PapR6, a putative atypical response regulator, functions as a pathway-specific activator of pristinamycin II biosynthesis in Streptomyces pristinaespiralis.

There are up to seven regulatory genes in the pristinamycin biosynthetic gene cluster of Streptomyces pristinaespiralis, which infers a complicated regulation mechanism for pristinamycin production. In this study, we revealed that PapR6, a putative atypical response regulator, acts as a pathway-specific activator of pristinamycin II (PII) biosynthesis. Deletion of the papR6 gene resulted in significantly reduced PII production, and its overexpression led to increased PII formation, compared to that of the parental strain HCCB 10218. However, either papR6 deletion or overexpression had very little effect on pristinamycin I (PI) biosynthesis. Electrophoretic mobility shift assays (EMSAs) demonstrated that PapR6 bound specifically to the upstream region of snaF, the first gene of the snaFE1E2GHIJK operon, which is likely responsible for providing the precursor isobutyryl-coenzyme A (isobutyryl-CoA) and the intermediate C11 αß-unsaturated thioester for PII biosynthesis. A signature PapR6-binding motif comprising two 4-nucleotide (nt) inverted repeat sequences (5'-GAGG-4 nt-CCTC-3') was identified. Transcriptional analysis showed that inactivation of the papR6 gene led to markedly decreased expression of snaFE1E2GHIJK. Furthermore, we found that a mutant (snaFmu) with base substitutions in the identified PapR6-binding sequence in the genome exhibited the same phenotype as that of the ΔpapR6 strain. Therefore, it may be concluded that pathway-specific regulation of PapR6 in PII biosynthesis is possibly exerted via controlling the provision of isobutyryl-CoA as well as the intermediate C11 αß-unsaturated thioester.

Absorption and translocation of selected pharmaceuticals in Pistia stratiotes: Spatial distribution analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Phytoremediation can eliminate pharmaceuticals from aquatic environments through absorption; however, understanding of absorption and transport processes in plants remains limited. In this study, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) method was developed to explore the absorption and translocation mechanisms of seven common pharmaceuticals in Pistia stratiotes. Results showed that 2,3-dicyanohydroquinone, an infrequently used matrix, exhibited outstanding performance in MALDI-MSI analysis, producing the highest signal intensity for four of the seven pharmaceuticals. Region of Interest (ROI) analysis revealed that charge speciation of pharmaceuticals significantly influenced their ability to enter vascular bundle. Neutral and positively charged pharmaceuticals easily entered vascular bundle, while negatively charged pharmaceuticals faced difficulty. ROI results for neutral and negatively charged pharmaceuticals exhibited positive correlation with their transfer factor values, indicating that their translocation ability from root to shoot was related to their capacity to enter vascular bundle. However, no correlation was observed for positively charged pharmaceuticals, suggesting that these compounds, upon entering vascular bundle, encountered difficulties in upward translocation through the xylem. This study introduces an innovative approach and offers novel insights into the retention and migration of pharmaceuticals in plant tissues, aiming to enhance the understanding of pharmaceutical accumulation in plants. ENVIRONMENTAL IMPLICATION: Pharmaceuticals in aquatic environment can inflict detrimental effects on both human health and ecosystem. Phytoremediation can remove pharmaceuticals from aquatic environments through absorption. However, our understanding of absorption and transportation of pharmaceuticals in plants remains limited. This study developed a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) method for pharmaceuticals in plant roots, and to explore the absorption and translocation mechanisms of pharmaceuticals. The study offers direct evidence of differences in accumulation behavior of pharmaceuticals in plants, providing valuable insights for targeted and effective strategies in using plants for remediating the aquatic ecosystem from pharmaceuticals.

Estimation of the time of zolpidem intake and differentiation between consumption and external contamination using MALDI-MSI for investigations on single hair samples.

Estimation of drug ingestion time (event time) and distinguishing between drug ingestion and external contamination are important for interpreting hair analysis results in forensics practice. Here, we present a matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) method for in situ analysis of intact hair. We applied a longitudinal cutting method for a single hair to analysis authentic hair samples from a victim of a drug-facilitated sexual assault (DFSA) case and zolpidem-soaked hair. MALDI-MSI showed that zolpidem-positive segments distributed at 4-6 mm or 6-8 mm from the root in three single hairs of a DFSA victim collected 25 days after the event, at concentrations ranging from 0.1 to 5.7 pg mm-1, in agreement with the results from segmental analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS). The estimation of drug intake time was about 20-30 days before sampling, which was consistent with the known time of drug intake. This MALDI-MS method allows imaging analysis of trace substances in a single hair and can realize the intuitive reflection of drug taking time. In addition, zolpidem applied by soaking was mainly distributed on both sides of the longitudinal hair shaft, whereas ingested zolpidem was found only in the middle of the hair shaft of the DFSA victim. The MALDI-MS images of unwashed and washed hair suggested that the amount of externally applied drug was decreased by washing, it was still present on surface layer (cuticle) sides although. Visualization using MALDI-MSI could therefore distinguish between drug ingestion and contamination by reflecting the distribution and deposition site of the drug in hair.

Multi-omics combined with MALDI mass spectroscopy imaging reveals the mechanisms of biosynthesis of characteristic compounds in Tetrastigma hemsleyanum Diels et Gilg.

Tetrastigma hemsleyanum Diels et Gilg is recognized as a source of extracts with various desirable bioactivities. However, current knowledge regarding the mechanisms of biosynthesis of flavonoids, phenolic compounds, and other bioactive chemicals is limited. We conducted comprehensive tissue distribution studies and biosynthetic analyses of the 26 main bioactive compounds of this plant. The majority of flavonoids exhibited higher concentrations in the cortex (CT) compared to the vascular cylinder (VC). The expression levels of genes and proteins in CT and VC were quantified using mRNA sequencing and isobaric tags for relative and absolute quantification (iTRAQ). A total of 31,700 genes were identified, among which 4921 exhibited differential expression between CT and VC. A total of 13,996 proteins were identified in the proteomes of CT and VC, with 927 showing differential expression. Co-expression network analyses of DEGs and DEPs from multiple sites demonstrated substantial pathway variations linked to flavonoid biosynthesis. Through differential enrichment analysis, a total of 32 genes involved in the flavone biosynthesis pathway were identified, with iTRAQ specifically detecting C3'H, F3H and FLS. Pearson correlation analysis revealed a strong association between the expression levels of C3'H, F3H, and FLS and the concentrations of flavonoids. The validation of multiple genes encoding pivotal enzymes was conducted using real-time fluorescence quantitative PCR (RT-qPCR). The findings provide a foundation for future investigations into the molecular mechanisms and functional characterization of T. hemsleyanum candidate genes associated with characteristic compounds.

Schisandra lignans ameliorate nonalcoholic steatohepatitis by regulating aberrant metabolism of phosphatidylethanolamines.

Nonalcoholic steatohepatitis (NASH) is a spectrum of chronic liver disease characterized by hepatic lipid metabolism disorder. Recent reports emphasized the contribution of triglyceride and diglyceride accumulation to NASH, while the other lipids associated with the NASH pathogenesis remained unexplored. The specific purpose of our study was to explore a novel pathogenesis and treatment strategy of NASH via profiling the metabolic characteristics of lipids. Herein, multi-omics techniques based on LC-Q-TOF/MS, LC-MS/MS and MS imaging were developed and used to screen the action targets related to NASH progress and treatment. A methionine and choline deficient (MCD) diet-induced mouse model of NASH was then constructed, and Schisandra lignans extract (SLE) was applied to alleviate hepatic damage by regulating the lipid metabolism-related enzymes CES2A and CYP4A14. Hepatic lipidomics indicated that MCD-diet led to aberrant accumulation of phosphatidylethanolamines (PEs), and SLE could significantly reduce the accumulation of intrahepatic PEs. Notably, exogenous PE (18:0/18:1) was proved to significantly aggravate the mitochondrial damage and hepatocyte apoptosis. Supplementing PE (18:0/18:1) also deteriorated the NASH progress by up regulating intrahepatic proinflammatory and fibrotic factors, while PE synthase inhibitor exerted a prominent hepatoprotective role. The current work provides new insights into the relationship between PE metabolism and the pathogenesis of NASH.

How to sample a seizure plant: the role of the visualization spatial distribution analysis of Lophophora williamsii as an example.

Natural compounds in plants are often unevenly distributed, and determining the best sampling locations to obtain the most representative results is technically challenging. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can provide the basis for formulating sampling guideline. For a succulent plant sample, ensuring the authenticity and in situ nature of the spatial distribution analysis results during MSI analysis also needs to be thoroughly considered. In this study, we developed a well-established and reliable MALDI-MSI method based on preservation methods, slice conditions, auxiliary matrices, and MALDI parameters to detect and visualize the spatial distribution of mescaline in situ in Lophophora williamsii. The MALDI-MSI results were validated using liquid chromatography-tandem mass spectrometry. Low-temperature storage at -80°C and drying of "bookmarks" were the appropriate storage methods for succulent plant samples and their flower samples, and cutting into 40 µm thick sections at -20°C using gelatin as the embedding medium is the appropriate sectioning method. The use of DCTB (trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile) as an auxiliary matrix and a laser intensity of 45 are favourable MALDI parameter conditions for mescaline analysis. The region of interest semi-quantitative analysis revealed that mescaline is concentrated in the epidermal tissues of L. williamsii as well as in the meristematic tissues of the crown. The study findings not only help to provide a basis for determining the best sampling locations for mescaline in L. williamsii, but they also provide a reference for the optimization of storage and preparation conditions for raw plant organs before MALDI detection. Key Points: An accurate in situ MSI method for fresh water-rich succulent plants was obtained based on multi-parameter comparative experiments.Spatial imaging analysis of mescaline in Lophophora williamsii was performed using the above method.Based on the above results and previous results, a sampling proposal for forensic medicine practice is tentatively proposed.

Single-cell RNA sequencing reveals a hierarchical transcriptional regulatory network of terpenoid biosynthesis in cotton secretory glandular cells.

Plants can synthesize a wide range of terpenoids in response to various environmental cues. However, the specific regulatory mechanisms governing terpenoid biosynthesis at the cellular level remain largely elusive. In this study, we employed single-cell RNA sequencing to comprehensively characterize the transcriptome profile of cotton leaves and established a hierarchical transcriptional network regulating cell-specific terpenoid production. We observed substantial expression levels of genes associated with the biosynthesis of both volatile terpenes (such as ß-caryophyllene and ß-myrcene) and non-volatile gossypol-type terpenoids in secretory glandular cells. Moreover, two novel transcription factors, namely GoHSFA4a and GoNAC42, are identified to function downstream of the Gossypium PIGMENT GLAND FORMATION genes. Both transcription factors could directly regulate the expression of terpenoid biosynthetic genes in secretory glandular cells in response to developmental and environmental stimuli. For convenient retrieval of the single-cell RNA sequencing data generated in this study, we developed a user-friendly web server . Our findings not only offer valuable insights into the precise regulation of terpenoid biosynthesis genes in cotton leaves but also provide potential targets for cotton breeding endeavors.