Elucidation of CKAP4-remodeled cell mechanics in driving metastasis of bladder cancer through aptamer-based target discovery.

Metastasis contributes to the dismal prognosis of bladder cancer (BLCA). The mechanical status of the cell membrane is expected to mirror the ability of cell migration to promote cancer metastasis. However, the mechanical characteristics and underlying molecular profile associated with BLCA metastasis remain obscure. To study the unique cellular architecture and traits associated with cell migration, using a process called cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX) we generated an aptamer-based molecular probe, termed spl3c, which identified cytoskeleton-associated protein 4 (CKAP4). CKAP4 was associated with tumor metastasis in BLCA, but we also found it to be a mechanical regulator of BLCA cells through the maintenance of a central-to-peripheral gradient of stiffness on the cell membrane. Notably, such mechanical traits were transportable through exosome-mediated intercellular CKAP4 trafficking, leading to significant enhancement of migration in recipient cells and, consequently, aggravating metastatic potential in vivo. Taken together, our study shows the robustness of this aptamer-based molecular tool for biomarker discovery, revealing the dominance of a CKAP4-induced central-to-peripheral gradient of membrane stiffness that benefits cell migration and delineating the role of exosomes in mediating mechanical signaling in BLCA metastasis.

Sulfonated Polyimide Membranes Constructed by Main-Chain and Molecular-Network Engineering Strategy for Direct Methanol Fuel Cell.

Excessive swelling is one important factor that leads to high fuel permeability and limited operating concentration of methanol for proton exchange membranes. Herein, a collaborative strategy of main-chain and molecular-network engineering is applied to lower swelling ratio and improve methanol resistance for highly sulfonated polyimide. Two m-phenylenediamine monomers (4-(2,3,5,6-tetrafluoro-4-vinylphenoxy)benzene-1,3-diamine and 4,6-bis(2,3,5,6-tetrafluoro-4-vinylphenoxy)benzene-1,3-diamine) with tetrafluorostyrol groups are designed and synthesized. Two series of cross-linked sulfonated polyimides (CSPI-Ts, CSPI-Bs) are prepared from the two diamines, 4,4'-diaminostilbene-2,2'-disulfonic acid and 1,4,5,8-naphthalenetetracarboxylicdianhydride. The rigid main-chain structure is cornerstone for wet CSPI-Ts and CSPI-Bs remaining stable at elevated temperatures. The introduction of hydrophobic cross-linked network further improves their dimensional stability and methanol resistance. CSPI-Ts and CSPI-Bs show obviously improved performances containing high proton conductivity (121 ± 0.27-158 ± 0.35 S cm-1 ), low swelling ratio (9.6 ± 0.40%-16.1 ± 0.01%) and methanol permeability (4.14-7.69 × 10-7 cm2 s-1 ) at 80 °C. The direct methanol fuel cell (DMFC) is assembled from CSPI-T-10 with balanced properties, and it exhibits high maximum power density (PDmax ) of 82.3 and 72.6 mW cm-2 in 2 and 10 m methanol solution, respectively. The ratio of PDmax in 10 m methanol solution to the value in 2 m methanol solution is as high as 88%. The CSPI-T-10 is promising proton exchange membrane candidate for DMFC application.

Deciphering the prognostic landscape of osteosarcoma: Integrating the roles of hippo pathway genes, programmed cell death, and the tumor immune microenvironment.

Osteosarcoma is a highly aggressive cancer prevalent among adolescents and young adults, notorious for its tendency to metastasize to the lungs. This research delves into the molecular foundations of osteosarcoma by examining the role of the Hippo signaling pathway and its interaction with the tumor immune microenvironment (TME). Through analysis of transcriptomic data from the TARGET-OS dataset and control samples from GTEx, we identified a set of 131 genes that link high expression profiles in osteosarcoma with the Hippo pathway. A focused examination through univariate Cox regression analysis revealed eight key genes (DLG5, WNT11, TGFB2, DLG4, WNT16, ID2, WNT10B, and WNT10A) with a significant correlation to patient outcomes. Hierarchical clustering of these genes delineated two distinct patient groups with significantly different survival rates, a finding supported by Kaplan-Meier survival analysis. Further investigation into immune cell infiltration and expression profiles of immunoregulatory factors uncovered a notable pattern of immune evasion in the group with poorer prognosis, marked by reduced effector immune cell activity and lower levels of immunostimulatory factors. Single-cell sequencing highlighted the cellular diversity within osteosarcoma samples and identified markers differentiating malignant from nonmalignant cells, correlating these markers with prognostic risk scores. Our results emphasize the critical prognostic value of Hippo pathway genes and the TME in osteosarcoma, shedding light on new avenues for therapeutic intervention and patient-specific treatment strategies.

Aptamer-Mediated Enrichment of Rare Circulating Fetal Nucleated Red Blood Cells for Noninvasive Prenatal Diagnosis.

Isolation of circulating fetal nucleated red blood cells (cfNRBCs) from maternal peripheral blood provides a superior strategy for noninvasive prenatal genetic diagnosis. Recent technical advances in single-cell isolation and genetic analyses have promoted the clinical application of circulating fetal cell-based noninvasive prenatal diagnosis. However, the lack of highly specific ligands for rare circulating fetal cell enrichment from massive maternal cells significantly impedes the clinical transformation progress. In this work, aptamers specific to NRBCs were developed through clinical sample-based cell-SELEX. Herein, the complex clinical system provides natural selection stringency through binding competition between target and background cells, and it empowers aptamers with high specificity. An aptamer-based strategy was also established to isolate cfNRBCs from maternal peripheral blood. Results show the remarkable selectivity and affinity of developed aptamers, enabling efficient enrichment of cfNRBCs from abundant maternal cells. Moreover, screening for fetal sex and trisomy syndrome achieved high accuracy through chromosome analysis of enriched cfNRBCs. To the best of our knowledge, this is the first report to develop aptamer ligands for cfNRBC enrichment, providing an efficient strategy to screen cfNRBC-specific ligands and demonstrating broad application potential for cfNRBC-based noninvasive prenatal diagnosis.

Generation of DNA Aptamers with Functional Activity in Mammalian Cells by Mimicking Retroviruses.

DNA aptamers are single-stranded DNA oligonucleotide sequences that bind to specific targets with high affinity. Currently, DNA aptamers can be produced only by in vitro synthesis. It is difficult for DNA aptamers to have a sustained impact on intracellular protein activity, which limits their clinical application. In this study, we developed a DNA aptamer expression system to generate DNA aptamers with functional activity in mammalian cells by mimicking retroviruses. Using this system, DNA aptamers targeting intracellular Ras (Ra1) and membrane-bound CD71 (XQ2) were successfully generated in cells. In particular, the expressed Ra1 not only specifically bound to the intracellular Ras protein but also inhibited the phosphorylation of downstream ERK1/2 and AKT. Furthermore, by inserting the DNA aptamer expression system for Ra1 into a lentivirus vector, the system can be delivered into cells and stably produce Ra1 over time, resulting in the inhibition of lung cancer cell proliferation. Therefore, our study provides a novel strategy for the intracellular generation of DNA aptamers with functional activity and opens a new avenue for the clinical application of intracellular DNA aptamers in disease treatment.

Exploration on anti-hypoxia properties of peptides: a review.

Peptides are important components of human nutrition and health, and considered as safe, nontoxic, and easily absorbed potential drugs. Anti-hypoxia peptides are a kind of peptides that can prevent hypoxia or hypoxia damage. In this paper, the sources, preparations, and molecular mechanisms of anti-hypoxia peptides were systemically reviewed. The combination of bioinformatics, chemical synthesis, enzymatic hydrolysis, and microbial fermentation are recommended for efficient productions of anti-hypoxic peptides. The mechanisms of anti-hypoxic peptides include interference with glycolytic process and HIF-1α pathway, mitochondrial apoptosis, and inflammatory response. In addition, bioinformatics analysis, including virtual screening and molecular docking, provides an alternative or auxiliary method for exploring the potential anti-hypoxic activities and mechanisms of peptides. The potential challenges and prospects of anti-hypoxic peptides are also discussed. This paper can provide references for researchers in this field and promote further research and clinical applications of anti-hypoxic peptides in the future.

Nucleic acid aptamer controls mycoplasma infection for inhibiting the malignancy of esophageal squamous cell carcinoma.

Esophageal cancer is one of the most frequent malignant tumors of the digestive tract, among which esophageal squamous cell carcinoma (ESCC) is the main pathological type worldwide. Previous studies have shown microbial infections in the upper digestive tract to be a potential risk factor in ESCC etiology. In this study, we identified that Mycoplasma hyorhinis infection promoted the malignancy of ESCC. In response, we generated a single-stranded DNA aptamer, ZY3A, against M. hyorhinis using the cell-SELEX strategy. The underlying recognition mechanism of ZY3A on M. hyorhinis involves its binding to M. hyorhinis-specific p37 protein. This tool allowed us to provide the first proof-of-concept evidence using a nucleic acid aptamer to control mycoplasma infection. More specifically, we found that ZY3A could neutralize M. hyorhinis infection on ESCC cells by blocking the interaction between p37 protein and its receptor TLR4 on the ESCC cell membrane. As a result, ZY3A inhibited the migration and invasion of M. hyorhinis-infected ESCC cells in vitro and metastasis in vivo. Taken together, these findings indicate that aptamer ZY3A is a potential candidate for development into a novel molecular tool for treatment of M. hyorhinis infection and a safe first-in-class M. hyorhinis-targeting antitumor agent.

Identification of the metabolites of neocnidilide in rat, monkey and human liver microsomes by liquid chromatography coupled to benchtop Orbitrap mass spectrometry.

Neocnidilide, a bioactive component isolated from Angelica sinensis (Oliv.) Diels, displayed anti-inflammatory activity. The present work was performed to investigate in vitro metabolism of neocnidilide using liver microsomes. Neocnidilide (10 µM) was incubated with NADPH-supplemented rat monkey and human liver microsomes. To identify the reactive metabolites, glutathione (GSH) was included in the incubations. Liquid chromatography coupled to an Orbitrap mass spectrometer was used to detect and identify the metabolites. The structures of the metabolites were characterized by accurate masses and fragmentation patterns. A total of six hydroxylation metabolites and nine GSH conjugates were tentatively identified characterized. The metabolic pathways included hydroxylation, dehydrogenation and GSH conjugation. M6 was the major metabolite in human liver microsomes. CYP1A2 (25%), 2B6 (31.6%), 2C9 (10.5%) and 3A4 (18%) were the predominant isoenzymes governing its formation. This study provides valuable information on the in vitro metabolism of neocnidilide, which is indispensable for the further safety assessment of this compound.

A high-resolution mass spectrometry-based methodology for characterization and identification of methylophiopogonanone B metabolites from cryopreserved hepatocytes and liver microsomes.

Methylophiopogonanone B (MOB), one of the homoisoflavonoids isolated from Ophiopogon japonicus, has been demonstrated to possess antioxidative and antitumor activities. The aim of this work was to investigate the metabolism of MOB using liver microsomes and hepatocytes. MOB was individually incubated with rat, monkey, and human hepatocytes to generate the metabolites. To investigate the bioactivation pathways, MOB was incubated with liver microsomes in the presence of glutathione (GSH). All the metabolites were detected and identified using LC with a quadrupole Orbitrap mass spectrometer. Under the current conditions, nine metabolites were identified in hepatocyte incubations. Of these metabolites, M7 derived from hydroxylation was identified as the most abundant metabolite in hepatocyte incubation. MOB was metabolized via demethylation, hydroxylation, and glucuronidation. In liver microsomes, five GSH conjugates were detected and identified. MOB was subjected to bioactivation through demethylation yielding M9, which further formed quinone-methide and ortho-quinone intermediates, followed by GSH conjugation. This work is the first to study the metabolism of MOB, which will help us understand its disposition and efficacy.

Multi-Omics Elucidates Difference in Accumulation of Bioactive Constituents in Licorice (Glycyrrhiza uralensis) under Drought Stress.

Licorice is a frequently applied herb with potential edible and medicinal value based on various flavonoids and triterpenes. However, studies on detailed flavonoid and triterpene metabolism and the molecular basis of their biosynthesis in licorice are very limited, especially under drought conditions. In the present study, we carried out transcriptome, proteome, and metabolome experiments. To ultimately combine three omics for analysis, we performed a bioinformatics comparison, integrating transcriptome data and proteome data through a Cloud platform, along with a simplified biosynthesis of primary flavonoids and triterpenoids in the KEGG pathway based on metabolomic results. The biosynthesis pathways of triterpenes and flavonoids are enriched at both gene and protein levels. Key flavonoid-related genes (PAL, 4CL, CHS, CHI, CYP93C, HIDH, HI4OMT, and CYP81E1_7) and representative proteins (HIDH, CYP81E1_7, CYP93C, and VR) were obtained, which all showed high levels after drought treatment. Notably, one R2R3-MYB transcription factor (Glyur000237s00014382.1), a critical regulator of flavonoid biosynthesis, achieved a significant upregulated expression as well. In the biosynthesis of glycyrrhizin, both gene and protein levels of bAS and CYP88D6 have been found with upregulated expression under drought conditions. Most of the differentially expressed genes (DEGs) and proteins (DEPs) showed similar expression patterns and positively related to metabolic profiles of flavonoid and saponin. We believe that suitable drought stress may contribute to the accumulation of bioactive constituents in licorice, and our research provides an insight into the genetic study and quality breeding in this plant.

The regulation of NONO by USP11 via deubiquitination is linked to the proliferation of melanoma cells.

Ubiquitin-specific protease 11 (USP11) has been implicated in the regulation of DNA repair, apoptosis, signal transduction and cell cycle. It belongs to a USP subfamily of deubiquitinases. Although previous research has shown that USP11 overexpression is frequently found in melanoma and is correlated with a poor prognosis, the potential molecular mechanism of USP11 in melanoma remains indefinitive. Here, we report that USP11 and NONO colocalize and interact with each other in the nucleus of melanoma cells. As a result, the knockdown of USP11 decreases NONO levels. Whereas, overexpression of USP11 increases NONO levels in a dose-dependent manner. Furthermore, we reveal that USP11 protects NONO protein from proteasome-mediated degradation by removing poly-ubiquitin chains conjugated onto NONO. Functionally, USP11 mediated melanoma cell proliferation via the regulation of NONO levels because ablation of USP11 inhibits the proliferation which could be rescued by ectopic expression of NONO protein. Moreover, a significant positive correlation between USP11 and NONO concentrations was found in clinical melanoma samples. Collectively, these results demonstrate that USP11 is a new deubiquitinase of NONO and that the signalling axis of USP11-NONO is significantly involved in melanoma proliferation.

Admission Hemoglobin Is Prognostic for In-Hospital Mortality in Oldest-Old Patients with Acute Ischemic Stroke.

INTRODUCTION: Anemia is a common condition encountered in acute ischemic stroke, and only a few pieces of evidence has been produced suggesting its possible association with short-term mortality have been produced. The study sought to assess whether admission anemia status had any impact on short-term clinical outcome among oldest-old patients with acute ischemic stroke. MATERIALS AND METHODS: A retrospective review of Electronic Medical Recording System was performed in 2 tertiary hospitals. Data, from the oldest-old patients aged > = 80 years consecutively admitted with a diagnosis of acute ischemic stroke between January 1, 2015, and December 31, 2019, were analyzed. Admission hemoglobin was used as indicator for anemia and severity. Univariate and multivariate regression analyses were used to compare in-hospital mortality and length of in-hospital stay in different anemia statuses and normal hemoglobin patients. RESULTS: A total of 705 acute ischemic stroke patients were admitted, and 572 were included in the final analysis. Of included patients, 240 of them were anemic and 332 nonanemic patients. A statistical difference between the 2 groups was found in in-hospital mortality (p < 0.001). After adjustment for baseline characteristics, the odds ratio value of anemia for mortality were 3.91 (95% confidence intervals (CI) 1.60-9.61, p = 0.003) and 7.15 (95% CI: 1.46-34.90, p = 0.015) in moderate and severely anemic patients, respectively. Similarly, length of in-hospital stay was longer in anemic patients (21.64 ± 6.17 days) than in nonanemic patients (19.08 ± 5.48 days, p < 0.001). CONCLUSIONS: Increased severity of anemia may be an independent risk factor for increased in-hospital mortality and longer length of stay in oldest-old patients with acute ischemic stroke.

GABA negatively regulates adventitious root development in poplar.

γ-Aminobutyric acid (GABA) influences plant growth, but little is known about how this metabolite regulates adventitious root (AR) development. Here, we investigate the effects of GABA on ARs using poplar lines overexpressing glutamate decarboxilase 2 (GAD2) and by treating poplar stem cuttings with exogenous GABA or vigabatrin (VGB; a specific GABA transaminase inhibitor). Endogenous GABA accumulation not only inhibited AR growth, but it also suppressed or delayed AR formation. Anatomical observations revealed that the GABA and VGB treatments resulted in a 1 d delay in the formation of AR primordia and the appearance of ARs. This delay coincided with changes in primary metabolism, including transient increases in hexose and amino acid levels. GABA-dependent changes in the expression of genes related to hormone synthesis and signalling, as well as analysis of hormone levels revealed that ethylene-dependent pathways were decreased at the earliest stage of AR formation. In contrast, auxin and abscisic acid were increased at 1-5 d as well as GA4 over a 5 d period of AR formation. These results demonstrate that GABA plays a crucial role in AR development. Evidence is presented demonstrating that GABA can interact with hormone-related pathways as well as carbon/nitrogen metabolism. These findings also elucidate the functions of GABA in plant development.

Admission serum cholinesterase concentration for prediction of in-hospital mortality in very elderly patients with acute ischemic stroke: a retrospective study.

BACKGROUND: Cholinesterase as a sensitive biomarker for prognosis in a variety of conditions but it is rare in stroke studies. The very elderly (≥ 80 years of age) represent the most susceptible group of ischemic stroke. We aimed to determine whether admission serum cholinesterase concentration had any effect on clinical outcome in very elderly patients (individuals aged ≥ 80 years) with acute ischemic stroke. METHODS: A retrospective record review was conducted in two tertiary university hospitals. Elderly patients aged ≥ 80 years admitted with a diagnosis of acute ischemic stroke from January 1, 2014 to November 30, 2019, who had a cholinesterase concentration drawn, were included. The patients were grouped based on the inflection points of the locally weighted regression and smoothing scatterplot (LOESS) curve between cholinesterase levels and in-hospital mortality (study outcome) with lower concentration as reference group. RESULTS: A total of 612 patients were admitted with a diagnosis of acute ischemic stroke, and 569 met the inclusion criteria. A threshold effect was identified using regression smoothing scatterplot (LOESS), with one cutoff point of 4.0 KU/L. There was a significant difference in-hospital mortality was observed (P < 0.001). After adjusted demographic and clinical features, the OR of cholinesterase for mortality was 0.43 (95% CI 0.34-0.54, P < 0.001), suggesting that lower admission cholinesterase level was an independent risk factors for all-cause mortality among patients with AIS. CONCLUSIONS: We have demonstrated a significant association between admission cholinesterase concentration and in-hospital mortality in very elderly patients with AIS.

Responses of GABA shunt coupled with carbon and nitrogen metabolism in poplar under NaCl and CdCl2 stresses.

The γ-aminobutyric acid (GABA) shunt is closely associated with plant tolerance; however, little is known about its mechanism. This study aimed to decipher the responses of the GABA shunt and related carbon-nitrogen metabolism in poplar seedlings (Populus alba × Populus glandulosa) treated with different NaCl and CdCl2 concentrations for 30 h. The results showed that the activities of glutamate decarboxylase (GAD) and GABA-transaminase (GABA-T) were activated, as well as α-ketoglutarate dehydrogenase (α-KGDH) and succinate dehydrogenase (SDH) activities were enhanced by NaCl and CdCl2 stresses, except for SDH under CdCl2 stress. Meanwhile, the expression levels of GADs, GABA-Ts SDHs, succinyl-CoA ligases (SCSs), and succinic acid aldehyde dehydrogenases (SSADHs) were also increased. Notably, significant increases in the key components of GABA shunt, Glu and GABA, were observed under both stresses. Soluble sugars and free amino acids were enhanced, whereas citrate, malate and succinate were almost inhibited by both NaCl and CdCl2 stresses except that citrate was not changed or just increased by 50-mM NaCl stress. Thus, these results suggested that the carbon-nitrogen balance could be altered by activating the GABA shunt when main TCA-cycle intermediates were inhibited under NaCl and CdCl2 stresses. This study can enhance the understanding about the functions of the GABA shunt in woody plants under abiotic stresses and may be applied to the genetic improvement of trees for phytoremediation.

Label- and modification-free-based in situ selection of bovine serum albumin specific aptamer.

Systematic evolution of ligands by exponential enrichment is a traditional approach to select aptamer, which has a great potential in biosensing field. However, chemical modifications of DNA library or targets before selection might block the real recognition and binding sites between aptamers and their targets. In this study, a label- and modification-free-based in situ selection strategy was developed to overcome this limitation. The strategy is an attempt to screen bovine serum albumin aptamers according to the principle of electrophoretic mobility shift assay, and allowed single-stranded DNA sequence to be fully exposed to interact with bovine serum albumin which was mixed with the agarose gel beforehand. After eight rounds of selection, specific aptamer with low dissociation constant (Kd ) value of 69.44 ± 7.60 nM was selected and used for subsequent establishment of fluorescence biosensor. After optimization, the optimal aptasensor exhibited a high sensitivity toward bovine serum albumin with a limit of detection of 0.24 ng/mL (linear range from 1 to 120 ng/mL). These results indicated that the label- and modification-free-based in situ selection strategy proposed in this work could effectively select specific aptamer to develop aptasensor for sensitive detection of bovine serum albumin or other targets in actual complicated samples.

Characterization of ginsenoside compound K metabolites in rat urine and feces by ultra-performance liquid chromatography with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Ginsenoside compound K (CK) is an active metabolite of ginsenoside and has been shown to have ameliorative property in various diseases. However, the detailed in vivo metabolism of this compound has rarely been reported. In the present study, a method using liquid chromatography quadrupole time-of-flight tandem mass spectrometry together with multiple data processing techniques, including extracted ion chromatogram, multiple mass defect filter and MS/MS scanning, was developed to detect and characterize the metabolites of CK in rat urine and feces. After oral administration of CK at a dose of 50 mg/kg, urine and feces were collected for a period of time and subjected to a series of pretreatment. A total of 12 metabolites were tentatively or conclusively identified, comprising 11 phase I metabolites and a phase II metabolite. Metabolic pathways of CK has been proposed, including oxidation, deglycosylation, deglycosylation with sequential oxidation and dehydrogenation and deglycosylation with sequential glucuronidation. Relative quantitative analyses suggested that deglycosylation was the main metabolic pathway. The result could offer insights for better understanding of the mechanism of its pharmacological activities.

Inhibition of α-ketoglutarate dehydrogenase activity affects adventitious root growth in poplar via changes in GABA shunt.

MAIN CONCLUSION: Blocking α-ketoglutarate dehydrogenase results in up-regulation of γ-aminobutyric acid (GABA) shunt activity, and inhibits the growth of poplar adventitious roots (ARs), indicating that AR growth is closely associated with GABA shunt. γ-Aminobutyric acid (GABA) shunt starts from α-ketoglutarate in the tricarboxylic acid cycle, which is thought to represent the cross road between carbon and nitrogen metabolism. Previous studies (Araújo et al. 2012b, Plant Cell 24: 2328-2351) have shown that blocking α-ketoglutarate dehydrogenase (α-KGDH) affects the GABA shunt activity, and inhibits growth. However, its effects on the growth of adventitious roots (ARs) are unclear. In this study, the growth of ARs in tissue-cultured 84K poplar (Populus alba × Populus glandulosa cv. '84K') was significantly inhibited when succinyl phosphate (SP), a specific inhibitor of α-KGDH, was supplied. The inhibition of ARs was associated with significant changes in the levels of soluble sugars, organic acids, and amino acids, and was coupled with the up-regulation of the GABA shunt activity at the transcriptional and translational levels. Exogenous GABA also inhibited AR growth following the increase of the endogenous GABA level. Transcriptomic analyses further showed that genes related to cell wall carbon metabolism and phytohormone (indoleacetic acid, ABA, and ethylene) signaling were affected by the changes of GABA shunt activity, resulting from the α-KGDH inhibition. Thus, our study indicates that the inhibition of poplar AR growth by blocking α-KGDH is closely associated with GABA shunt, which would benefit a better understanding of GABA's roles in plant development and stress response.

Roles of γ-aminobutyric acid on salinity-responsive genes at transcriptomic level in poplar: involving in abscisic acid and ethylene-signalling pathways.

MAIN CONCLUSION: γ-Aminobutyric acid (GABA) affected ABA and ethylene metabolic genes and signal components in salt-treated poplar, indicating its potential role in signal pathways of ABA and ethylene during salt stress. GABA is a small signalling molecule that accumulates rapidly in plants exposed to various stresses. However, the relationship between GABA and other signalling molecules, such as hormones, remains unclear. Here, in the poplar woody plant under 200-mM NaCl conditions, the application of low (0.25 mM) and high (10 mM) exogenous GABA, compared to 0 mM, affected the accumulation of hydrogen peroxide and hormones, including ABA and ethylene, in different manners. Transcriptomic analysis demonstrated that 1025 differentially expressed genes (DEGs; |log2Ratio| ≥ 1.5) were widely affected by exogenous GABA under salt stress. A clustering analysis revealed that GABA could rescue or promote the effects of salt stress on gene expression. Among them, 146 genes involved in six hormone-signalling pathways were enriched, including 22 ABA- and 50 ethylene-related genes. Quantitative expression of selected genes involved in hormone-related pathways showed that ABA metabolic genes (ABAG, ABAH2, and ABAH4), ethylene biosynthetic genes (ACO1, ACO2, ACO5, ACOH1, ACS1, and ACS7) and receptor genes (PYL1, PYL2, PYL4, and PYL6) were regulated by exogenous GABA, even at a 0.1 mM level. The production of ABA was negatively correlated with ABAH expression levels at different GABA concentrations. The increase of endogenous GABA, resulting from inhibitor (succinyl phosphonate) of α-ketoglutarate dehydrogenase, affected the PYLs levels. Thus, GABA may be involved in ABA- and ethylene-signalling pathways. Our data provide a better understanding of GABA's roles in the plant responses to environmental stresses.

Fabrication of a biochar-doped monolithic adsorbent and its application for the extraction and determination of coumarins from Angelicae Pubescentis Radix.

A monolithic adsorbent was designed aiming to the structure of osthole and columbianadin, and fabricated using diallyl phthalate as the monomer and ethylene dimethacrylate as the crosslinker with the addition of bamboo biochar, via polymerization reaction in a stainless-steel tube. The prepared composite adsorbent packed in the tube was used as a solid-phase extraction column for the extraction and determination of two coumarins (osthole and columbianadin) in Angelicae Pubescentis Radix, combing with a C18 analytical column through an HPLC instrument, which show excellent matrix-removal ability and good selectivity to osthole and columbianadin. Furthermore, the present adsorbent shows good applicability, which was used for the extraction of osthole from Duhuo Jisheng Pill. Compared to the commercial C18 and phenyl adsorbent, the present adsorbent own better selectivity and higher resolution. These results attributed to the enhanced specific surface area (141 m2/g) and enriched interaction sites of the resulting composite adsorbent, due to the doping of bamboo biochar, which can produce hydrogen bond, dipole-dipole, π-π and hydrophobic force interactions with the osthole and columbianadin. The methodology validation indicated that the present method showed good precision and good accuracy, and the composite adsorbent showed good preparative repeatability, which can be reused for no less than 100 times with the relative standard deviation ≤4.6 % (n = 100). The present work provided a simple and efficient method for the extraction and determination osthole and columbianadin from Angelicae Pubescentis Radix.