximately 88.6%-271.1% at different temperatures. The tapered structure demonstrated superior tooth-fixing strength. The maximum fastening force is the smallest at 300 °C, approximately 23.7%-61.2% lower than at normal temperature. Under the same interference conditions, the maximum fastening force increased with greater taper. With interference values of 0.075, 0.095, and 0.115, the maximum fastening force increased by 48.9%-175.1%, 14%-141.6%, and 53%-271.1%, respectively, when compared to cylindrical teeth with C300, C200, C100, and C50 tapers. The tapered structure exhibited superior tooth-fixing strength and significantly enhanced tooth retention strength at high temperatures.

STGNNks: Identifying cell types in spatial transcriptomics data based on graph neural network, denoising auto-encoder, and k-sums clustering.

BACKGROUND: Spatial transcriptomics technologies fully utilize spatial location information, tissue morphological features, and transcriptional profiles. Integrating these data can greatly advance our understanding about cell biology in the morphological background. METHODS: We developed an innovative spatial clustering method called STGNNks by combining graph neural network, denoising auto-encoder, and k-sums clustering. First, spatial resolved transcriptomics data are preprocessed and a hybrid adjacency matrix is constructed. Next, gene expressions and spatial context are integrated to learn spots' embedding features by a deep graph infomax-based graph convolutional network. Third, the learned features are mapped to a low-dimensional space through a zero-inflated negative binomial (ZINB)-based denoising auto-encoder. Fourth, a k-sums clustering algorithm is developed to identify spatial domains by combining k-means clustering and the ratio-cut clustering algorithms. Finally, it implements spatial trajectory inference, spatially variable gene identification, and differentially expressed gene detection based on the pseudo-space-time method on six 10x Genomics Visium datasets. RESULTS: We compared our proposed STGNNks method with five other spatial clustering methods, CCST, Seurat, stLearn, Scanpy and SEDR. For the first time, four internal indicators in the area of machine learning, that is, silhouette coefficient, the Davies-Bouldin index, the Caliniski-Harabasz index, and the S_Dbw index, were used to measure the clustering performance of STGNNks with CCST, Seurat, stLearn, Scanpy and SEDR on five spatial transcriptomics datasets without labels (i.e., Adult Mouse Brain (FFPE), Adult Mouse Kidney (FFPE), Human Breast Cancer (Block A Section 2), Human Breast Cancer (FFPE), and Human Lymph Node). And two external indicators including adjusted Rand index (ARI) and normalized mutual information (NMI) were applied to evaluate the performance of the above six methods on Human Breast Cancer (Block A Section 1) with real labels. The comparison experiments elucidated that STGNNks obtained the smallest Davies-Bouldin and S_Dbw values and the largest Silhouette Coefficient, Caliniski-Harabasz, ARI and NMI, significantly outperforming the above five spatial transcriptomics analysis algorithms. Furthermore, we detected the top six spatially variable genes and the top five differentially expressed genes in each cluster on the above five unlabeled datasets. And the pseudo-space-time tree plot with hierarchical layout demonstrated a flow of Human Breast Cancer (Block A Section 1) progress in three clades branching from three invasive ductal carcinoma regions to multiple ductal carcinoma in situ sub-clusters. CONCLUSION: We anticipate that STGNNks can efficiently improve spatial transcriptomics data analysis and further boost the diagnosis and therapy of related diseases. The codes are publicly available at https://github.com/plhhnu/STGNNks.

The combined effect of titanium dioxide nanoparticles and cypermethrin on male reproductive toxicity in rats.

Titanium nanoparticles and pyrethroid pesticides are now being widely used in industrial, agriculture, and biomedical applications. In recent years, their health safety profiles have aroused concerns among health scientists. This study mainly explored the combined effects of titanium dioxide nanoparticles (TiO2NPs) and cypermethrin (CYP) on reproductive toxicity in male rats by gavage for 90 days. Thirty-two male Sprague-Dawley rats were assigned to four groups: the control group, the TiO2NPs group, the CYP group, and the combined titanium dioxide nanoparticles with cypermethrin (TiO2NPs + CYP) group. The results of biochemical analysis on testicular tissue homogenate showed that TiO2NPs and CYP mixtures decreased the activities of glutathione peroxidase (GSH-Px) and catalase (CAT) while increasing the activity of malondialdehyde (MDA) and lactate dehydrogenase (LDH). Meanwhile, the results of two-way factorial analysis of variance (ANOVA) showed a significant effect on GSH-Px, CAT, LDH, testicular cell apoptosis, and sperm quality in rats after exposure. Furthermore, the combined exposure group exhibited apoptosis of testicular cells and DNA damage. The results indicated that exposure to a mixture of TiO2NPs and CYP had adverse effects on the reproductive status of male rats.

Functional characterization of the switchgrass (Panicum virgatum) R2R3-MYB transcription factor PvMYB4 for improvement of lignocellulosic feedstocks.

• The major obstacle for bioenergy production from switchgrass biomass is the low saccharification efficiency caused by cell wall recalcitrance. Saccharification efficiency is negatively correlated with both lignin content and cell wall ester-linked p-coumarate: ferulate (p-CA : FA) ratio. In this study, we cloned and functionally characterized an R2R3-MYB transcription factor from switchgrass and evaluated its potential for developing lignocellulosic feedstocks. • The switchgrass PvMYB4 cDNAs were cloned and expressed in Escherichia coli, yeast, tobacco and switchgrass for functional characterization. Analyses included determination of phylogenetic relations, in situ hybridization, electrophoretic mobility shift assays to determine binding sites in target promoters, and protoplast transactivation assays to demonstrate domains active on target promoters. • PvMYB4 binds to the AC-I, AC-II and AC-III elements of monolignol pathway genes and down-regulates these genes in vivo. Ectopic overexpression of PvMYB4 in transgenic switchgrass resulted in reduced lignin content and ester-linked p-CA : FA ratio, reduced plant stature, increased tillering and an approx. threefold increase in sugar release efficiency from cell wall residues. • We describe an alternative strategy for reducing recalcitrance in switchgrass by manipulating the expression of a key transcription factor instead of a lignin biosynthetic gene. PvMYB4-OX transgenic switchgrass lines can be used as potential germplasm for improvement of lignocellulosic feedstocks and provide a platform for further understanding gene regulatory networks underlying switchgrass cell wall recalcitrance.

A de novo regulation design shows an effectiveness in altering plant secondary metabolism.

Introduction: Transcription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants. Objectives: We selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering. Methods: G-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual-luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism. Results: PAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P + T and PAP1 genotypes. Conclusion: G-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

A genomic approach to isoflavone biosynthesis in kudzu (Pueraria lobata).

Roots of kudzu (Pueraria lobata) are a rich source of isoflavone O- and C-glycosides. Although O-glycosylation of (iso)flavonoids has been well characterized at the molecular level, no plant isoflavonoid C-glycosyltransferase genes have yet been isolated. To address the biosynthesis of kudzu isoflavonoids, we generated 6,365 high-quality expressed sequence tags (ESTs) from a subtraction cDNA library constructed using RNA from roots that differentially accumulate puerarin. The ESTs were clustered into 722 TCs and 3,913 singletons, from which 15 family I glycosyltransferases (UGTs) were identified. Hierarchical clustering analysis of the expression patterns of these UGTs with isoflavone synthase (IFS) in a range of tissues identified UGTs with potential functions in isoflavone glycosylation. The open reading frames of these UGTs were expressed in E. coli for functional analysis, and one was shown to preferentially glycosylate isoflavones at the 7-O-position. In addition, ESTs corresponding to chalcone synthase, chalcone reductase, chalcone isomerase (CHI) and 2-hydroxyisoflavanone dehydratase were identified. Recombinant CHI proteins had high activities with both 6'-deoxy- and 6'-hydroxy chalcones, typical of Type II CHIs. Establishment of this EST database and identification of genes associated with kudzu isoflavone biosynthesis and glycosylation provide a new resource for metabolic engineering of bioactive kudzu isoflavones.

Phenylalanine ammonia-lyase (PAL) from tobacco (Nicotiana tabacum): characterization of the four tobacco PAL genes and active heterotetrameric enzymes.

PAL (L-phenylalanine ammonia-lyase), the first enzyme of phenylpropanoid biosynthesis, is often encoded by multigene families in plants. A PCR-based approach was used to isolate cDNA clones corresponding to the four PAL genes of tobacco (Nicotiana tabacum). By careful comparison of cDNA and genomic clones, a new PAL gene (PAL4) was defined. PCR amplification of PAL sequences from cDNA led to the generation of chimaeric clones between PAL1 and PAL4, and incorrect annotation of PAL4 ESTs (expressed sequence tags) as PAL1 in the EST database has given rise to a randomly shuffled tentative consensus sequence. The PAL2 previously described in the literature was shown, by domain swapping experiments with PAL1, to possess a single nucleotide substitution leading to an inactive enzyme. The altered amino acid resulting from this substitution maps to the base of the active site pocket in the three-dimensional structure of PAL. The inactive PAL2 allele could not be recovered from 13 different tobacco cultivars examined. PALs 1-4 were co-expressed in multiple plant organs, and were also co-induced following exposure of cell cultures to yeast elicitor or methyl jasmonate. All four tobacco PAL proteins expressed in Escherichia coli displayed normal Michaelis-Menten kinetics, with Km values between 36 and 60 muM. Co-expression of different PAL proteins in E. coli resulted in formation of heterotetramers, which possessed kinetic properties within the same range as those of the individual homotetramers. The potential physiological function of heterotetrameric PAL forms is discussed.

Elicitor-induced transcription factors for metabolic reprogramming of secondary metabolism in Medicago truncatula.

BACKGROUND: Exposure of Medicago truncatula cell suspension cultures to pathogen or wound signals leads to accumulation of various classes of flavonoid and/or triterpene defense molecules, orchestrated via a complex signalling network in which transcription factors (TFs) are essential components. RESULTS: In this study, we analyzed TFs responding to yeast elicitor (YE) or methyl jasmonate (MJ). From 502 differentially expressed TFs, WRKY and AP2/EREBP gene families were over-represented among YE-induced genes whereas Basic Helix-Loop-Helix (bHLH) family members were more over-represented among the MJ-induced genes. Jasmonate ZIM-domain (JAZ) transcriptional regulators were highly induced by MJ treatment. To investigate potential involvement of WRKY TFs in signalling, we expressed four Medicago WRKY genes in tobacco. Levels of soluble and wall bound phenolic compounds and lignin were increased in all cases. WRKY W109669 also induced tobacco endo-1,3-beta-glucanase (NtPR2) and enhanced the systemic defense response to tobacco mosaic virus in transgenic tobacco plants. CONCLUSION: These results confirm that Medicago WRKY TFs have broad roles in orchestrating metabolic responses to biotic stress, and that they also represent potentially valuable reagents for engineering metabolic changes that impact pathogen resistance.

Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).

Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.

Construction and characterization of VL-VH tail-parallel genetically engineered antibodies against staphylococcal enterotoxins.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have increasingly given rise to human health and food safety. Genetically engineered small molecular antibody is a useful tool in immuno-detection and treatment for clinical illness caused by SEs. In this study, we constructed the V(L)-V(H) tail-parallel genetically engineered antibody against SEs by using the repertoire of rearranged germ-line immunoglobulin variable region genes. Total RNA were extracted from six hybridoma cell lines that stably express anti-SEs antibodies. The variable region genes of light chain (V(L)) and heavy chain (V(H)) were cloned by reverse transcription PCR, and their classical murine antibody structure and functional V(D)J gene rearrangement were analyzed. To construct the eukaryotic V(H)-V(L) tail-parallel co-expression vectors based on the "5'-V(H)-ivs-IRES-V(L)-3'" mode, the ivs-IRES fragment and V(L) genes were spliced by two-step overlap extension PCR, and then, the recombined gene fragment and V(H) genes were inserted into the pcDNA3.1(+) expression vector sequentially. And then the constructed eukaryotic expression clones termed as p2C2HILO and p5C12HILO were transfected into baby hamster kidney 21 cell line, respectively. Two clonal cell lines stably expressing V(L)-V(H) tail-parallel antibodies against SEs were obtained, and the antibodies that expressed intracytoplasma were evaluated by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry. SEs can stimulate the expression of some chemokines and chemokine receptors in porcine IPEC-J2 cells; mRNA transcription level of four chemokines and chemokine receptors can be blocked by the recombinant SE antibody prepared in this study. Our results showed that it is possible to get functional V(L)-V(H) tail-parallel genetically engineered antibodies in same vector using eukaryotic expression system.