Overexpressing PagGS1;2 maintains carbon and nitrogen balance under high-ammonium conditions and shows increased tolerance to ammonium toxicity in 84K Poplar.

In plants, the glutamine synthetase - glutamic acid synthetase (GS/GOGAT) cycle plays important roles in nitrogen metabolism, growth, development, and stress resistance. Excess ammonium (NH4+) restricts plant growth, but GS can help to alleviate NH4+ toxicity. In this study, 84K poplar (Populus alba × P. glandulosa) showed reduced biomass accumulation and leaf chlorosis under high-NH4+ stress. These symptoms were less severe in lines overexpressing the gene encoding glutamine synthetase (PagGS1;2-OE), and more severe in lines with inhibition of PagGS1;2 expression (PagGS1;2-RNAi). Compared with the wild type（WT）, the PagGS1;2-OE lines showed significantly increased GS and GOGAT activities and higher contents of free amino acids, soluble protein, total nitrogen, and chlorophyll under high-NH4+ stress. In contrast, the antioxidant capacity and NH4+ assimilation capacity of PagGS1;2-RNAi lines were decreased under high-NH4+ stress. The total carbon (C) content and C/N ratio (C/N) of roots and leaves of PagGS1;2-OE lines were significantly higher than those of WT under high-NH4+ stress. Overexpression of PagGS1;2 led to increased accumulation of various amino acids (3-methylaspartic acid, glutamic acid, proline, serine, and histidine); reduced contents of carbohydrates (fructose, starch, galactose, glucose 1-phosphate, fructose 6-phosphate); and increased contents of sugar alcohols (sedum heptanose, maltose, mannitol, galactose, sorbitol) in the roots under high-NH4+ stress. Under high-NH4+ stress, genes related to amino acid biosynthesis, sucrose and starch degradation, galactose metabolism, and the antioxidant system were significantly up-regulated in the roots of PagGS1;2-OE lines, compared with those of wild type. Thus, PagGS1;2 overexpression affected C metabolism and amino acid metabolism pathways under high-NH4+ stress, which helped to maintain the balance of C and N metabolism and alleviate the symptoms of NH4+ toxicity. Modification of the GS/GOGAT cycle by genetic engineering is a promising strategy to improve NH4+ tolerance of forest trees.

Network Analysis of Metabolome and Transcriptome Revealed Regulation of Different Nitrogen Concentrations on Hybrid Poplar Cambium Development.

Secondary development is a key biological characteristic of woody plants and the basis of wood formation. Exogenous nitrogen can affect the secondary growth of poplar, and some regulatory mechanisms have been found in the secondary xylem. However, the effect of nitrogen on cambium has not been reported. Herein, we investigated the effects of different nitrogen concentrations on cambium development using combined transcriptome and metabolome analysis. The results show that, compared with 1 mM NH4NO3 (M), the layers of hybrid poplar cambium cells decreased under the 0.15 mM NH4NO3 (L) and 0.3 mM NH4NO3 (LM) treatments. However, there was no difference in the layers of hybrid poplar cambium cells under the 3 mM NH4NO3 (HM) and 5 mM NH4NO3 (H) treatments. Totals of 2365, 824, 649 and 398 DEGs were identified in the M versus (vs.) L, M vs. LM, M vs. HM and M vs. H groups, respectively. Expression profile analysis of the DEGs showed that exogenous nitrogen affected the gene expression involved in plant hormone signal transduction, phenylpropanoid biosynthesis, the starch and sucrose metabolism pathway and the ubiquitin-mediated proteolysis pathway. In M vs. L, M vs. LM, M vs. HM and M vs. H, differential metabolites were enriched in flavonoids, lignans, coumarins and saccharides. The combined analysis of the transcriptome and metabolome showed that some genes and metabolites in plant hormone signal transduction, phenylpropanoid biosynthesis and starch and sucrose metabolism pathways may be involved in nitrogen regulation in cambium development, whose functions need to be verified. In this study, from the point of view that nitrogen influences cambium development to regulate wood formation, the network analysis of the transcriptome and metabolomics of cambium under different nitrogen supply levels was studied for the first time, revealing the potential regulatory and metabolic mechanisms involved in this process and providing new insights into the effects of nitrogen on wood development.

Structure and Expression Analysis of PtrSUS, PtrINV, PtrHXK, PtrPGM, and PtrUGP Gene Families in Populus trichocarpa Torr. and Gray.

Exogenous nitrogen and carbon can affect plant cell walls, which are composed of structural carbon. Sucrose synthase (SUS), invertase (INV), hexokinase (HXK), phosphoglucomutase (PGM), and UDP-glucose pyrophosphorylase (UGP) are the key enzymes of sucrose metabolism involved in cell wall synthesis. To understand whether these genes are regulated by carbon and nitrogen to participate in structural carbon biosynthesis, we performed genome-wide identification, analyzed their expression patterns under different carbon and nitrogen treatments, and conducted preliminary functional verification. Different concentrations of nitrogen and carbon were applied to poplar (Populus trichocarpa Torr. and Gray), which caused changes in cellulose, lignin, and hemicellulose contents. In poplar, 6 SUSs, 20 INVs, 6 HXKs, 4 PGMs, and 2 UGPs were identified. Moreover, the physicochemical properties, collinearity, and tissue specificity were analyzed. The correlation analysis showed that the expression levels of PtrSUS3/5, PtrNINV1/2/3/5/12, PtrCWINV3, PtrVINV2, PtrHXK5/6, PtrPGM1/2, and PtrUGP1 were positively correlated with the cellulose content. Meanwhile, the knockout of PtrNINV12 significantly reduced the cellulose content. This study could lay the foundation for revealing the functions of SUSs, INVs, HXKs, PGMs, and UGPs, which affected structural carbon synthesis regulated by nitrogen and carbon, proving that PtrNINV12 is involved in cell wall synthesis.

Genome-Wide Identification and Characterization of WRKY Transcription Factors in Betula platyphylla Suk. and Their Responses to Abiotic Stresses.

The WRKY transcription factor (TF) family is one the largest plant-specific transcription factor families. It has been proven to play significant roles in multiple plant biological processes, especially stress response. Although many WRKY TFs have been identified in various plant species, WRKYs in white birch (Betula platyphylla Suk.) remain to be studied. Here, we identified a total of 68 BpWRKYs, which could be classified into four main groups. The basic physiochemical properties of these TFs were analyzed using bioinformatics tools, including molecular weight, isoelectric point, chromosome location, and predicted subcellular localization. Most BpWRKYs were predicted to be located in the nucleus. Synteny analysis found 17 syntenic gene pairs among BpWRKYs and 52 syntenic gene pairs between BpWRKYs and AtWRKYs. The cis-acting elements in the promoters of BpWRKYs could be enriched in multiple plant biological processes, including stress response, hormone response, growth and development, and binding sites. Tissue-specific expression analysis using qRT-PCR showed that most BpWRKYs exhibited highest expression levels in the root. After ABA, salt (NaCl), or cold treatment, different BpWRKYs showed different expression patterns at different treatment times. Furthermore, the results of the Y2H assay proved the interaction between BpWRKY17 and a cold-responsive TF, BpCBF7. By transient expression assay, BpWRKY17 and BpWRKY67 were localized in the nucleus, consistent with the previous prediction. Our study hopes to shed light for research on WRKY TFs and plant stress response.

Overexpression of 2-Cys Peroxiredoxin alleviates the NaHCO3 stress-induced photoinhibition and reactive oxygen species damage of tobacco.

Plant 2-cysteine peroxiredoxin (2-Cys Prx) is a mercaptan peroxidase localized in chloroplasts and has unique catalytic properties. To explore the salt stress tolerance mechanisms of 2-Cys Prx in plants, we analyzed the effects of overexpressing the 2-CysPrx gene on the physiological and biochemical metabolic processes of tobacco under NaHCO3 stress through joint physiological and transcriptomic analysis. These parameters included growth phenotype, chlorophyll, photosynthesis, and antioxidant system. After NaHCO3 stress treatment, a total of 5360 differentially expressed genes (DEGs) were identified in 2-Cysprx overexpressed (OE) plants, and the number of DEGs was significantly lower than 14558 in wild-type (WT) plants. KEGG enrichment analysis showed that DEGs were mainly enriched in photosynthetic pathways, photosynthetic antenna proteins, and porphyrin and chlorophyll metabolism. Overexpressing 2-CysPrx significantly reduced the growth inhibition of tobacco induced by NaHCO3 stress, alleviating the down-regulation of the DEGs related to chlorophyll synthesis, photosynthetic electron transport and the Calvin cycle and the up-regulation of those related to chlorophyll degradation. In addition, it also interacted with other redox systems such as thioredoxins (Trxs) and the NADPH-dependent Trx reductase C (NTRC), and mediated the positive regulation of the activities of antioxidant enzymes such as peroxidase (POD) and catalase (CAT) and the expression of related genes, thereby reducing the accumulation of superoxide anion (O2·-), hydrogen peroxide (H2O2) and malondialdehyde (MDA). In conclusion, 2-CysPrx overexpression could alleviate the NaHCO3 stress-induced photoinhibition and oxidative damage by regulating chlorophyll metabolism, promoting photosynthesis and participating in the regulation of antioxidant enzymes, and thus improve the ability of plants to resist salt stress damage.

LC-MS/MS quantification of ropivacaine and local analgesic and adverse effects of Long-acting Ropivacaine Injection based on pharmacokinetic-pharmacodynamic modelling in Bama minipigs.

The local analgesic efficacy and adverse effects of a new Long-acting Ropivacaine formulation were examined based on pharmacokinetic-pharmacodynamic (PK-PD) modelling in Bama minipigs. 24 Bama minipigs, 12 males and 12 females, were randomly and equally divided into the following treatment groups: normal saline injection, drug vehicle injection, Long-acting Ropivacaine Injection and Ropivacaine Hydrochloride Injection. After routine disinfection, a skin incision about 3 cm long and 3 cm deep was produced in the leg of each pig, and mechanical withdrawal threshold (MWT) measured at various times pre- and post-injection as an index of analgesia against incision pain. Plasma ropivacaine concentrations were also measured at the same times using a novel liquid chromatography-tandem mass spectroscopy (LC-MS/MS) method. Minipigs were sacrificed 24 h post-injection and hearts collected for drug concentration measurements by LC-MS/MS. The LC-MS/MS method demonstrated high sensitivity, linearity and precision. The Long-acting Ropivacaine formulation produced a longer analgesic effect (∼12 h) at a lower plasma concentration than Ropivacaine Hydrochloride (∼4h), suggesting a better side-effects profile. A PK-PD model revealed a direct relationship between plasma ropivacaine concentration and MWT, with peak analgesia at about 1000 ng/mL and behaved good prediction ability. Long-acting Ropivacaine Injection is a superior local anaesthetic-analgesic treatment due to longer-lasting efficacy at lower concentrations compared to Ropivacaine Hydrochloride, which will reduce the risk of side effects such as cardiotoxicity.

Genome-wide identification of glutamate synthase gene family and expression patterns analysis in response to carbon and nitrogen treatment in Populus.

Glutamate synthase (GOGAT) is a key enzyme in glutamine synthetase (GS)/GOGAT cycle and at the hub of carbon and nitrogen metabolism, catalyzing the formation of glutamate from α-oxoglutarate and glutamine. In this study, members of GOGAT family in Populus trichocarpa were identified and analyzed by bioinformatics. The four PtGOGATs were divided into two subgroups: subgroup A (Fd-GOGAT1 and Fd-GOGAT2) and subgroup B (NADH-GOGAT1 and NADH-GOGAT2). Many important elements have been identified in the promoters of different PtGOGATs, including hormone- and light-responsive elements. Meanwhile, the transcript levels of PxGOGATs were affected by light and diurnal cycle. Quantitative real-time PCR showed PxFd-GOGATs and PxNADH-GOGATs were mainly expressed in leaves and roots in Populus × xiaohei T. S. Hwang et Liang, respectively. Under elevated CO2, PxGOGATs were suppressed in all tissues except the stem. And PxFd-GOGATs and PxNADH-GOGATs were strongly induced by nitrogen in leaves and roots, respectively. In addition, PxGOGATs were stimulated significantly in roots in response to NH4+and glutamine directly. Our results provide new insights about GOGATs in poplar and their expression patterns under exogenous substances, to lay molecular basis for studying gene function and provide a reference for exploring putative roles of GOGATs in carbon-nitrogen balance.

Effects of intracoronary low-dose prourokinase administration on ST-segment elevation in patients with myocardial infarction and a high thrombus burden: a randomized controlled trial.

OBJECTIVE: To evaluate the efficacy and safety of low-dose prourokinase (pro-UK) administration during primary percutaneous coronary intervention (PCI) for the treatment of acute ST-segment elevation myocardial infarction (STEMI) in patients with a high thrombus burden. METHODS: A prospective, randomized controlled trial was conducted at the Inner Mongolia People's Hospital, China. Patients with STEMI and a high thrombus burden who underwent thrombus aspiration and primary PCI were randomly allocated to pro-UK administration or control groups. The primary endpoint was corrected thrombolysis in myocardial infarction (TIMI) frame count (CTFC). RESULTS: There were no significant differences in the baseline demographics or clinical characteristics of the two groups. The CTFC, tissue myocardial perfusion grade, ST-segment resolution, and myocardial blush grade of the pro-UK group were significantly better than those of the control group. In addition, after 30 days of follow-up, the pro-UK group had better cardiac function and perfusion than the control group. There were no differences in the clinical outcomes or incidence of hemorrhage. CONCLUSIONS: Intracoronary low-dose pro-UK improves myocardial perfusion and cardiac function in patients with a high thrombus burden. Major hemorrhages still occur in patients administered pro-UK, but are no more frequent.Study registration: Chinese Clinical Trial Registry (ChiCTR1900022290).

Pharmacokinetic and gut microbiota analyses revealed the effect of Lactobacillus acidophilus on the metabolism of Olsalazine in ulcerative colitis rats.

Olsalazine is a typical 5-aminosalicylic acid (5-ASA) drug that depends on gut microbiota to liberate its anti-inflammatory moiety 5-ASA in the treatment of ulcerative colitis (UC). In recent decades, 5-ASA drugs combined with probiotics have achieved a better effective treatment for UC. Mechanisms of combination therapy have been widely discussed from a pharmacodynamic perspective. However, it is still unclear whether the better therapeutic efficacy of combination therapy was made by changing the metabolism of 5-ASA drugs in the colon under the regulation of probiotics. In the present study, combined with pharmacokinetic and gut microbiota analyses, we systematically evaluated the potential effect of Lactobacillus acidophilus (L. acidophilus) on the metabolism of Olsalazine at three levels (pharmacokinetic characteristics, metabolic microbiota, and metabolic enzymes) to offer some insights into this issue. As pharmacokinetic results showed, L. acidophilus barely had an influence on the pharmacokinetic parameters of Olsalazine, 5-ASA, and N-Ac-5-ASA. Notably, the colonic exposure of 5-ASA was not affected by L. acidophilus. Gut microbiota results also illustrated that L. acidophilus did not change the total abundance of azoreductase (azoR) and N-acetyltransferase (NAT) associated gut microbiota and enzymes, which are involved in the metabolism of Olsalazine. Both pharmacokinetic and gut microbiota results revealed that L. acidophilus did not increase the colonic exposure of 5-ASA to improve the efficacy of combination therapy. L. acidophilus played its role in UC treatment by regulating gut microbiota composition and amino acid, phenolic acid, oligosaccharide, and peptidoglycan metabolic pathways. There was no potential medication risk of combination therapy of Olsalazine and L. acidophilus. In summary, this research provided strong evidence of medication safety and a comprehensive understanding of therapeutic advantages for combination therapy of probiotics and 5-ASA drugs from the pharmacokinetic and gut microbiota perspectives.

Simultaneous determination of vitamin D metabolites 25(OH)D3 and 1α,25(OH)2D3 in human plasma using liquid chromatography tandem mass spectrometry.

Background: Although measurement of 25(OH)D3 is a routine analytical method to determine plasma vitamin D status, 1α,25(OH)2D3 is the biologically active form. Hence, simultaneous measurement of 25(OH)D3 and 1α,25(OH)2D3 could provide better insight into vitamin D status and pharmacokinetics. However, 1α,25(OH)2D3 has a low plasma concentration, making its quantification challenging for most analytical techniques. Here, we demonstrate use of liquid chromatography tandem mass spectrometry (LC-MSMS) for the development of a simple and rapid method for the simultaneous quantification of 25(OH)D3 and 1α,25(OH)2D3. Methods: Samples were purified from 250 µL human plasma. Chromatography was performed on an analytical column, under gradient conditions using a mobile phase consisting of methanol-lithium acetate. The mass detector was operated in positive multiple reaction monitoring mode. The established method was validated according to the guidance issued by ICH and FDA. Furthermore, a clinical study was performed using this method to detect the plasma concentrations of 1α,25(OH)2D3 after oral administration of calcitriol. Results and conclusion: The method was acceptably linear over the concentration ranges of 20-1200 pg/mL for 1α,25(OH)2D3 and 1-60 ng/mL for 25(OH)D3, respectively, with correlation coefficients of r2 > 0.993. Both the inter-assay and intra-assay precision was < 15%, and the analytical recoveries were within 100% ± 10%, with no significant matrix effect or carryover. Thereby, we, provide a facile method for the simultaneous detection of vitamin D metabolites in plasma.

Identification and expression analysis of the PtGATL genes under different nitrogen and carbon dioxide treatments in Populus trichocarpa.

Pectin is one of the most important components of the plant cell wall. Galacturonosyltransferase-like (GATL) is an important enzyme involved in forming pectin in Arabidopsis thaliana. In this study, 12 PtGATL genes were identified and characterized based on the Populus trichocarpa genome using bioinformatics methods. The results showed that the PtGATLs contained four typical motifs, including DXD, LPPF, GLG, and HXXGXXKPW. According to phylogenetic analysis, PtGATLs were divided into six groups. Chromosome distribution and genome synteny analysis showed that there were 11 segmental-duplicated gene pairs with repeated fragments on chromosomes 2, 5, 7, 8, 10, and 14. Tissue-specific expression profiles indicated that these PtGATLs had different expression patterns. The transcription level of PtGATLs was regulated by different carbon dioxide and nitrogen concentrations. In conclusion, the identification and analysis of PtGATL genes in poplar provide important information on the gene function. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03129-y.

New Ultrasound-Controlled Paclitaxel Releasing Balloon vs. Asymmetric Drug-Eluting Stent in Primary ST-Segment Elevation Myocardial Infarction　- A Prospective Randomized Trial.

BACKGROUND: Application of drug-coated balloons (DCBs) is popular for the treatment of percutaneous coronary intervention (PCI). A new DCB has been designed as ultrasound-controlled paclitaxel releasing. This study was conducted to determine whether a DCB-only strategy has a similar safety profile and equal angiographic and clinical outcomes to DES implantation in primary ST-elevation myocardial infarction (STEMI) patients, as well as determine the efficiency and safety of this new DCB.Methods and Results:Overall, 184 pretreated STEMI patients were randomized into DCB and DES groups with a 1:1 allocation. The main study end-point was late lumen loss (LLL) during the 9 months after PCI. Late lumen loss was reported to be 0.24±0.39 mm in the DCB group and 0.31±0.38 mm in the DES group (P=0.215). Diameter stenosis was 28.27±15.35% in the DCB group and 25.73±15.41% in the DES group (P=0.312). Major adverse cardiovascular events (MACEs) were reported in 3 patients (3.4%) in the DCB group and 4 patients (4.7%) in the DES group (P=0.718). TLR and TVR in the DCB group was 2.3%, 3.4% and 2.4%, 3.5% in the DES group (P=1.000), respectively. No cardiac death and stent thrombosis (ST) was found in the DCB group at 12 months clinical follow up. CONCLUSIONS: The DCB-only strategy showed good angiographic and clinical outcomes in the 9- and 12-month follow-up periods, respectively. The VasoguardTM DCB is safe and feasible to treat STEMI patients.

Genome Identification and Expression Profiles in Response to Nitrogen Treatment Analysis of the Class I CCoAOMT Gene Family in Populus.

Lignin is essential for the characteristics and quality of timber. Nitrogen has significant effects on lignin contents in plants. Nitrogen has been found to affect wood quality in plantations and lignin content in plants. Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is an important methyltransferase in lignin biosynthesis. However, the classification of woody plant CCoAOMT gene family members and the regulation mechanism of nitrogen are not clear. Bioinformatics methods were used to predict the members, classification, and transcriptional distribution of the CCoAOMT gene family in Populus trichocarpa. The results showed that there were five PtCCoAOMTs identified, and they could be divided into three sub-groups according to their structural and phylogenetic features. The results of tissue expression specificity analysis showed that: PtCCoAOMT1 was highly expressed in roots and internodes; PtCCoAOMT2 was highly expressed in roots, nodes, and internodes, PtCCoAOMT3 was highly expressed in stems; PtCCoAOMT4 was highly expressed in young leaves, and, PtCCoAOMT5 was highly expressed in roots. Different forms and concentrations of nitrogen had varying effects on the expression patterns of genes in different plant tissue types. The results of real-time PCR showed that the expression levels of PtCCoAOMT1 and PtCCoAOMT2 in stems increased significantly under different forms of nitrogen. PtCCoAOMT3 and PtCCoAOMT4 were induced by nitrate nitrogen in upper stems and lower leaves, respectively. PtCCoAOMT4 and PtCCoAOMT5 were induced by different concentrations of nitrate nitrogen in lower stems and roots, respectively. These results could provide valuable information for revealing the differences between functions and expression patterns of the various CCoAOMT gene family members under different forms and concentrations of exogenous nitrogen in poplar.

Determination of Fentanyl, Alpha-Methylfentanyl, Beta-Hydroxyfentanyl and the Metabolite Norfentanyl in Rat Urine by LC-MS-MS.

Fentanyl and its analogs are potent synthetic opioids with a high potential for abuse and dependence. They have become major contributors to opioid deaths. This study aimed to determine whether the metabolites of fentanyl, alpha-methylfentanyl and beta-hydroxyfentanyl, excreted in the urine, can demonstrate historical drug exposure. Fentanyl is primarily metabolized via CYP3A4 into norfentanyl, although there is little research on its metabolization into alpha-methylfentanyl and beta-hydroxyfentanyl. We conducted in vitro experiments with human liver microsomes (HLMs) and rat liver microsomes (RLMs) to elucidate the major metabolic pathways of alpha-methylfentanyl and beta-hydroxyfentanyl using ultra-high-performance liquid chromatography coupled with mass spectrometry. The results showed that both alpha-methylfentanyl and beta-hydroxyfentanyl were predominantly metabolized into norfentanyl in HLM and RLM. Urine samples were collected at different intervals from 0 h to 72 h after intravenous administration of alpha-methylfentanyl and beta-hydroxyfentanyl (20 µg/kg) to Sprague-Dawley rats. We prepared the samples by liquid-liquid extraction, and the internal standard (IS) was cariprazine. A sensitive, rapid liquid chromatography-tandem mass spectrometry method was developed and validated to determine four analytes in the urine. The lower limit of qualification in urine was 2 pg/mL for fentanyl, 5 pg/mL for alpha-methylfentanyl, 10 pg/mL for beta-hydroxyfentanyl and 40 pg/mL for norfentanyl. The analytical range was 0.002-2 ng/mL for fentanyl, 0.005-5 ng/mL for alpha-methylfentanyl, 0.01-10 ng/mL for beta-hydroxyfentanyl and 0.04-40 ng/mL for norfentanyl. All analytes demonstrated good linearity (R2 > 0.99). The extraction recoveries were in the 67.8%-92.1% range, and the IS-normalized matrix effects were between 55.5% and 74.0% (coefficient of variance < 15%). Our data indicated that norfentanyl has a higher concentration in rat urine and was detectable for at least 3 days after exposure to these compounds. This developed method may be useful in various fields, including forensic analysis, workplace drug testing and monitoring drug abuse.

Genome-wide identification of FRK genes in Populus trichocarpa and their expression under different nitrogen treatments.

Fructokinase (FRK) is the main fructose phosphorylase and plays an important role in catalyzing the irreversible reaction of free fructose phosphorylation. In order to study the regulatory effect of different forms and concentrations of nitrogen on PtFRK genes in Populus trichocarpa, seven genes encoding the hypothetical FRK proteins were identified in Populus trichocarpa genome by bioinformatics method. Phylogenetic analysis revealed that PtFRK family genes can be divided into two subgroups: SI (PtFRK 1, 3, 4, 6) and SII (PtFRK 2, 5, 7). The tissue-specific expression data obtained from PopGenIE indicate that PtFRK2, 3, 4 and 5 are expressed highly in the stem. Quantitative real-time RT-PCR illustrate that PtFRK1-7 showed different expression patterns in different tissues under different concentrations and morphological nitrogen application. Under high nitrate treatment, the expression levels of PtFRK1, 2, 3 and 6 in stem increased significantly, while under low nitrate treatment, only the expression of PtFRK1, 4 in the upper stem and the expression of PtFRK3, 5 in the lower stem increased significantly. In contrast, ammonium tends to inhibit the expression of PtFRKs in lower stems, the expression levels of PtFRK2, 3, 4 and 5 are significantly reduced under ammonium treatment. However, high ammonium had significant effects on PtFRK6 in the apical bud and upper leaves, which were 6 and 8 times of the control, respectively. These results laid the foundation for the study of the PtFRK gene family of poplar and provided a theoretical basis for the molecular mechanism of nitrogen regulating cell wall development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01055-6.

Rapid quantitation of three synthetic cathinones in urine by magnetic dispersive solid-phase extraction combined with DART-HRMS.

For the rapid quantitation of three synthetic cathinones, namely 1-(4-chlorophenyl)-2-(1-pyrrolidinyl)pentan-1-one (4-Cl-α-PVP), 1-(4-methylphenyl)-2-(methylamino)pentan-1-one (4-MPD), and 1-(5,6,7,8-tetrahydronaphthalen-2-yl)-2-(1-pyrrolidinyl)pentan-1-one (ß-TH-naphyrone), in urine, a new method was established using magnetic dispersive solid-phase extraction (MDSPE) combined with direct analysis in real time and high-resolution mass spectrometry (DART-HRMS). Methcathinone-D3 and proadifen (SKF525A) were used as the internal standards. Hydrophobic magnetic adsorbents were used and consisted of hydrophobic functional group (divinylbenzene) and hydrophilic functional group (vinylpyrrolidone) at a ratio of 3 : 1, and NaH2PO4//NaOH buffer (0.2 M, pH 7) was used in MDSPE. Detection was conducted by DART-HRMS in less than 1 min. For 4-Cl-α-PVP, 4-MPD and ß-TH-Naphyrone, the limits of detection were 0.1 ng mL-1, 0.05 ng mL-1 and 0.1 ng mL-1, and the linear ranges were 0.5-100 ng mL-1, 0.2-100 ng mL-1 and 0.2-100 ng mL-1, respectively. The correlation coefficients were all greater than 0.99. The precision and deviation of accuracy were all within ±15%, and the stability of the samples was high under various conditions. The method was successfully applied to detect 4-Cl-α-PVP, 4-MPD and ß-TH-naphyrone in rat urine after subcutaneous administration. In summary, a fast and convenient detection method was established, providing new and effective technical support for the rapid quantitation of three synthetic cathinones (4-Cl-α-PVP, 4-MPD and ß-TH-Naphyrone) for forensic purposes.

Bioinformatics analysis of PAE family in Populus trichocarpa and responsiveness to carbon and nitrogen treatment.

Plant Pectin acetylesterase (PAE) belongs to family CE13 of carbohydrate esterases in the CAZy database. The ability of PAE to regulate the degree of acetylation of pectin, an important polysaccharide in the cell wall, affects the structure of plant cell wall. In this study, ten PtPAE genes were identified and characterized in Populus trichocarpa genome using bioinformatics methods, and the physiochemical properties such as molecular weight, isoelectric points, and hydrophilicity, as well as the secondary and tertiary structure of the protein were predicted. According to phylogenetic analysis, ten PtPAEs can be divided into three evolutionary clades, each of which had similar gene structure and motifs. Tissue-specific expression profiles indicated that the PtPAEs had different expression patterns. Real-time quantitative PCR (RT-qPCR) analysis showed that transcription level of PtPAEs was regulated by different CO2 and nitrogen concentrations. These results provide important information for the study of the phylogenetic relationship and function of PtPAEs in Populus trichocarpa. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02918-1.

Genome-wide analysis of UGDH genes in Populus trichocarpa and responsiveness to nitrogen treatment.

Plant UDP-glucose 6-dehydrogenase (UGDH) is an important enzyme for the formation of hemicellulose and pectin. Previous studies on UGDH have primarily focused on the biosynthesis of cell wall polysaccharides, while few studies have focused on their regulation by exogenous nitrogen. In the present study, four genes encoding PtUGDH proteins were analyzed by bioinformatics methods. And, the expression profiles of PtUGDH genes under different nitrogen treatments were evaluated with qRT-PCR. The results showed that PtUGDHs have conserved NAD coenzyme binding motif GAGYVGG and the catalytic motif GFGGSCFQKDIL. According to the phylogenetic analysis, PtUGDHs were divided into two subgroups. PtUGDH3 and PtUGDH4 were closely related to AtUGDH1 (important for normal development of Arabidopsis cell wall structure). Chromosomal distribution and genome synteny analysis revealed four segmental-duplicated gene pairs on chr4, 8, 10 and 17. Tissue-specific data from PlantGenIE showed that PtUGDH3 and PtUGDH4 were highly expressed in stems. The qRT-PCR detection showed that the expression of PtUGDH3 in the lower stem and PtUGDH2 of upper leaves were significantly increased induced by low ammonium or nitrate condition. This comprehensive analysis of the UGDH family in poplar provides new insights into their regulation by nitrogen, and would increase our understanding of the roles of UGDHs in hemicellulose and pectin biosynthesis in the cell wall and during poplar development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02697-9.

Identification and Characterization of the APX Gene Family and Its Expression Pattern under Phytohormone Treatment and Abiotic Stress in Populus trichocarpa.

Ascorbate peroxidase (APX) is a member of class I of the heme-containing peroxidase family. The enzyme plays important roles in scavenging reactive oxygen species for protection against oxidative damage and maintaining normal plant growth and development, as well as in biotic stress responses. In this study, we identified 11 APX genes in the Populus trichocarpa genome using bioinformatic methods. Phylogenetic analysis revealed that the PtrAPX proteins were classifiable into three clades and the members of each clade shared similar gene structures and motifs. The PtrAPX genes were distributed on six chromosomes and four segmental-duplicated gene pairs were identified. Promoter cis-elements analysis showed that the majority of PtrAPX genes contained a variety of phytohormone- and abiotic stress-related cis-elements. Tissue-specific expression profiles indicated that the PtrAPX genes primarily function in roots and leaves. Real-time quantitative PCR (RT-qPCR) analysis indicated that PtrAPX transcription was induced in response to drought, salinity, high ammonium concentration, and exogenous abscisic acid treatment. These results provide important information on the phylogenetic relationships and functions of the APX gene family in P. trichocarpa.

Acute exposure to N-Ethylpentylone induces developmental toxicity and dopaminergic receptor-regulated aberrances in zebrafish larvae.

N-Ethylpentylone (NEP) is one of the most recent novel stimulants, and there is limited understanding of its toxicity. Here we employed zebrafish model for analyzing the effects of NEP on early embryos and cardiovascular and nervous systems at late developmental stages. We first observed multi-malformations in early embryos and larvae after NEP administration, together with significant deregulations of brain and heart development-associated genes (neurog1, her6, elavl3, nkx2.5, nppa, nppb, tnnt2a) at transcriptional level. Low-dosed NEP treatment induced an anxiety-like phenotype in zebrafish larvae, while higher doses of NEP exerted an inhibitory effect on locomotion and heart rate. Besides, the expression of th (tyrosine hydroxylase) and th2 (tyrosine hydroxylase 2), identifying dopamine (DA) release, were significantly increased during one-hour free swimming after effective low-dosed NEP administration, along with the upregulation of gene fosab and fosb related to stress and anxiety response. D1R antagonist SCH23390 and D2R antagonist sulpiride partially alleviated the aberrances of locomotion and heart rate, indicating dopaminergic receptors were involved in the bidirectional dosage-dependent pattern of NEP-induced performance. Meanwhile, sulpiride offset the upregulated expression of th, th2 and fosab in the group of 1.5 µM NEP, which highlighted the significant role of D2R in NEP-induced locomotive effects. This study systematically described the developmental, neuronal and cardiac toxicity of NEP in zebrafish, and identified the dopaminergic receptors as one of the downstream effectors of NEP administration.