Simultaneous determination of choline, L-carnitine, betaine, trimethylamine, trimethylamine N-oxide, and creatinine in plasma, liver, and feces of hyperlipidemic rats by UHPLC-MS/MS.

BACKGROUND: Due to the close correlation between choline, L-carnitine, betaine and their intestinal microbial metabolites, including trimethylamine (TMA) and trimethylamine N-oxide (TMAO), and creatinine, there has been an increasing interest in the study of these compounds in vivo. METHODS: In this study, a rapid stable isotope dilution (SID)-UHPLC-MS/MS method was developed for the simultaneous determination of choline, L-carnitine, betaine, TMA, TMAO and creatinine in plasma, liver and feces of rats. The method was validated using quality control (QC) samples spiked at low, medium and high levels. Second, we applied the method to quantify the effects of Rosa Roxburghii Tratt juice (RRTJ) on plasma, liver, and fecal levels of choline, L-carnitine, betaine, TMA, TMAO, and creatinine in high-fat diet-induced hyperlipidemic rats, demonstrating the utility of the method. RESULTS: The limits of detection (LOD) were 0.04-0.027 µM and the limits of quantification (LOQ) were 0.009-0.094 µM. The linear ranges for each metabolite in plasma were choline1.50-96 µM; L-carnitine: 2-128 µM; betaine: 3-192 µM; TMA: 0.01-40.96 µM; TMAO: 0.06-61.44 µM and creatinine: 1-64 µM (R2 ≥ 0.9954). The linear ranges for each metabolite in liver were Choline: 12-768 µM; L-carnitine: 1.5-96 µM; betaine: 10-640 µM; TMA: 0.5-32 µM; TMAO: 0.02-81.92 µM and creatinine: 0.2-204.8 µM (R2 ≥ 0.9938). The linear ranges for each metabolite in feces were choline: 1.5-96 µM; L-carnitine: 0.01-40.96 µM; Betaine: 1.5-96 µM; TMA: 1-64 µM; TMAO: 0.02-81.92 µM and Creatinine: 0.02-81.92 µM (R2 ≥ 0.998). The intra-day and inter-day coefficients of variation were < 8 % for all analytes. The samples were stabilized after multiple freeze-thaw cycles (3 freeze-thaw cycles), 24 h at room temperature, 24 h at 4 °C and 20 days at -80 °C. The samples were stable. The average recovery was 89 %-99 %. This method was used to quantify TMAO and its related metabolites and creatinine levels in hyperlipidemic rats. The results showed that high-fat diet led to the disorder of TMAO and its related metabolites and creatinine in rats, which was effectively improved after the intervention of Rosa Roxburghii Tratt juice（RRTJ）. CONCLUSIONS: A method for the determination of choline, L-carnitine, betaine, TMA, TMAO and creatinine in plasma, liver and feces samples was established, which is simple, time-saving, high precision, accuracy and recovery.

Effects of Distracting Behaviors on Driving Workload and Driving Performance in a City Scenario.

Distractors faced by drivers grow continuously, and concentration on driving becomes increasingly difficult, which has detrimental influences on road traffic safety. The present study aims to investigate changes in driving workload and driving performance caused by distracting tasks. The recruited subjects were requested to drive along a city route in a real vehicle and perform three secondary tasks sequentially. Electrocardiography and driving performance were measured. Heart rate variability (HRV) was adopted to quantitatively analyze the driving workload. Findings show that: (i) increments are noticed in the root mean square differences of successive heartbeat intervals (RMSSD), the standard deviation of normal-to-normal peak (SDNN), the heart rate growth rate (HRGR), and the ratio of low-frequency to high-frequency powers (LF/HF) compared to undistracted driving; (ii) the hands-free phone conversation task has the most negative impacts on driving workload; (iii) vehicle speed reduces due to secondary tasks while changes in longitudinal acceleration exhibit inconsistency; (iv) the experienced drivers markedly decelerate during hands-free phone conversation, and HRGR shows significant differences in both driving experience and gender under distracted driving conditions; (v) correlations exist between HRV and driving performance, and LF/HF correlates positively with SDNN/RMSSD in the hands-free phone conversation and chatting conditions while driving.

Genome assembly of the JD17 soybean provides a new reference genome for comparative genomics.

Cultivated soybean (Glycine max) is an important source for protein and oil. Many elite cultivars with different traits have been developed for different conditions. Each soybean strain has its own genetic diversity, and the availability of more high-quality soybean genomes can enhance comparative genomic analysis for identifying genetic underpinnings for its unique traits. In this study, we constructed a high-quality de novo assembly of an elite soybean cultivar Jidou 17 (JD17) with chromosome contiguity and high accuracy. We annotated 52,840 gene models and reconstructed 74,054 high-quality full-length transcripts. We performed a genome-wide comparative analysis based on the reference genome of JD17 with 3 published soybeans (WM82, ZH13, and W05), which identified 5 large inversions and 2 large translocations specific to JD17, 20,984-46,912 presence-absence variations spanning 13.1-46.9 Mb in size. A total of 1,695,741-3,664,629 SNPs and 446,689-800,489 Indels were identified and annotated between JD17 and them. Symbiotic nitrogen fixation genes were identified and the effects from these variants were further evaluated. It was found that the coding sequences of 9 nitrogen fixation-related genes were greatly affected. The high-quality genome assembly of JD17 can serve as a valuable reference for soybean functional genomics research.

Chromosomal-level genome and multi-omics dataset of Pueraria lobata var. thomsonii provide new insights into legume family and the isoflavone and puerarin biosynthesis pathways.

Pueraria lobata var. thomsonii (hereinafter abbreviated as Podalirius thomsonii), a member of legumes, is one of the important traditional Chinese herbal medicines, and its puerarin extraction is widely used in health and pharmaceutical industry. Here, we assembled a high-quality genome of P. thomsonii using long-read single-molecule sequencing and Hi-C technologies. The genome assembly is approximately 1.37 Gb in size and consists of 5145 contigs with a contig N50 of 593.70 Kb, further clustered into 11 pseudochromosomes. The genome structural annotation resulted in about 869.33 Mb (about 62.70% of the genome) repeat regions and 45 270 protein-coding genes. Genome evolution analysis revealed that P. thomsonii is most closely related to soybean and underwent two ancient whole-genome duplication events, one was in the common ancestor shared by legume species, the other occurred independently at around 7.2 million years ago after its specification. A total of 2373 gene families were found unique in P. thomsonii comparing to five other legume species. Genes and metabolites related to puerarin content in tuberous tissues were characterized. A total of 572 genes upregulated in the puerarin biosynthesis pathway were identified, and 235 candidate genes were further enriched by omics data. Furthermore, we identified 6 8-C-glucosyltransferase (8-C-GT) candidate genes significantly involved in puerarin metabolism. Our study filled in a key genomic gap in legume family, and provided valuable multi-omic resources for the genetic improvement of P. thomsonii.

Arthrobacter wenxiniae sp. nov., a novel plant growth-promoting rhizobacteria species harbouring a carotenoids biosynthetic gene cluster.

A bacterial strain, designated AETb3-4T was isolated from the rhizosphere of lily. Comparison of 16S rRNA gene sequences showed that the sequence from strain AETb3-4T exhibits high sequence similarity with those of Arthrobacter silviterrae KIS14-16T (97.9%), Arthrobacter livingstonensis LI2T (97.2%) and Arthrobacter stackebrandtii CCM 2783T (97.0%). Whole genome average nucleotide identity (ANI) and the digital DNA-DNA hybridization (dDDH) values between strain AETb3-4T and the reference strains A. silviterrae DSM 27180T, A. livingstonensis L12T and A. stackebrandtii DSM 16005T were below 83.6% and 27.7%, respectively, values which are considerably below the proposed thresholds for the species delineation, consistent with the proposal that strain AETb3-4T represents a novel species. The genome size of strain AETb3-4T is 4.33 Mb and the genomic DNA G + C content is 67.3%. The main polar lipids were identified as phosphatidylglycerol, diphosphatidylglycero, phosphatidylinositol and an unidentified glycolipid. The major fatty acids (> 10%) were identified as anteiso-C15: 0 and anteiso-C17: 0. The predominant menaquinone was found to be menaquinone 9 (MK-9) (H2) (82.2%). Phenotypic tests allowed the strain to be differentiated from its close phylogenetic neighbors. Based on the results obtained, it is proposed that the strain AETb3-4T (= CFCC 16390T = LMG 31708T) represents a novel species in the genus Arthrobacter, for which the names Arthrobacter wenxiniae sp. nov. is proposed. In addition, the novel strain AETb3-4T has multiple plant growth-promoting characters including ACC-deaminase activity and production of IAA. Furthermore, the genome contains secondary metabolite biosynthesis gene clusters, including a carotenoid biosynthetic gene cluster, suggesting potential capacities for secondary metabolite synthesis. These data suggest that strain AETb3-4T may have potential applications both in medicine and sustainable agriculture.

Insights into the Ancient Adaptation to Intertidal Environments by Red Algae Based on a Genomic and Multiomics Investigation of Neoporphyra haitanensis.

Colonization of land from marine environments was a major transition for biological life on Earth, and intertidal adaptation was a key evolutionary event in the transition from marine- to land-based lifestyles. Multicellular intertidal red algae exhibit the earliest, systematic, and successful adaptation to intertidal environments, with Porphyra sensu lato (Bangiales, Rhodophyta) being a typical example. Here, a chromosome-level 49.67 Mb genome for Neoporphyra haitanensis comprising 9,496 gene loci is described based on metagenome-Hi-C-assisted whole-genome assembly, which allowed the isolation of epiphytic bacterial genome sequences from a seaweed genome for the first time. The compact, function-rich N. haitanensis genome revealed that ancestral lineages of red algae share common horizontal gene transfer events and close relationships with epiphytic bacterial populations. Specifically, the ancestor of N. haitanensis obtained unique lipoxygenase family genes from bacteria for complex chemical defense, carbonic anhydrases for survival in shell-borne conchocelis lifestyle stages, and numerous genes involved in stress tolerance. Combined proteomic, transcriptomic, and metabolomic analyses revealed complex regulation of rapid responses to intertidal dehydration/rehydration cycling within N. haitanensis. These adaptations include rapid regulation of its photosynthetic system, a readily available capacity to utilize ribosomal stores, increased methylation activity to rapidly synthesize proteins, and a strong anti-oxidation system to dissipate excess redox energy upon exposure to air. These novel insights into the unique adaptations of red algae to intertidal lifestyles inform our understanding of adaptations to intertidal ecosystems and the unique evolutionary steps required for intertidal colonization by biological life.

Whole-genome resequencing of Osmanthus fragrans provides insights into flower color evolution.

Osmanthus fragrans is a well-known ornamental plant that has been domesticated in China for 2500 years. More than 160 cultivars have been found during this long period of domestication, and they have subsequently been divided into four cultivar groups, including the Yingui, Jingui, Dangui, and Sijigui groups. These groups provide a set of materials to study genetic evolution and variability. Here, we constructed a reference genome of O. fragrans 'Liuyejingui' in the Jingui group and investigated its floral color traits and domestication history by resequencing a total of 122 samples, including 119 O. fragrans accessions and three other Osmanthus species, at an average sequencing depth of 15×. The population structure analysis showed that these 119 accessions formed an apparent regional cluster. The results of linkage disequilibrium (LD) decay analysis suggested that varieties with orange/red flower color in the Dangui group had undergone more artificial directional selection; these varieties had the highest LD values among the four groups, followed by the Sijigui, Jingui, and Yingui groups. Through a genome-wide association study, we further identified significant quantitative trait loci and genomic regions containing several genes, such as ethylene-responsive transcription factor 2 and Arabidopsis pseudoresponse regulator 2, that are positively associated with petal color. Moreover, we found a frameshift mutation with a 34-bp deletion in the first coding region of the carotenoid cleavage dioxygenase 4 gene. This frameshift mutation existed in at least one site on both alleles in all varieties of the Dangui group. The results from this study shed light on the genetic basis of domestication in woody plants, such as O. fragrans.

The chromosome-level Hemerocallis citrina Borani genome provides new insights into the rutin biosynthesis and the lack of colchicine.

Hemerocallis citrina Borani (huang hua cai in Chinese) is an important horticultural crop whose flower buds are widely consumed as a delicious vegetable in Asia. Here we assembled a high-quality reference genome of H. citrina using single-molecule sequencing and Hi-C technologies. The genome assembly was 3.77 Gb and consisted of 3183 contigs with a contig N50 of 2.09 Mb, which were further clustered into 11 pseudochromosomes. A larger portion (3.25 Gb or 86.20%) was annotated as a repetitive content and 54,295 protein-coding genes were annotated in the genome. Genome evolution analysis showed that H. citrina experienced a recent whole-genome duplication (WGD) event at ~15.73 million years ago (Mya), which was the main factor leading to many multiple copies of orthologous genes. We used this reference genome to predict 20 genes involved in the rutin biosynthesis pathway. Moreover, our metabolomics data revealed neither colchicine nor its precursors in H. citrina, challenging the long-standing belief that this alkaloid causes poisoning by the plant. The results of our disruptive research are further substantiated by our genomic finding that H. citrina does not contain any genes involved in colchicine biosynthesis. The high-quality genome lays a solid foundation for genetic research and molecular breeding of H. citrina.

CRISPR/Cas9-induced nos2b mutant zebrafish display behavioral abnormalities.

The immunomodulatory function of nitric oxide synthase (NOS2) has been extensively studied. However, some behavioral abnormalities caused by its mutations have been found in a few rodent studies, of which the molecular mechanism remains elusive. In this research, we generated nos2b gene knockout zebrafish (nos2bsou2/sou2 ) using CRISPR/Cas9 approach and investigated their behavioral and molecular changes by doing a series of behavioral detections, morphological measurements, and molecular analyses. We found that, compared with nos2b+/+ zebrafish, nos2bsou2/sou2 zebrafish exhibited enhanced motor activity; additionally, nos2bsou2/sou2 zebrafish were characterized by smaller brain size, abnormal structure of optic tectum, reduced mRNA level of presynaptic synaptophysin and postsynaptic homer1, and altered response to sodium nitroprusside/methylphenidate hydrochloride treatment. These findings will likely contribute to future studies of behavioral regulation.

Genomics insights into different cellobiose hydrolysis activities in two Trichoderma hamatum strains.

BACKGROUND: Efficient biomass bioconversion is a promising solution to alternative energy resources and environmental issues associated with lignocellulosic wastes. The Trichoderma species of cellulolytic fungi have strong cellulose-degrading capability, and their cellulase systems have been extensively studied. Currently, a major limitation of Trichoderma strains is their low production of ß-glucosidases. RESULTS: We isolated two Trichoderma hamatum strains YYH13 and YYH16 with drastically different cellulose degrading efficiencies. YYH13 has higher cellobiose-hydrolyzing efficiency. To understand mechanisms underlying such differences, we sequenced the genomes of YYH13 and YYH16, which are essentially identical (38.93 and 38.92 Mb, respectively) and are similar to that of the T. hamatum strain GD12. Using GeneMark-ES, we annotated 11,316 and 11,755 protein-coding genes in YYH13 and YYH16, respectively. Comparative analysis identified 13 functionally important genes in YYH13 under positive selection. Through examining orthologous relationships, we identified 172,655, and 320 genome-specific genes in YYH13, YYH16, and GD12, respectively. We found 15 protease families that show differences between YYH13 and YYH16. Enzymatic tests showed that exoglucanase, endoglucanase, and ß-glucosidase activities were higher in YYH13 than YYH16. Additionally, YYH13 contains 10 families of carbohydrate-active enzymes, including GH1, GH3, GH18, GH35, and GH55 families of chitinases, glucosidases, galactosidases, and glucanases, which are subject to stronger positive selection pressure. Furthermore, we found that the ß-glucosidase gene (YYH1311079) and pGEX-KG/YYH1311079 bacterial expression vector may provide valuable insight for designing ß-glucosidase with higher cellobiose-hydrolyzing efficiencies. CONCLUSIONS: This study suggests that the YYH13 strain of T. hamatum has the potential to serve as a model organism for producing cellulase because of its strong ability to efficiently degrade cellulosic biomass. The genome sequences of YYH13 and YYH16 represents a valuable resource for studying efficient production of biofuels.

Optimization of submerged fermentation of Thelephora ganbajun zang.

The effects of the fermentation conditions on both the biomass yield and the organic selenium yield of Thelephora ganbajun zang were studied. The components most suitable for the submerged fermentation medium were examined using the orthogonal array method; they comprised sucrose at 30 g L(-1) , carbamide 1 g L(-1) , corn steep liquor 8 g L(-1) , MgSO4 ·7H2 O 0.3 g L(-1) , KH2 PO4 0.5 g L(-1) , and NaCl 5 g L(-1) . The optimum cultivation conditions that resulted in maximal biomass yield were obtained using the response surface methodology (RSM). The conditions were as follows: initial pH, 5.84; temperature, 26.16 °C; and rotation speed, 170 rpm. Feeding sucrose led to a higher biomass yield, with a maximum of 21.20 g L(-1) . The biomass yield and the organic Se yield of T. ganbajun could reach 10.8 g L(-1) and 3256.07 mg kg(-1) , respectively, in a culture medium supplemented with 200 mg L(-1) sodium selenite (Na2 SeO3 ), which was added to the medium at 36 h after inoculation. Application of the orthogonal array method and RSM gave rise to a significant enhancement in the biomass yield of T. ganbajun. The results of these experiments indicate that T. ganbajun is a promising microorganism for selenium enrichment.

Microbial floral dynamics of Chinese traditional soybean paste (doujiang) and commercial soybean paste.

Traditional soybean paste from Shandong Liangshan and Tianyuan Jiangyuan commercial soybean paste were chosen for analysis and comparison of their bacterial and fungal dynamics using denaturing gel gradient electrophoresis and 16S rRNA gene clone libraries. The bacterial diversity results showed that more than 20 types of bacteria were present in traditional Shandong soybean paste during its fermentation process, whereas only six types of bacteria were present in the commercial soybean paste. The predominant bacteria in the Shandong soybean paste were most closely related to Leuconostoc spp., an uncultured bacterium, Lactococcus lactis, Bacillus licheniformis, Bacillus spp., and Citrobacter freundii. The predominant bacteria in the Tianyuan Jiangyuan soybean paste were most closely related to an uncultured bacterium, Bacillus licheniformis, and an uncultured Leuconostoc spp. The fungal diversity results showed that 10 types of fungi were present in the Shandong soybean paste during the fermentation process, with the predominant fungi being most closely related to Geotrichum spp., an uncultured fungal clone, Aspergillus oryzae, and yeast species. The predominant fungus in the commercial soybean paste was Aspergillus oryzae.