Advancing Glucose Conjugated Gibberellins Discovery: A Structure-Oriented Screening and Identification Method for Unraveling Gibberellin Metabolites in Plants.

Gibberellins (GAs) play a pivotal role in modulating plant growth and development. Glucose-conjugated gibberellins (Glc-GAs), a prevalent conjugated form of GAs, regulate intracellular GA levels by the coupling and decoupling of glucose groups. However, the diversity of Glc-GAs identified within individual species remains limited, hinting at a multitude of yet undiscovered gibberellin metabolites. This lacuna poses considerable impediments to research efforts dedicated to comprehensively delineating the GA metabolic pathway. In this study, we developed a structure-oriented screening and identification method for Glc-GAs in plant species by employing LC-MS/MS coupled with chemical derivatization. Through the application of chemical derivatization technique, carboxyl groups on Glc-GAs were labeled which effectively enhanced the sensitivity and selectivity of mass spectrometry detection for these compounds. Concurrently, the integration of mass spectrometry fragmentation and chromatographic retention behavior facilitated the efficient screening and identification of potential Glc-GAs. With this strategy, we screened and identified 12 potential Glc-GAs from six plant species. These findings expand the Glc-GA diversity in plants and contribute to understanding GA metabolic pathways.

The feedback regulation of carbohydrates intake on food intake and appetite in grass carp (Ctenopharyngodon idella).

Improving carbohydrate utilization can contribute to sustainability of aquaculture. In order to explore the feedback mechanism of glucose homeostasis in fish, one control diet (25% carbohydrate and 40% protein), one relatively high carbohydrate diet named HG (42% carbohydrate and 40% protein), and one high dietary carbohydrate coupled with relatively low protein diet named HGP (42% carbohydrate and 25% protein) were fed to grass carp for 40 days. After the feeding trial, HG group impeded the food intake and growth performance of fish compared with the other two groups. Meanwhile, the serum glucose and insulin level were both significantly elevated under the condition of high carbohydrates intake when compared HG with control group. However, although no significant difference was observed in peripheral glucose or insulin between HG and HGP groups, fish fed with HGP diet increased growth performance and food intake compared with the HG group. Gene expression data indicated that fish selectively regulated the expressions of the cerebral anorexigenic genes (cart and pomc) to adapt to the HG and HGP intake. Therefore, the HGP diet with high carbohydrate and low protein was more suitable for grass carp feeding and growth when compared with the other two diets, possibly because the diet composition was closer to the natural food of this fish. In addition, the serum leptin level was highly consistent with changes in food intake and anorexigenic gene expressions when comparing the three experimental diets, indicating that leptin might be the key to mediate the feedback regulation of carbohydrates intake on food intake and appetite in fish.

A structure-guided screening strategy for the discovery and identification of potential gibberellins from plant samples using liquid chromatography-mass spectrometry assisted by chemical isotope labeling.

Gibberellins (GAs) play crucial roles in plant growth and development, and their regulatory functions rely on complex metabolic networks and signaling pathways. Therefore, the exploration of GAs metabolic network is of great importance. However, limited GAs have been found in given plant species, which makes it difficult to comprehensively study the GAs metabolic network. Herein, a structure-guided strategy for GAs screening based on liquid chromatography-mass spectrometry analysis assisted by chemical isotope labeling (CIL-LC-MS) was developed. In the proposed strategy, N,N-dimethyl ethylenediamine (DMED) and its isotopologue d4-DMED were used to label GAs. In light of the characteristic fragmentation patterns exhibited by the labeled GAs, four principles were summarized to screen the potential GAs from plant tissues. Subsequently, the MS/MS fragmentation behavior and quantitative structure-retention relationship (QSRR) model were employed to assist in deciphering structures of GA candidates. With this strategy, thirty potential GAs were screened out and identified from five plant species. Seven of them were confirmed by the authentic standards. Twenty-two of them have not been reported before in the five plant species used in this study, including thirteen that have been reported in other plant species and nine that have never been reported in any plant species. Noteworthily, a total of nine potential GAs were speculated to be novel 16, 17-double hydrated GAs, which indicated that the 16, 17-double hydration may be a ubiquitous metabolic pathway of GAs in plants. This study was the first attempt to establish a structure-guided screening strategy for GAs. Our findings have enriched the GA species in plants and expanded the GAs family, which may be helpful for study of the metabolic pathway and physiological function of GAs.

Differential Roles of Two Leptin Gene Paralogues on Food Intake and Hepatic Metabolism Regulation in Mandarin Fish.

Leptin affects food intake regulation and energy homeostasis in mammals, as opposed to mammals who have a single leptin gene, fish have duplicated leptin gene paralogues. Until now, most functional studies on fish focused on the first reported paralogue without much explanation on specific gene paralogue. This study successfully expressed two homologous recombinant mandarin fish leptin genes (LepA and LepB) for the first time. To explore the differential roles of these two gene paralogues involved in food intake and energy homeostasis, mandarin fish were treated with homologous recombinant LepA and LepB proteins by acute IP administration. The results showed that LepB inhibited the food intake of mandarin fish after acute IP administration through modifying the expressions of hypothalamic orexigenic genes, while LepA had no significant effect on its food intake. In addition, LepB administration decreased the hepatic glycogen level through regulating the gene expressions of glycogen synthase and glycogen phosphorylase in mandarin fish until 4 d, while LepA did not change the hepatic glycogen level as it failed to change the expressions of these regulatory genes. Moreover, LepA and LepB downregulated the expressions of key gluconeogenic genes (phosphofructokinase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase), indicating both mandarin fish leptins could regulate the rate of glucose production. However, these two gene paralogues presented secondary effects on lipid metabolism as they only enhanced the triglyceride level by modifying the gene expressions of adipose triglyceride lipase or acetyl CoA carboxylase just for 1 d after IP. Therefore, LepB played an important role in food intake and glucose homeostasis regulation, while LepA showed a limited role in gluconeogenesis and lipid metabolism.

Changes of DNA Methylation Pattern in Metabolic Pathways Induced by High-Carbohydrate Diet Contribute to Hyperglycemia and Fat Deposition in Grass Carp (Ctenopharyngodon idellus).

Although studies have determined that epigenetics plays an essential role in regulating metabolism in mammals, research on nutrition-related DNA methylation remains to be lacking in teleosts. In the present study, we provided a hepatic whole-genome DNA methylation analysis in grass carp fed with moderate- or excessive-carbohydrate-level diet. Although a high-carbohydrate (HC) diet significantly changed the mRNA expression levels of metabolic genes, it did not affect the global genomic DNA methylation levels in grass carp liver. However, compared with the control group, 3,972 genes were hyper-methylated and 2,904 genes were hypo-methylated in the promoter region. Meanwhile, 10,711 genes were hyper-methylated and 6,764 genes were hypo-methylated in the gene body region in the HC group. These differentially methylated genes (DMGs) were enriched in multiple pathways, including carbohydrate metabolism, insulin pathway, lipid metabolism, and adipocytokine signaling pathway. In addition, the variations in DNA methylation significantly regulated the transcription levels of key genes of metabolism, which could affect the glucose concentrations and the lipid deposition of grass carp. Furthermore, we compared the DNA methylation alterations of genes in glucose metabolism and obesity pathways of grass carp with those of mammalian models in different nutritional states. The results showed that most of the DMGs in grass carp were also regulated by DNA methylation in mammals when the nutritional state changed. The findings revealed more differentially methylated regions and candidate genes for glucose metabolism and broken species boundaries.

Mandarin fish (Sinipercidae) genomes provide insights into innate predatory feeding.

Mandarin fishes (Sinipercidae) are piscivores that feed solely on live fry. Unlike higher vertebrates, teleosts exhibit feeding behavior driven mainly by genetic responses, with no modification by learning from parents. Mandarin fishes could serve as excellent model organisms for studying feeding behavior. We report a long-read, chromosomal-scale genome assembly for Siniperca chuatsi and genome assemblies for Siniperca kneri, Siniperca scherzeri and Coreoperca whiteheadi. Positive selection analysis revealed rapid adaptive evolution of genes related to predatory feeding/aggression, growth, pyloric caeca and euryhalinity. Very few gill rakers are observed in mandarin fishes; analogously, we found that zebrafish deficient in edar had a gill raker loss phenotype and a more predatory habit, with reduced intake of zooplankton but increased intake of prey fish. Higher expression of bmp4, which could inhibit edar expression and gill raker development through binding of a Xvent-1 site upstream of edar, may cause predatory feeding in Siniperca.

Expansion of sweet taste receptor genes in grass carp (Ctenopharyngodon idellus) coincided with vegetarian adaptation.

BACKGROUND: Taste is fundamental to diet selection in vertebrates. Genetic basis of sweet taste receptor in the shaping of food habits has been extensively studied in mammals and birds, but scarcely studied in fishes. Grass carp is an excellent model for studying vegetarian adaptation, as it exhibits food habit transition from carnivory to herbivory. RESULTS: We identified six sweet taste receptors (gcT1R2A-F) in grass carp. The four gcT1R2s (gcT1R2C-F) have been suggested to be evolved from and paralogous to the two original gcT1R2s (gcT1R2A and gcT1R2B). All gcT1R2s were expressed in taste organs and mediated glucose-, fructose- or arginine-induced intracellular calcium signaling, revealing they were functional. In addition, grass carp was performed to prefer fructose to glucose under a behavioral experiment. Parallelly, compared with gcT1R2A-F/gcT1R3 co-transfected cells, gcT1R2C-F/gcT1R3 co-transfected cells showed a higher response to plant-specific fructose. Moreover, food habit transition from carnivory to herbivory in grass carp was accompanied by increased gene expression of certain gcT1R2s. CONCLUSIONS: We suggested that the gene expansion of T1R2s in grass carp was an adaptive strategy to accommodate the change in food environment. Moreover, the selected gene expression of gcT1R2s might drive the food habit transition from carnivory to herbivory in grass carp. This study provided some evolutional and physiological clues for the formation of herbivory in grass carp.

Rapid Analysis of Monosaccharides in Sub-milligram Plant Samples Using Liquid Chromatography-Mass Spectrometry Assisted by Post-column Derivatization.

Monosaccharides play important roles in plant growth and development, and their biofunctions are closely related to their endogenous contents. Therefore, the determination of monosaccharides is beneficial for the further study of monosaccharide biofunction. In this work, we developed a liquid chromatography-mass spectrometry analytical method assisted by a post-column derivatization technique (LC-PCD-MS) for the fast and automatic determination of 16 monosaccharides in samples. Post-column chemical derivatization of monosaccharides was performed by a reaction of monosaccharides with 4-benzylaminobenzeneboronic acid (4-PAMBA) through boronate ester formation in a three-way connector. 4-PAMBA worked as a derivatization reagent to improve the selectivity and sensitivity of monosaccharide detection by MS. The developed LC-PCD-MS method integrates LC separation, chemical derivatization, and MS detection in one run, thus greatly reducing the analysis time for each sample. The limits of detection and limits of quantification for 16 monosaccharides were in the range of 0.002-0.1 and 0.007-0.5 ng/mL, respectively. Good linearity was obtained from the linear regression, with a determination coefficient (R2) ranging from 0.9928 to 1.0000. The relative recoveries were in the range of 80.7-117.8%, with the intra- and interday relative standard deviations less than 19.7 and 16.5%, respectively, indicating good accuracy and acceptable reproducibility of the method. Finally, the method was successfully applied to investigate the spatial and temporal distribution of 16 monosaccharides in the developing flower and germinating seed of Arabidopsis thaliana.

Two SLENDER AND CRINKLY LEAF dioxygenases play an essential role in rice shoot development.

It is clear that 2-oxoglutarate-dependent dioxygenases have critical functions in salicylic acid (SA) metabolism in plants, yet their role in SA biosynthesis is poorly understood. Here, we report that two dioxygenase-encoding genes, SLENDER AND CRINKLY LEAF1 (SLC1) and SLC2, play essential roles in shoot development and SA production in rice. Overexpression of SLC1 (SLC1-OE) or SLC2 (SLC2-OE) in rice produced infertile plants with slender and crinkly leaves. Disruption of SLC1 or SLC2 led to dwarf plants, while simultaneous down-regulation of SLC1 and SLC2 resulted in a severe defect in early leaf development. Enhanced SA levels in SLC1-OE plants and decreased SA levels in slc1 and slc2 mutants were observed. Accordingly, these lines all showed altered expression of a set of SA-related genes. We demonstrated that SLC1 interacts with homeobox1 (OSH1), and that either the knotted1-like homeobox (KNOX1) or glutamate, leucine, and lysine (ELK) domain of OSH1 is sufficient for accomplishing this interaction. Collectively, our data reveal the importance of SLC1 and SLC2 in rice shoot development.

Stable isotope labeling combined with liquid chromatography-tandem mass spectrometry for comprehensive analysis of short-chain fatty acids.

Short-chain fatty acids (SCFAs) are one class of bacterial metabolites mainly formed by gut microbiota from undigested fibers and proteins. These molecules are able to mediate signal conduction processes of cells, acting as G protein-coupled receptors (GPR) activators and histone deacetylases (HDAC) inhibitors. It was reported that SCFAs were closely associated with various human diseases. However, it is still challenging to analyze SCFAs because of their diverse structures and broad range of concentrations. In this study, we developed a highly sensitive method for simultaneous detection of 34 SCFAs by stable isotope labeling coupled with ultra-high performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS/MS) analysis. In this respect, a pair of isotope labeling reagents, N-(4-(aminomethyl)benzyl)aniline (4-AMBA) and N-(4-(aminomethyl)benzyl)aniline-d5 (4-AMBA-d5), were synthesized to label SCFAs from the feces of mice and SCFA standards, respectively. The 4-AMBA-d5 labeled SCFAs were used as internal standards to compensate the ionization variances resulting from matrix effect and thus minimize quantitation deviation in MS detection. After 4-AMBA labeling, the retention of SCFAs on the reversed-phase column increased and the separation resolution of isomers were improved. In addition, the MS responses of most SCFAs were enhanced by up to three orders of magnitude compared to unlabeled SCFAs. The limits of detection (LODs) of SCFAs were as low as 0.005 ng/mL. Moreover, good linearity for 34 SCFAs was obtained with the coefficient of determination (R2) ranging from 0.9846 to 0.9999 and the intra- and inter-day relative standard deviations (RSDs) were <17.8% and 15.4%, respectively, indicating the acceptable reproducibility of the developed method. Using the developed method, we successfully quantified 21 SCFAs from the feces of mice. Partial least squares discriminant analysis (PLS-DA) and t-test analysis showed that the contents of 9 SCFAs were significantly different between Alzheimer's disease (AD) and wide type (WT) mice fecal samples. Compared to WT mice, the contents of propionic acid, isobutyric acid, 3-hydroxybutyric acid, and 3-hydroxyisocaleric acid were decreased in AD mice, while lactic acid, 2-hydroxybutyric acid, 2-hydroxyisobutyric acid, levulinic acid, and valpronic acid were increased in AD mice. These significantly changed SCFAs in the feces of AD mice may afford to a better understanding of the pathogenesis of AD. Taken together, the developed UHPLC-ESI-MS/MS method could be applied for the sensitive and comprehensive determination of SCFAs from complex biological samples.

Simultaneous Determination of Multiclass Phytohormones in Submilligram Plant Samples by One-Pot Multifunctional Derivatization-Assisted Liquid Chromatography-Tandem Mass Spectrometry.

Phytohormones play crucial roles in every aspect of plant life, and their regulatory functions rely on complex crosstalk networks among the different classes of phytohormones in an antagonistic or synergistic manner. Therefore, the simultaneous determination of multiclass phytohormones is important for studies of phytohormone functions and networks. However, due to the heterogeneity of the sensitivity resulting from structural diversity and the low content in the plant samples, simultaneous determination of multiclass phytohormones is challenging, especially in very tiny plant tissues or organs. Here, we describe a novel method for the simultaneous determination of 31 phytohormones from different classes in a single run. This uses a one-pot multifunctional derivatization coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS). N, N-Diethyl ethylenediamine (DEED) and 2-methyl-4-phenylaminomethyl-benzeneboronic acid (2-methy-4-PAMBA) were used for derivatization of carboxylated phytohormones and brassinosteroids (BRs), respectively, to simultaneously improve detection sensitivities of carboxylated phytohormones and BRs. The method was fully validated for the 31 targeted phytohormones and could quantify multiclass endogenous phytohormones in small amounts of fresh plant samples (0.02-5 mg, FW). Finally, we used this method to investigate the spatial-temporal distribution of multiple phytohormones in reproductive organs of a single flower of Arabidopsis thaliana for the first time. This method could be a powerful auxiliary tool for studies of phytohormone functions and regulatory networks.

A new boronic acid reagent for the simultaneous determination of C27-, C28-, and C29-brassinosteroids in plant tissues by chemical labeling-assisted liquid chromatography-mass spectrometry.

Brassinosteroids (BRs) are endogenous plant growth-promoting hormones affecting growth and development during the entire life cycle of plants. Naturally occurring BRs can be classified into C27-, C28-, or C29-BRs based on the nature of the alkyl groups occupying the C-24 position in the side chain of the 5a-cholestane carbon skeleton. However, while C27-BRs exhibit similar bioactivities to C28- and C29-BRs, the biosynthetic pathways of C27-BRs in plants have not yet been clearly characterized. In addition to a lack of biochemical and enzymatic evidence regarding the biosynthetic pathways of C27-BRs, even most of the intermediate compounds on their pathways have not been explored and identified due to the lower endogenous levels of C27-BRs. Therefore, the development of highly sensitive analytical methods is essential for studying the biosynthetic pathways and physiological functions of C27-BRs. Accordingly, this study establishes qualitative and quantitative methods for identifying and detecting C27-, C28-, and C29-BRs using a newly synthesized boronic acid reagent denoted as 2-methyl-4-phenylaminomethylphenylboronic acid (2-methyl-4-PAMBA) in conjunction with liquid chromatography-mass spectrometry (LC-MS). Labeling with 2-methyl-4-PAMBA provides derivatives with excellent stability, and the detection sensitivities of BRs, particularly for C27-BRs, are dramatically improved. The limits of detection (with a signal-to-noise ratio of 3) for six BRs, including 2 C27-BRs (28-norCS and 28-norBL), 3 C28-BRs (CS, BL, and TY), and a single C29-BR (28-homoBL), are found to be 0.10-1.68 pg/mL after labeling with 2-methyl-4-PAMBA. Finally, the proposed analytical method is successfully applied for the detection of endogenous BRs in small mass samples of Oryza sativa seedlings, Rape flowers, Arabidopsis shoots, and Arabidopsis flowers. In addition, a method for profiling potential BRs in plants is also developed using LC-MS in multiple reaction monitoring scan mode assisted by 2-methyl-4-PAMBA and 2-methyl-4-PAMBA-d5 labeling. The developed method is able to identify 10 potential BRs in a Rape flower extract. The proposed quantitative and qualitative methods established by 2-methyl-4-PAMBA labeling are helpful for facilitating an understanding of the physiological functions and biosynthetic pathways of BRs, particularly for C27-BRs. Graphical abstract.

Metabolic analysis of the melatonin biosynthesis pathway using chemical labeling coupled with liquid chromatography-mass spectrometry.

Characterization of the melatonin (MLT) biosynthesis pathway in plants is still limited. Additionally, a metabolomic analysis of MLT biosynthesis in plants is still a challenge due to analyte structural and chemical diversity, low analyte abundances, and plant matrix complexities. Herein, a sensitive liquid chromatography-mass spectrometry (LC-MS) method enabling the simultaneous determination of seven plant MLT biosynthetic metabolites was developed. In the proposed strategy, the targeted metabolites, which included tryptophan (Trp), tryptamine (TAM), 5-hydroxytryptophan (5HTP), serotonin (5HT), N-acetylserotonin (NAS), 5-methoxytryptamine (5MT), and MLT, were purified from plant extracts using a one-step dispersive solid-phase extraction (DSPE). The samples were then chemically labeled with dansyl chloride (DNS-Cl), followed by analysis using LC-MS. The limit of detection (LOD) values ranged from 0.03 to 1.36 pg/mL and presented a 22- to 469-fold decrease when compared to the unlabeled metabolites. Due to the high sensitivity of the proposed method, the consumption of plant materials was reduced to 10 mg FW. Ultimately, the established method was utilized to examine the distributions of MLT and its intermediates in rice shoots and roots with or without cadmium (Cd) stress. The results suggested that under normal condition, MLT may also be generated via a Trp/TAM/5HT/5MT/MLT path (Pathway II) in addition to the previously reported Trp/TAM/5HT/NAS/MLT path (Pathway I), although Pathway I was shown to be dominant. During Cd stress, MLT was also shown to be produced through these two pathways, with Pathway II shown to be dominant in rice shoots and roots.

Profiling of potential brassinosteroids in different tissues of rape flower by stable isotope labeling - liquid chromatography/mass spectrometry analysis.

Brassinosteroids (BRs) play crucial roles in a variety of physiological processes in plants. The full elucidation of the functions of RBs relies on sensitive detection and accurate measurement of BRs in plants. However, the identification and quantification of BRs are challenging due to their low abundance as well as poor ionization efficiencies during mass spectrometry-based analysis. Herein, we developed a highly sensitive and selective strategy for profiling potential BRs in plants by stable isotope labeling liquid chromatography multiple reaction monitoring scan mass spectrometry (SIL-LC-MRM-MS) analysis. In the strategy, we used a pair of stable isotope labeling reagents 4-phenylaminomethyl-benzeneboronic acid (4-PAMBA) and d5-4-phenylaminomethyl-benzeneboronic acid (4-PAMBA-d5) that can react with C22-C23 cis-diol on BRs for profiling potential BRs in plant tissues. The 4-PAMBA and 4-PAMBA-d5 labeled BRs could generate two characteristic neutral loss under collision induced dissociation (CID), respectively, which is used to establish the MRM-based detection and screening. The precursor ions of BRs labeled with 4-PAMBA and 4-PAMBA-d5 were set according to the reported structures of BRs, and the corresponding product ions were predicted by subtracting the lost neutral loss. In this respect, corresponding precursor ions and product ions in MRM transitions are formed. The peak pairs with a fixed mass difference, similar retention times and intensities were assigned as potential BRs. Using the developed SIL-LC-MRM-MS strategy, we successfully found 13 potential BR in different tissues of rape flower. Taken together, the SIL-LC-MRM-MS analytical strategy is promising for profiling potential BRs as well as other compounds that have the same functional moiety from complex biological samples.

Two isostructural Ln-MOFs showing luminescent sensing (Eu) and slow magnetic relaxation (Dy) properties.

Two isostructural lanthanide-based MOFs [Ln(HL)L]·H2O (1-Ln, Ln = Eu, Dy, H2L = 5-((pyridin-4-ylthio)methyl)isophthalic acid) were successfully obtained via the solvothermal reaction. 1-Eu exhibits high fluorescence quenching efficiency for C2O72- and Fe3+, which can be potentially used as a luminescent probe; 1-Dy behaves as a typical single-molecule magnet with an energy barrier (ΔUeff) of 54(2) K.

Spatio-temporal profiling of abscisic acid, indoleacetic acid and jasmonic acid in single rice seed during seed germination.

Abscisic acid (ABA), indoleacetic acid (IAA) and jasmonic acid (JA) are plant hormones that were reported to play indispensable roles during seed germination. However, the interactions between these plant hormones during rice seed germination have still not been explored clearly. A sensitive method for determination of these plant hormones would be beneficial for the exploration of such interactions. Herein, we present a liquid chromatography coupled with mass spectrometry (LC-MS) method for the quantification of ABA, IAA and JA in a single tissue of rice seed to investigate the spatio-temporal distribution of these plant hormones during rice seed germination. To this end, an in silico strategy was developed in order to select a derivatization reagent with an ideal sensitivity of MS detection. This strategy was confirmed with experimental studies on three reagents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), N,N-dimethylethylenediamine (DMED), and N-(acridin-9-yl)-2-bromoacetamide (AYBA) and their formic acid derivatives. Our results from the in silico and LC-MS experiments show that AYBA is a good derivatization reagent for ABA, IAA and JA due to its reasonable ionization efficiency in electrospray ionization mass spectrometry (ESI-MS) and excellent hydrophobicity. Finally, a sensitive LC-MS method upon AYBA was established for the determination of ABA, IAA and JA in germinated seeds. Good linearities for ABA, IAA, and JA were obtained with correlation coefficients greater than 0.99. The limits of detection (LODs) were in the range of 0.14-0.16 pg mL-1. The method exhibits good precisions with RSD 1.5%-13.8% (intra-day) and 1.2%-7.3% (inter-day) and acceptable recoveries (88.6%-102.9%, n = 6). Finally, the method was successfully employed in the spatio-temporal profiling of ABA, IAA and JA in a single tissue of rice seed during rice seed germination.

Macular integrity assessment to determine the association between macular microstructure and functional parameters in diabetic macular edema.

AIM: To respectively evaluate macular morphological features and functional parameters by using spectral-domain optical coherence tomography (SD-OCT) and macular integrity assessment (MAIA) in patients with diabetic macular edema (DME). METHODS: This prospective, non-controlled, open study included 61 eyes of 38 consecutive patients with DME. All patients underwent best-corrected visual acuity (BCVA) measurement, MAIA microperimetry, and SD-OCT. DME morphology, including central retinal thickness (CRT) and central retinal volume (CRV); integrity of the external limiting membrane (ELM) and photoreceptor inner segment/outer segment (IS/OS) junction; and the deposition of hard macular exudates were assessed within a 1000-µm central subfield area. MAIA microperimetry parameters evaluated were average threshold (AT)-retinal sensitivity, macular integrity index (MI), fixation points within a circle of radius 1° (P1) and 2° (P2), and bivariate contour ellipse area considering 63% and 95% of the fixation points (A63 and A95, respectively). RESULTS: MI was significantly higher in eyes with disrupted ELM or IS/OS, compared with eyes with intact ELM and IS/OS. Values of BCVA (logMAR), total AT, AT within 1000-µm diameter, P2, A63, A95, and CRT were significantly worse in eyes with disrupted IS/OS, compared with eyes with intact IS/OS. The values of BCVA (logMAR), AT within 1000-µm diameter, and CRT were significantly worse in eyes with disrupted ELM, compared with eyes with intact ELM. These parameters were not significantly different between eyes with or without hard macular exudate deposition. CRV was not significantly different in the presence or absence of the integrity of ELM, IS/OS, or deposition of hard macular exudates. At the center, nasal and temporal sectors of the fovea, significant negative correlations were observed between retinal thickness and AT of the corresponding area. At the inferior and superior sectors of the fovea, no correlations were observed between retinal thickness and AT of the corresponding area. In the intact IS/OS group, significant negative correlations were observed between CRT and central AT. There was no correlation between retinal sensitivity and thickness when the IS/OS layer was disrupted. Multiple linear regression analyses revealed that IS/OS integrity was an independent factor affecting MI. CONCLUSION: Functional (BCVA and visual field) and morphological parameters (retinal thickness) were significantly associated with an intact IS/OS. Local photoreceptor integrity was a strong predictor of local visual function throughout the retina. MI revealed the functional status in DME, reflecting the IS/OS juction status in the macula.

Comprehensive Profiling of Phytohormones in Honey by Sequential Liquid-Liquid Extraction Coupled with Liquid Chromatography-Mass Spectrometry.

Honey exhibits various nutritional and medicinal functions, which are highly related to the active components; thus, the exploration of new compounds in honey is of great importance. Because honey is a byproduct of flower nectar, which is rich in phytohormones, the existence of phytohormones in honey is anticipated. In this research, a method for comprehensive profiling of 49 phytohormones in honey was developed by sequential liquid-liquid extraction (LLE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Good linearities for 49 phytohormones were obtained with correlation coefficients (R) larger than 0.9913. The limits of detection (LODs) were in the range of 0.2-628.2 pg/mL. Satisfied reproducibility and reliability were achieved by evaluation of the intra- and interday precisions with relative standard deviations (RSDs) less than 15.8% and relative recoveries ranging from 80.4 to 123.7%. The method was further applied to analyze the phytohormones in 14 monofloral raw honey samples and 3 commercial honey samples. The existence of 34 phytohormones was confirmed, including 14 cytokinins (CKs), 8 gibberellins (GAs), 5 brassinosteroids (BRs), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), jasmonoyl-leucine (JA-Leu), and jasmonoyl-phenylalanine (JA-Phe). In addition, the content and species of phytohormones varies in different kinds of honey. The study is beneficial to fully illustrate the phytohormone profile of honey and contributive to elucidate the mechanism of its nutritional and medicinal functions.

A rapid approach to investigate spatiotemporal distribution of phytohormones in rice.

BACKGROUND: Phytohormones play crucial roles in almost all stages of plant growth and development. Accurate and simultaneous determination of multiple phytohormones enabled us to better understand the physiological functions and the regulatory networks of phytohormones. However, simultaneous determination of multiple phytohormones in plant is still a challenge due to their low concentrations, structural and chemical diversity, and complex matrix of plant tissues. Therefore, development of a simple and selective method for the simultaneous determination of multiple phytohormones is highly needed. RESULTS: We developed a clean-up strategy for profiling of multiple phytohormones, which can overcome the challenge of structural and chemical diversity. By using a one-step dispersive solid-phase extraction (DSPE) combined with UPLC-MS/MS, 54 phytohormones including auxins, ABA, SA, JA, GAs and CKs were simultaneously analyzed from a single rice sample extract. Using the developed method, we investigated the spatiotemporal distribution of phytohormones in rice. The profiling of various tissues of rice at different growth stages revealed the complexity of metabolic regulation and allocations of phytohormone species. CONCLUSION: A rapid one-step method was developed for the simultaneous analysis of six groups of phytohormones, including cytokinins, auxins, salicylic acid, jasmonates, abscisic acid and gibberellins in a single run, using UPLC-ESI-MS/MS. The proposed method was successfully applied to investigate spatiotemporal distribution of multiple phytohormones in rice. The spatiotemporal information obtained may be helpful for better understanding of phytohormones functions throughout life cycle of rice when integrated into transcriptome and other omics data.

Molecular cloning, expression and single nucleotide polymorphisms of protein phosphatase 1 (PP1) in mandarin fish (Siniperca chuatsi).

In the wild, mandarin fish (Siniperca chuatsi) only feed on live prey fish, refusing dead prey. When reared in ponds, training will result in some mandarin fish accepting artificial diets. However, little is currently known about the molecular mechanism of the individual difference. Serine/threonine protein phosphatase 1 (PP1) is a suppressor of learning and long-term memory (LTM) in mammals. In the present study, the relationship between PP1 and the individual difference in acceptance of artificial diets in mandarin fish was investigated. The complete CDS (coding sequence) of four PP1 isoforms (PP1caa, PP1cab, PP1cb and PP1cc) were cloned in mandarin fish. The amino acid sequences of these PP1 isoforms are highly conserved in different species. The mRNA expressions of PP1caa and PP1cb in brain of artificial diet feeders were significantly higher than those in nonfeeders, suggesting the deficiency in the maintenance of long-term memory of its natural food habit (live prey fish). The SNP loci in PP1caa and PP1cb were also found to be associated with the individual difference in acceptance of artificial diets in mandarin fish. These SNPs of PP1caa and PP1cb genes could be useful markers for gene-associated breeding of mandarin fish, which could accept artificial diets. In conclusion, different mRNA expression and SNPs of PP1caa and PP1cb genes in feeders and nonfeeders of artificial diets might contribute to understanding the molecular mechanism of individual difference in acceptance of artificial diets in mandarin fish.