Direct Observation of Anionic Yields from the Liquid-Vapor Interface by Electron Irradiation.

Dissociative electron attachment (DEA) is widely believed to play a high-profile role in ionizing radiation damages of bioorganic molecules, and its fundamentals are mainly learned from the gas-phase studies. However, the DEA process in aqueous solution is still in debate. Here we provide experimental evidence about the DEA processes of liquid methanol by using electron-impact-time-delayed mass spectrometry. In contrast to the gas- and solid-phase DEAs, methoxide ion CH3O- is the predominant product from the liquid interface. Furthermore, this anion can be produced with both the primary low-energy electrons and the inelastically scattered and secondary low-energy electrons. On the contrary, the primary low-energy electrons in the liquid bulk are more likely to be solvated, rather than directly participating in the DEA process. Our study provides new insights into radiation chemistry, particularly of bioorganic relevance.

Associations between Parental Educational Attainment, Children's 24-h Behaviors and Children's Hyperactivity Behavior in the COVID-19 Pandemic.

BACKGROUND: Parental Educational Attainment and children's 24-h behaviors significantly influenced children's hyperactivity symptoms. This study aimed to examine the mediating role of children's 24-h behavior changes due to the COVID-19 pandemic between Parental Educational Attainment and children's hyperactivity index. It also aimed to investigate the associations between Children's Physical Activity, digital media use, sleep, and hyperactivity index between two clusters of Parental Educational Attainments. The goal was to provide targeted behavioral optimization recommendations for caregivers to reduce the risk of children's hyperactivity. METHODS: The study was a collaborative extension of the International iPreschooler Surveillance Study Among Asians and otheRs project and the Chinese Children and Adolescent Sports Health Promotion Action Project. The Parent-Surveillance of Digital Media in Childhood Questionnaire® and the Abbreviated Rating Scales from the Conners Parent Symptom Questionnaire were used to measure Parental Educational Attainment, children's behavior changes during the COVID-19 pandemic, and hyperactivity indexes. A total of 11,190 parents of 6-to-12-year-old children completed the online surveys in mainland China. A structural equation model was established by using Smart-PLS, and the linear regression model, and isotemporal substitution models were established by using a Compositional Data Analysis package with R program to achieve the research objectives. RESULTS: Changes in children's 24-h behaviors due to the COVID-19 pandemic had a significant mediation effect on the negative associations between Parental Educational Attainment and children's hyperactivity index (ß = 0.018, T = 4.521, p < 0.001) with a total effect (ß = -0.046, T = 4.521, p < 0.001) and a direct effect (ß = -0.064, T = 6.330, p < 0.001). Children's Digital Media use was significantly and negatively associated with hyperactivity index among all children. Reallocated time from digital media use to both sleep and physical activity decreased the hyperactivity index, and vice-versa. For parents without tertiary education (R2 = 0.09, p < 0.001), sleep was significantly and negatively associated with the hyperactivity index (ßilr-CSL = -0.06, p < 0.001); for parents with tertiary education (R2 = 0.07, p < 0.001), physical activity was significantly and negatively associated with the hyperactivity index (ßilr-CPA = -0.05, p < 0.001), and sleep was significantly and positively associated with the hyperactivity index (ßilr-CSL = 0.03, p < 0.001). A significant increase in the hyperactivity index was detected when physical activity time was reallocated to sleep, with a significant decrease in the opposite direction. CONCLUSIONS: Parental Educational Attainment and children's 24-h behaviors directly influenced children's hyperactivity index. However, a purposeful and targeted optimization of children's 24-h behaviors-namely, physical activity, digital media use, and sleep-could assist parents with different educational attainments to reduce their children's hyperactivity index and mitigate the risk of hyperactivity.

Hydroxyapatite-based nano-drug delivery system for nicotinamide mononucleotide (NMN): significantly enhancing NMN bioavailability and replenishing in vivo nicotinamide adenine dinucleotide (NAD+) levels.

OBJECTIVES: This study addresses the bioavailability challenges associated with oral nicotinamide mononucleotide (NMN) administration by introducing an innovative NMN formulation incorporated with hydroxyapatite (NMN-HAP). METHODS: The NMN-HAP was developed using a wet chemical precipitation and physical adsorption method. To assess its superiority over conventional free NMN, we examined NMN, nicotinamide adenine dinucleotide (NAD+), and nicotinamide riboside (NR) levels in mouse plasma and tissues following oral administration of NMN-HAP. KEY FINDINGS: NMN-HAP nanoparticles demonstrated a rod-shaped morphology, with an average size of ~50 nm, along with encapsulation efficiency and drug loading capacity exceeding 40%. In vitro, drug release results indicated that NMN-HAP exhibited significantly lower release compared with free NMN. In vivo studies showed that NMN-HAP extended circulation time, improved bioavailability compared with free NMN, and elevated plasma levels of NMN, NAD+, and NR. Moreover, NMN-HAP administration displayed tissue-specific distribution with a substantial accumulation of NMN, NAD+, and NR in the brain and liver. CONCLUSION: NMN-HAP represents an ideal formulation for enhancing NMN bioavailability, enabling tissue-specific delivery, and ultimately elevating in vivo NAD+ levels. Considering HAP's biocompatible nature and versatile characteristics, we anticipate that this system has significant potential for various future applications.

ANAC087 transcription factor positively regulates age-dependent leaf senescence through modulating the expression of multiple target genes in Arabidopsis.

Leaf senescence is the final stage of leaf development and appropriate onset and progression of leaf senescence are critical for reproductive success and fitness. Although great progress has been made in identifying key genes regulating leaf senescence and elucidating the underlining mechanisms in the model plant Arabidopsis, there is still a gap to understanding the complex regulatory network. In this study, we discovered that Arabidopsis ANAC087 transcription factor (TF) positively modulated leaf senescence. Expression of ANAC087 was induced in senescing leaves and the encoded protein acted as a transcriptional activator. Both constitutive and inducible overexpression lines of ANAC087 showed earlier senescence than control plants, whereas T-DNA insertion mutation and dominant repression of the ANAC087 delayed senescence rate. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) profiling showed that the expression of an array of senescence-associated genes was upregulated in inducible ANAC087 overexpression plants including BFN1, NYE1, CEP1, RbohD, SAG13, SAG15, and VPEs, which are involved in programmed cell death (PCD), chlorophyll degradation and reactive oxygen species (ROS) accumulation. In addition, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assays demonstrated that ANAC087 directly bound to the canonical NAC recognition sequence (NACRS) motif in promoters of its target genes. Moreover, mutation of two representative target genes, BFN1 or NYE1 alleviated the senescence rate of ANAC087-overexpression plants, suggesting their genetic regulatory relationship. Taken together, this study indicates that ANAC087 serves as an important regulator linking PCD, ROS, and chlorophyll degradation to leaf senescence.

Identification of Cannabidivarin Metabolites in Different Mouse Organs Using Ultra-Performance Liquid Chromatography Coupled to a Quadrupole Time-of-Flight Mass Spectrometer.

Background and Objectives: As a natural analog of cannabidiol (CBD), nonpsychoactive cannabidivarin (CBDV) has therapeutic potential. However, the precise metabolism of CBDV either in vivo or in vitro has not been fully understood. Objective and Experimental Approach: Therefore, mice were intragastrically administered CBDV, and metabolite-rich and potential target organs and tissues were collected and analyzed by ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. The metabolic pathways of CBDV in mice were illustrated more comprehensively for the first time. Results: Twenty-one metabolites were found, all of which, except decarbonylated CBDV, were initially identified. Compared with CBD, the newly identified metabolic pathways were single dehydrogenation, combined decarbonylation and monohydroxylation, and glutathione conjugations of CBDV and its phase I metabolite. Conclusions: According to the very low response in plasma and the extremely high response in intestinal contents 1 h later after the administration, it was assumed that the oral bioavailability of CBDV was as poor as that of CBD, and the major forms to excrete were conjugates of glutathione and glucuronic acid. In contrast to CBDV, decarbonylated CBDV in the keto form and enol form had considerable responses in plasma and preferred to target fatty tissues and organs owing to their higher lipophilicity. Whether these forms can function as genuine active substances in vivo instead of CBDV is worthy of investigation. These results and supposes contribute notable information regarding the pharmacokinetics and pharmacodynamics of CBDV.

The Effect of the KDL Active School Plan on Children and Adolescents' Physical Fitness in China.

The development of physical fitness among Chinese children and adolescents is not fundamentally improving, and an exploration of effective ways to promote it is an urgent need. Research into physical fitness promotion in schools is increasingly deepening worldwide. However, the implementation and verification of intervention programs with local characteristics in accordance with China's national conditions are relatively weak. This study conducted a randomized controlled trial to examine the effects of the KDL (Know it, Do it, Love it) Active School Plan (KDL-ASP) on children and adolescents' physical fitness. A total of 596 students from level two (2nd-grade students) to five (11th-grade students) in China were assessed in terms of their physical fitness. Of these, 308 students were randomly selected to participate in the KDL-ASP, which uses a combination of indoor and outdoor sports activities in which teachers, parents, and students participate together. The remaining 288 students performed conventional physical activities. After one school year of intervention with the KDL-ASP, the physical fitness of the children and adolescents improved. The improvements in the speed of level two girls, the strength of level four boys, and the lung capacity of level five boys were the most obvious. These results demonstrate the viability of indigenized intervention in schools to improve physical fitness and suggest that KDL-ASP needs to be considered throughout the whole progress of physical education learning for children and adolescents.

Improvement of tissue-specific distribution and biotransformation potential of nicotinamide mononucleotide in combination with ginsenosides or resveratrol.

Decreased Nicotinamide adenine dinucleotide (NAD+ ) level has received increasing attention in recent years since it plays a critical role in many diseases and aging. Although some research has proved that supplementing nicotinamide mononucleotide (NMN) could improve the level of NAD+ , it is still uncertain whether the NAD+ level in specific tissues could be improved in combination with other nutrients. So far, a variety of nutritional supplements have flooded the market, which contains the compositions of NMN coupled with natural products. However, the synergy and transformation process of NMN has not been fully elucidated. In this study, oral administration of NMN (500 mg/kg) combined with resveratrol (50 mg/kg) or ginsenoside Rh2&Rg3 (50 mg/kg) was used to validate the efficacy of appropriate drug combinations in mice. Compared with NMN alone, NMN combined with resveratrol could increase the levels of NAD+ in the heart and muscle by about 1.6 times and 1.7 times, respectively, whereas NMN coupled with ginsenoside Rh2&Rg3 could effectively improve the level of NAD+ in lung tissue for approximately 2.0 times. Our study may provide new treatment ideas for aging or diseases in cardiopulmonary caused by decreased NAD+ levels.

Electron-Induced Synthesis of Dimethyl Ether in the Liquid-Vapor Interface of Methanol.

Ether synthesis from alcohol is known to be acid-catalyzed. Such a process could happen in the acidified liquid of alcohol, but hitherto lacking the experimental evidence. Here we demonstrate that dimethyl ether is spontaneously synthesized in the liquid-vapor interface of pure methanol after ionizing radiation with electrons. Using time-delayed tandem mass spectrometry measurements in combination with theoretical calculations, we further confirm that the protonated dimethyl ether is produced from the ion-molecule reactions not only in the dense vapor above the interface but also within the molecular clusters of the acidic interface. Our finding provides a convincing piece of evidence about the liquid-vapor interfacial acidification by the electron-impact ionizing radiation, exhibiting a promising way to control the chemical reactions in the liquid surface.

Characterization of deglycosylated metabolites of platycosides reveals their biotransformation after oral administration.

Platycodon grandiflorus is a well-known edible and medicinal plant that has been developed for dietary supplements or functional foods to relieve pulmonary disorders. Platycosides are the main active constituents of P. grandiflorus with multiple pharmacological activities. However, their metabolic fates after dietary consumption are still unclear. Herein, 25 deglycosylated metabolites of platycosides were identified, most of which were identified in vivo for the first time. Notably, 3-O-ß-d-glucopyranosyl platycosides could be absorbed into the bloodstream, and their structures were unambiguously characterized with the aid of chemically prepared standards, including two new compounds (M3 and M11). These findings reveal that both intestinal bacterial metabolism and hydrolysis of ester linkage at C-28 by carboxylesterases in liver are the possible in vivo deglycosylation metabolism pathway of platycosides. This study greatly facilitated our understanding of the fate of the platycosides after dietary consumption of P. grandiflorus products.

Glycomic Analysis Reveals That Sialyltransferase Inhibition Is Involved in the Antiviral Effects of Arbidol.

Due to the high mutation rate of influenza virus and the rapid increase of drug resistance, it is imperative to discover host-targeting antiviral agents with broad-spectrum antiviral activity. Considering the discrepancy between the urgent demand of antiviral drugs during an influenza pandemic and the long-term process of drug discovery and development, it is feasible to explore host-based antiviral agents and strategies from antiviral drugs on the market. In the current study, the antiviral mechanism of arbidol (ARB), a broad-spectrum antiviral drug with potent activity at early stages of viral replication, was investigated from the aspect of hemagglutinin (HA) receptors of host cells. N-glycans that act as the potential binding receptors of HA on 16-human bronchial epithelial (16-HBE) cells were comprehensively profiled for the first time by using an in-depth glycomic approach based on TiO2-PGC chip-Q-TOF MS. Their relative levels upon the treatment of ARB and virus were carefully examined by employing an ultra-high sensitive qualitative method based on Chip LC-QQQ MS, showing that ARB treatment led to significant and extensive decrease of sialic acid (SA)-linked N-glycans (SA receptors), and thereby impaired the virus utilization on SA receptors for rolling and entry. The SA-decreasing effect of ARB was demonstrated to result from its inhibitory effect on sialyltransferases (ST), ST3GAL4 and ST6GAL1 of 16-HBE cells. Silence of STs, natural ST inhibitors, as well as sialidase treatment of 16-HBE cells, resulted in similar potent antiviral activity, whereas ST-inducing agent led to the diminished antiviral effect of ARB. These observations collectively suggesting the involvement of ST inhibition in the antiviral effect of ARB. IMPORTANCE This study revealed, for the first time, that ST inhibition and the resulted destruction of SA receptors of host cells may be an underlying mechanism for the antiviral activity of ARB. ST inhibition has been proposed as a novel host-targeting antiviral approach recently and several compounds are currently under exploration. ARB is the first antiviral drug on the market that was found to possess ST inhibiting function. This will provide crucial evidence for the clinical usages of ARB, such as in combination with neuraminidase (NA) inhibitors to exert optimized antiviral effect, etc. More importantly, as an agent that can inhibit the expression of STs, ARB can serve as a novel lead compound for the discovery and development of host-targeting antiviral drugs.

Ectopic overexpression of a membrane-tethered transcription factor gene NAC60 from oilseed rape positively modulates programmed cell death and age-triggered leaf senescence.

Senescence is an integrative final stage of plant development that is governed by internal and external cues. The NAM, ATAF1/2, CUC2 (NAC) transcription factor (TF) family is specific to plants and membrane-tethered NAC TFs (MTTFs) constitute a unique and sophisticated mechanism in stress responses and development. However, the function of MTTFs in oilseed rape (Brassica napus L.) remains unknown. Here, we report that BnaNAC60 is an MTTF associated with the endoplasmic reticulum (ER) membrane. Expression of BnaNAC60 was induced during the progression of leaf senescence. Translocation of BnaNAC60 into nuclei was induced by ER stress and oxidative stress treatments. It binds to the NTLBS motif, rather than the canonical NAC recognition site. Overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, induces significant reactive oxygen species (ROS) accumulation and hypersensitive response-like cell death in both tobacco (Nicotiana benthamiana) and oilseed rape protoplasts. Moreover, ectopic overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, in Arabidopsis also induces precocious leaf senescence. Furthermore, screening and expression profiling identified an array of functional genes that are significantly induced by BnaNAC60 expression. Further it was found that BnaNAC60 can activate the promoter activities of BnaNYC1, BnaRbohD, BnaBFN1, BnaZAT12, and multiple BnaVPEs in a dual-luciferase reporter assay. Electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative PCR assays revealed that BnaNAC60 directly binds to the promoter regions of these downstream target genes. To summarize, our data show that BnaNAC60 is an MTTF that modulates cell death, ROS accumulation, and leaf senescence.

Chemical Comparison of Ophiopogonis radix and Liriopes radix Based on Quantitative Analysis of Multiple Components by HPLC Coupled with Electrospray Ionization Tandem Triple Quadrupole Mass Spectrometry.

BACKGROUND: Ophiopogonis radix and Liriopes radix are well known for the treatment of dry coughs and phthisis. Liriopes radix is occasionally used as a substitute for Ophiopogonis radix in various prescriptions due to the extremely similar pharmacological activities and clinical efficacies, but they are regarded as two different remedies in the Chinese Pharmacopoeia. Accordingly, the establishment of a reliable analytical approach for the discrimination and quality evaluation of Ophiopogonis and Liriopes is required. OBJECTIVE: To establish a simple, accurate, and reliable method that can simultaneously determine multiple components in Ophiopogonis radix and Liriopes radix. To comprehensively compare the chemical compositions of the two herbs and find markers for discrimination and quality assessments. METHOD: An HPLC-ESI-triple quadrupole (QQQ)-MS/MS method was developed for simultaneous characterization and quantification of chemical components in the two herbs. The results were further analyzed by PLS discriminant analysis to provide more information about the chemical differences, as well as to evaluate the quality of each sample. RESULTS: A total of 23 compounds have been characterized and quantified in 31 batches of herbs from different geographical regions, among which liriopesides B, sprengerinin A, ophiopogonin B, and ophiopogonanone E contribute mostly. The contents of homoisoflavonoids were much higher in Ophiopogonis radix than in Liriopes radix, but the levels of steroidal saponins followed a contrary trend. CONCLUSIONS: Simultaneous determination of multiple components by HPLC-QQQ-MS/MS coupled with chemometrics analysis is an acceptable strategy to evaluate and control the quality of Ophiopogonis radix and Liriope radix. HIGHLIGHTS: Simultaneous determination of 12 steroidal saponins and 11 homoisoflavonoids in both Ophiopogonis radix and Liriope radix by using HPLC-QQQ-MS/MS in positive ion mode, as well as the quality control study.

Chemerin isoform analysis in human biofluids using an LC/MRM-MS-based targeted proteomics approach with stable isotope-labeled standard.

Targeted proteomics has advantages over earlier conventional technologies for protein detection. We developed and validated an LC/MRM-MS-based targeted proteomic method combined with immunoaffinity precipitation for the enrichment and detection of low abundance chemerin isoforms in human biofluids. After tryptic digestion, each chemerin isoform was characterized by isoform-specific peptides, and the absolute quantification was achieved by using stable isotope-labeled peptides as internal standards. In serum, follicular fluid and synovial fluid, a total of 6 chemerin isoforms were identified and quantified, among which a novel natural isoform 153Q was discovered for the first time. The relative content of the six chemerin isoforms in human serum was 157S ≫ 156F ≫ 158K > 154F ≥ 155A > 153Q in the ratio of 25:17:5:2.5:2.2:1, respectively. The absolute contents were in the range of 88-3.5 ng/mL. This distribution remained consistent among the 3 biofluids analyzed. Total chemerin were found to be increased in both polycystic ovary syndrome (serum and follicular fluid) and rheumatoid arthritis (serum) patients. However, chemerin isoform analysis revealed that only 156F & 157S were increased in the former, while 155A, 156F & 157S were increased in the latter. This demonstrates the potential of this method in detailed characterization of changes in chemerin isoforms that may be of clinical relevance.

Comprehensive Glycomic Profiling of Respiratory Tract Tissues of Tree Shrews by TiO2-PGC Chip Mass Spectrometry.

Due to its relatively small size, homology to humans, and susceptibility to human viruses, the tree shrew becomes an ideal alternative animal model for the study of human viral infectious diseases. However, there is still no report for the comprehensive glycan profile of the respiratory tract tissues in tree shrews. In this study, we characterized the structural diversity of N-glycans in the respiratory tract of tree shrews using our well-established TiO2-PGC chip-Q-TOF-MS method. As a result, a total of 219 N-glycans were identified. Moreover, each identified N-glycan was quantitated by a high sensitivity and accurate MRM method, in which 13C-labeled internal standards were used to correct the inherent run-to-run variation in MS detection. Our results showed that the N-glycan composition in the turbinate and lung was significantly different from the soft palate, trachea, and bronchus. Meanwhile, 28 high-level N-glycans in turbinate were speculated to be correlated with the infection of H1N1 virus A/California/04/2009. This study is the first to reveal the comprehensive glycomic profile of the respiratory tract of tree shrews. Our results also help to better understand the role of glycan receptors in human influenza infection and pathogenesis.

Micro-PET Imaging Demonstrates 3-O-ß-D-Glucopyranosyl Platycodigenin as an Effective Metabolite Affects Permeability of Cell Membrane and Improves Dosimetry of [18F]-Phillygenin in Lung Tissue.

Platycodon grandiflorum, as a traditional medicinal plant, is commonly used in the treatment of pulmonary disease. Platycodon saponins are proposed as active ingredients. However, the role of secondary saponin metabolites (SSM) in the traditional use of Platycodon has not yet been fully clarified. In this study, [18F]-phillygenin ([18F]-PH) probe was synthesized and thereby used as a tracer for micro-positron emission tomography scanning to explore the effects of platycodon saponins. The membrane permeability with different SSM was evaluated in vitro based on the dye-carrying capacity of fluorescein isothiocyanate. The results showed that total platycodon saponins improved the dosimetry of [18F]-PH in the lung tissue, and an SSM named 3-O-ß-D-glucopyranosyl platycodigenin (GPD682) appreciably changed the distribution of drugs both in vitro and in vivo. We propose that GPD682 could be utilized as an important ingredient to help drug delivery to the lung tissue and improve the treatment of respiratory disease.

Improved approach for comprehensive profiling of gangliosides and sulfatides in rat brain tissues by using UHPLC-Q-TOF-MS.

Gangliosides (GAs) and sulfatides (STs) are major acidic glycosphingolipids (GSLs) that are particularly abundant in the central nervous system and associated with substantial neurodegenerative diseases. In this study, we developed an improved approach for the comprehensive profiling of GAs and STs in rat brain tissues by adopting a pre-fractionation step before the LC-MS analysis. The pre-fractionation step allows the efficient enrichment of different types of acidic GSLs and the removal of high-abundance interferences, thereby greatly enhanced the detection sensitivity and accuracy of low-abundance acidic GSLs. By using this improved approach, a total of 340 acidic GSLs (from 281 compositions) were characterized in rat brain tissues, including 277 GAs (from 230 compositions) and 63 STs (from 51 compositions), among which 57 GAs and 14 STs were novel acidic GSLs that have not been reported previously. This study represented the most comprehensive profiling of acidic GSLs in rat brain tissues. The result of this study greatly enlarged our understanding of the structural diversity of natural acidic GSLs, and provided important chemical information for the exploration of biological function of acidic GSLs in the central nervous system.

LC-MS based sphingolipidomic study on A549 human lung adenocarcinoma cell line and its taxol-resistant strain.

BACKGROUND: Resistance to chemotherapy drugs (e.g. taxol) has been a major obstacle in successful cancer treatment. In A549 human lung adenocarcinoma, acquired resistance to the first-line chemotherapy taxol has been a critical problem in clinics. Sphingolipid (SPL) controls various aspects of cell growth, survival, adhesion, and motility in cancer, and has been gradually regarded as a key factor in drug resistance. To better understand the taxol-resistant mechanism, a comprehensive sphingolipidomic approach was carried out to investigate the sphingolipid metabolism in taxol-resistant strain of A549 cell (A549T). METHODS: A549 and A549T cells were extracted according to the procedure with optimal condition for SPLs. Sphingolipidomic analysis was carried out by using an UHPLC coupled with quadrupole time-of-flight (Q-TOF) MS system for qualitative profiling and an UHPLC coupled with triple quadrupole (QQQ) MS system for quantitative analysis. The differentially expressed sphingolipids between taxol-sensitive and -resistant cells were explored by using multivariate analysis. RESULTS: Based on accurate mass and characteristic fragment ions, 114 SPLs, including 4 new species, were clearly identified. Under the multiple reaction monitoring (MRM) mode of QQQ MS, 75 SPLs were further quantified in both A549 and A549T. Multivariate analysis explored that the levels of 57 sphingolipids significantly altered in A549T comparing to those of A549 (p < 0.001 and VIP > 1), including 35 sphingomyelins (SMs), 14 ceramides (Cers), 3 hexosylceramides (HexCers), 4 lactosylceramides (LacCers) and 1 sphingosine. A significant decrease of SM and Cer levels and overall increase of HexCer and LacCer represent the major SPL metabolic characteristic in A549T. CONCLUSIONS: This study investigated sphingolipid profiles in human lung adenocarcinoma cell lines, which is the most comprehensive sphingolipidomic analysis of A549 and A549T. To some extent, the mechanism of taxol-resistance could be attributed to the aberrant sphingolipid metabolism, "inhibition of the de novo synthesis pathway" and "activation of glycosphingolipid pathway" may play the dominant role for taxol-resistance in A549T. This study provides insights into the strategy for clinical diagnosis and treatment of taxol resistant lung cancer.

Mechanistic studies of DepR in regulating FK228 biosynthesis in Chromobacterium violaceum no. 968.

DepR, a LysR-type transcriptional regulator encoded by the last gene of the putative min operon (orf21-20-19-depR) located at the downstream region of the anticancer agent FK228 biosynthetic gene cluster in Chromobacterium violaceum No. 968, positively regulates the biosynthesis of FK228. In this work, the mechanism underlining this positive regulation was probed by multiple approaches. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay (DIFA) identified a conserved 35-nt DNA segment in the orf21-orf22 intergenic region where the purified recombinant DepR binds to. Quantitative reverse transcription PCR (RT-qPCR) and green fluorescent protein (GFP) promoter probe assays established that transcription of phasin gene orf22 increases in the depR deletion mutant of C. violaceum (CvΔdepR) compared to the wild-type strain. FK228 production in the orf22-overexpressed strain C. violaceum was reduced compared with the wild-type strain. DepR has two conserved cysteine residues C199 and C208 presumed to form a disulfide bridge upon sensing oxidative stress. C199X point mutations that locked DepR in a reduced conformation decreased the DNA-binding affinity of DepR; T232A or R278A mutation also had a negative impact on DNA binding of DepR. Complementation of CvΔdepR with any of those versions of depR carrying a single codon mutation was not able to restore FK228 production to the level of wild-type strain. All evidences collectively suggested that DepR positively regulates the biosynthesis of FK228 through indirect metabolic networking.

A Novel NAC-Type Transcription Factor, NAC87, from Oilseed Rape Modulates Reactive Oxygen Species Accumulation and Cell Death.

Reactive oxygen species (ROS) are thought to play a dual role in plants by functioning as signaling molecules and toxic by-products of aerobic metabolism. The hypersensitive response (HR) is a typical feature of immune responses in plants and also a type of programmed cell death (PCD). How these two processes are regulated in oilseed rape (Brassica napus L.) at the transcriptional level remains largely unknown. In this study, we report that an oilseed rape (Brassica napus L.) NAM-ATAF-CUC (NAC)-type transcription factor NAC87 modulates ROS and cell death accompanied by typical changes at the morphological and cellular levels. The BnaNAC87 gene was induced by multiple stress and hormone treatments and was highly expressed in senescent leaves by quantitative reverse transcription-PCR (qRT-PCR). BnaNAC87 is located in nuclei and has transcriptional activation activity. Expression of BnaNAC87 promoted significant ROS production, cell death as well as death of protoplasts, as indicated by histological staining. In addition, putative downstream target genes of NAC87 were identified through both qRT-PCR and dual luciferase reporter assays. We found that genes implicated in ROS generation (RbohB), cell death (VPE1a, ZEN1), leaf senescence (WRKY6, ZAT12) and defense (PR2, PR5 and HIN1) were significantly induced. Through an electrophoretic mobility shift assay (EMSA), we confirmed that BnaNAC87 directly binds to the NACRS-containing promoter fragments of ZEN1, ZAT12, HIN1 and PR5 genes. From these results, we conclude that oilseed rape NAC87 is a novel NAC transcription factor that acts as a positive regulator of ROS metabolism and cell death.