Mass spectrometry detection of organophosphorus pesticide adducts on butyrylcholinesterase and albumin.

This study established a method to prepare and detect OPs adducts on butyrylcholinesterase (BChE) and human serum albumin (HSA). OPs (methyl paraoxon, ethyl paraoxon, methyl parathion, parathion) were incubated with BChE or HSA in vitro, and the adducts of OPs-BChE or OPs-HSA were prepared and qualitatively analyzed by ultra-performance liquid chromatography data-dependent high-resolution tandem mass spectrometry (UPLC-ddHRMS/MS). The amounts of BChE and HSA in the incubating systems were varied and the resulting amounts of the adducts were determined using linear regression. OPs-BChE in the blood were isolated by immunomagnetic separation (IMS), and then digested into the OPs-nonapeptide adduct by pepsin. The proteins in the remaining blood plasma were precipitated and digested by pronase to OPs-tyrosines(OPs-Tyr), which were quantified by UPLC-ddHRMS/MS. 4 OPs-nonapeptides and 4 OPs-Tyr adducts were obtained through the process above. The relative mass deviation of incubated adducts between the actual and theoretical exact masses was less than 10 ppm, and further confirmed by fragmentation mass spectra analysis. Calibration curves were linear for all adducts with a coefficient of determination value (R2) ≥0.995. The limits of detection (LOD) and limits of quantification (LOQ) for adducts detected by MS ranged from 0.05 to 1.0 ng/mL, and from 0.1 to 2.0 ng/mL, respectively. The recovery percentages for adducts ranged from 76.1 % to 107.1 %, matrix effects ranged from 83.4 % to 102.1 %. The inter-day and intra-day precision were 6.1-10.1 % and 6.9-12.9 % for adducts. This study provides a new reference method for the detection of organophosphorus pesticide poisoning. In addition, two blood samples with organophosphorus poisoning were tested by the designed method, and the corresponding adducts were detected in both samples.

Liquid chromatography with tandem mass spectrometric method for determination of 52 antibiotics in human whole blood and urine and application to forensic cases.

PURPOSE: A rapid and reliable method was developed and validated for the simultaneous analysis of 52 antibiotics (cephalosporins, penicillins, carbapenems, lincosamides, quinolones, nitroimidazoles, macrolides, sulfonamides, tetracyclines, glycopeptide) in urine and whole blood by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). METHOD: Analytes were extracted by dilution or protein precipitation and analyzed on an Agilent 1260 HPLC system coupled to an Agilent 6470 Triple Quadrupole Mass Spectrometer. RESULTS: The method attended method validation criteria. The limits of detection were equal or lower than 2.0 ng/mL, whereas the limits of quantification ranged from 0.1 to 10.0 ng/mL, from 0.1 to 5.0 ng/mL, in urine and whole blood, respectively. For all analytes, the bias and intra- and inter-day precision values were less than 14.7%. The ranges of recovery values of all antibiotics were 76.5-124.5% in whole blood and 76.3-121.8% in urine, values of the effects were lower than 25% in two matrices. No evidence of carryover was observed. The study of sample stability showed that almost all analytes were stable at 24 °C for 24 h, all analytes were stable at -20 °C for 14 days and at -80 °C for 30 days. Freeze-thaw cycles stability showed that antibiotics were stable except for imipenem. Autosampler stability study showed that all analytes were stable for 24 h, except for imipenem and amoxicillin. Applicability was proven by analyzing authentic whole blood (n = 86) and urine (n = 79) samples from patients under antibiotics treatment. Therefore, this method was applied to the analysis 3 forensic allergy cases, which were positive for at least one analyte. CONCLUSIONS: A simple, sensitive and high-throughput method for the simultaneous determination of different classes of antibiotics in urine and whole blood samples was developed and applied. This sensitive method was successfully applied to forensic cases.

Rapid Screening of 34 Emerging Contaminants in Surface Water by UHPLC-Q-TOF-MS.

OBJECTIVES: To establish a rapid screening method for 34 emerging contaminants in surface water by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF-MS). METHODS: The pretreatment conditions of solid phase extraction (SPE) were optimized by orthogonal experimental design and the surface water samples were concentrated and extracted by Oasis® HLB and Oasis® MCX SPE columns in series. The extracts were separated by Kinetex® EVO C18 column, with gradient elution of 0.1% formic acid aqueous solution and 0.1% formic acid methanol solution. Q-TOF-MS 'fullscan' and 'targeted MS/MS' modes were used to detect 34 emerging contaminants and to establish a database with 34 emerging contaminants precursor ion, product ion and retention times. RESULTS: The 34 emerging contaminants exhibited good linearity in the concentration range respectively and the correlation coefficients (r) were higher than 0.97. The limit of detection was 0.2-10 ng/L and the recoveries were 81.2%-119.2%. The intra-day precision was 0.78%-18.70%. The method was applied to analyze multiple surface water samples and 6 emerging contaminants were detected, with a concentration range of 1.93-157.71 ng/L. CONCLUSIONS: The method is simple and rapid for screening various emerging contaminants at the trace level in surface water.

Toxicokinetics of MDMA and Its Metabolite MDA in Rats.

OBJECTIVES: To investigate the toxicokinetic differences of 3,4-methylenedioxy-N-methylamphetamine (MDMA) and its metabolite 4,5-methylene dioxy amphetamine (MDA) in rats after single and continuous administration of MDMA, providing reference data for the forensic identification of MDMA. METHODS: A total of 24 rats in the single administration group were randomly divided into 5, 10 and 20 mg/kg experimental groups and the control group, with 6 rats in each group. The experimental group was given intraperitoneal injection of MDMA, and the control group was given intraperitoneal injection of the same volume of normal saline as the experimental group. The amount of 0.5 mL blood was collected from the medial canthus 5 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h after administration. In the continuous administration group, 24 rats were randomly divided into the experimental group (18 rats) and the control group (6 rats). The experimental group was given MDMA 7 d by continuous intraperitoneal injection in increments of 5, 7, 9, 11, 13, 15, 17 mg/kg per day, respectively, while the control group was given the same volume of normal saline as the experimental group by intraperitoneal injection. On the eighth day, the experimental rats were randomly divided into 5, 10 and 20 mg/kg dose groups, with 6 rats in each group. MDMA was injected intraperitoneally, and the control group was injected intraperitoneally with the same volume of normal saline as the experimental group. On the eighth day, 0.5 mL of blood was taken from the medial canthus 5 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h after administration. Liquid chromatography-triple quadrupole tandem mass spectrometry was used to detect MDMA and MDA levels, and statistical software was employed for data analysis. RESULTS: In the single-administration group, peak concentrations of MDMA and MDA were reached at 5 min and 1 h after administration, respectively, with the largest detection time limit of 12 h. In the continuous administration group, peak concentrations were reached at 30 min and 1.5 h after administration, respectively, with the largest detection time limit of 10 h. Nonlinear fitting equations for the concentration ratio of MDMA and MDA in plasma and administration time in the single-administration group and continuous administration group were as follows: T=10.362C-1.183, R2=0.974 6; T=7.397 3C-0.694, R2=0.961 5 (T: injection time; C: concentration ratio of MDMA to MDA in plasma). CONCLUSIONS: The toxicokinetic data of MDMA and its metabolite MDA in rats, obtained through single and continuous administration, including peak concentration, peak time, detection time limit, and the relationship between concentration ratio and administration time, provide a theoretical and data foundation for relevant forensic identification.

Melatonin attenuates fentanyl - induced behavioral sensitization and circadian rhythm disorders in mice.

Melatonin is a neurohormone synthesized by the pineal gland to regulate the circadian rhythms and has proven to be effective in treating drug addiction and dependence. However, the effects of melatonin to modulate the drug-seeking behavior of fentanyl and its underlying molecular mechanism is elusive. This study was designed to investigate the effects of melatonin on fentanyl - induced behavioral sensitization and circadian rhythm disorders in mice. The accompanying changes in the expression of Brain and Muscle Arnt-Like (BMAL1), tyrosine hydroxylase (TH), and monoamine oxidase A (MAO-A) in relevant brain regions including the suprachiasmatic nucleus (SCN), nucleus accumbens (NAc), prefrontal cortex (PFC), and hippocampus (Hip) were investigated by western blot assays to dissect the mechanism by which melatonin modulates fentanyl - induced behavioral sensitization and circadian rhythm disorders. The present study suggest that fentanyl (0.05, 0.1 and 0.2 mg/kg) could induce behavioral sensitization and melatonin (30.0 mg/kg) could attenuate the behavioral sensitization and circadian rhythm disorders in mice. Fentanyl treatment reduced the expression of BMAL1 and MAO-A and increased that of TH in relevant brain regions. Furthermore, melatonin treatment could reverse the expression levels of BMAL1, MAO-A, and TH. In conclusion, our study demonstrate for the first time that melatonin has therapeutic potential for fentanyl addiction.

Stability of diazepam's phase II metabolites in dried blood spots on filter paper.

Phase II metabolites play an important role in diazepam-related cases. The study aimed to assess the stability of diazepam's phase II metabolites in dried blood spots on filter paper. METHODS: A piece of filter paper was spotted with 100 µL of whole blood (added 1% sodium fluoride as needed) obtained from participant who received 5 mg diazepam orally, air dried for 2 h at room temperature, and then stored at different conditions. Whole spots were cut at 0.1 cm from the outer edge of blood spots at post-consumption time-points of prior (zero), 5, 16, 35, 61, 120 days and 1, 1.5 years. Analytes were extracted with methanol/water mixture (8:2, v/v) and determined using HPLC-MS/MS. Decomposition rules were analyzed by a statistical software "SPSS". RESULTS: Temazepam glucuronide remained stable (0.5-18.6% loss) at 20 â„ƒ and at 20 â„ƒ with 1% sodium fluoride for 16 days, while it was unstable after 5 days at 4 â„ƒ (21.1-26.2% loss) and - 20 â„ƒ (28.9 - 34.4% loss). After 35 days, temazepam glucuronide concentrations began to fluctuate significantly under all conditions, and an obvious increase (290.4-355.1%) was observed in 1.5 years. Oxazepam glucuronide was always unstable after 5 days, the percentage loss was even 100% when it was stored for 61 days and 1.5 years. CONCLUSIONS: Dried blood spots on ordinary filter paper are recommended to be stored at 20 â„ƒ or 20 â„ƒ with 1% sodium fluoride within 16 days. Samples should be analyzed immediately or stored in sterile and dry media.

Forensic toxicokinetics of difenidol hydrochloride in vivo verified by practical lethal cases.

A gas chromatography-mass spectrometry (GC-MS) method for the determination of difenidol hydrochloride in biological specimens has been developed. The method exhibited excellent recovery (> 90%) and precision (RSD < 10%), and the LOD was 0.05 µg/mL or µg/g, which met the requirements of bioanalytical method. Through the animal model of the forensic toxicokinetics, the dynamic distribution, postmortem redistribution (PMR) and stability in specimen preservation process of difenidol in animals were studied. The experimental results showed that after intragastric administration, the difenidol's concentrations in the heart-blood and various organs increased over time except stomach, and then decreased gradually after reaching the peaks of concentration. The toxicological kinetics equation and toxicokinetic parameters were established by processing the data of the mean drug concentration of difenidol changing with time. In PMR experiment, the concentrations of difenidol in some organs closer to the gastrointestinal tract (heart-blood, heart, liver, lung, kidney, and spleen) changed significantly at different time points. But the concentration of difenidol in brain tissues which were far away from the gastrointestinal tract and muscles with larger overall mass was relatively stable. PMR of difenidol was therefore confirmed. Thus, the effect of PMR on the concentration of difenidol in the specimens should be considered in cases involving difenidol poisoning or death. Furthermore, the stability of difenidol in heart-blood samples from poisoned rats was investigated at various time points and under different preservation conditions (20 °C, 4 °C, - 20 °C and 20 °C (1% NaF)) for a period of two months. Difenidol was stable and did not decompose in the preserved blood. Therefore, this study provided the experimental basis for the forensic identification of the cases of difenidol hydrochloride poisoning (death). PMR has been verified by practical lethal cases.

Effects of Different Nitrogen Forms on Blackberry Fruit Quality.

To study the optimal form of nitrogen (N) application and to determine the best harvest date for blackberries, different N fertilizers were applied during the critical growth period of blackberry plants. The results showed that NH4+-N significantly improved the appearance of blackberry fruits, including their size, firmness, and color, and promoted the accumulation of soluble solids, sugars, anthocyanin, ellagic acid, and vitamin C (VC), while fruit treated with NO3--N accumulated more flavonoids and organic acids and had improved antioxidant capacity. In addition, the fruit size, firmness, and color brightness decreased with the harvest period. While the contents of sugars, anthocyanin, ellagic acid, flavonoids, and VC were higher in the early harvests and then decreased as the season progressed, the total antioxidant capacity and DPPH radical scavenging capacity increased. In all, application of NH4+-N is recommended, as it is more beneficial to fruit appearance, taste, and nutritional quality. Harvests in the early stage help to obtain a good fruit appearance, while harvests in the middle and later stages are more beneficial to fruit taste and quality. This study may help growers to determine the best fertilization scheme for blackberries and choose the appropriate harvest time according to their needs.

Exploration and Practice of the "One Combination, Two Highlights, Three Combinations, Four in One" Innovative Talents Training Mode in Forensic Medicine.

Talent is one of the basic and strategic supports for building a modern socialist country in all aspects. Since the 1980s, the establishment of forensic medicine major and the cultivation of innovative talents in forensic medicine have become hot topics in higher education in forensic medicine. Over the past 43 years, the forensic medicine team of Shanxi Medical University has adhered to the joint education of public security and colleges, and made collaborative innovation, forming a training mode of "One Combination, Two Highlights, Three Combinations, Four in One" for innovative talents in forensic medicine. It has carried out "5+3/X" integrated reform, and formed a relatively complete talent training innovation mode and management system in teaching, scientific research, identification, major, discipline, team, platform and cultural construction. It has made a historic contribution to China's higher forensic education, accumulated valuable experience for the construction of first-class major and first-class discipline of forensic medicine, and provided strong support for the construction of the national new forensic talent training system. The popularization of this training mode is conducive to the rapid and sustainable development of forensic science, and provides more excellent forensic talents for national building, regional social development and the discipline construction of forensic science.

Effects of Different Light Wavelengths on Fruit Quality and Gene Expression of Anthocyanin Biosynthesis in Blueberry (Vaccinium corymbosm).

Different light wavelengths display diverse effects on fruit quality formation and anthocyanin biosynthesis. Blueberry is a kind of fruit rich in anthocyanin with important economic and nutritional values. This study explored the effects of different light wavelengths (white (W), red (R), blue (B) and yellow (Y)) on fruit quality and gene expression of anthocyanin biosynthesis in blueberry. We found that the B and W treatments attained the maximum values of fruit width, fruit height and fruit weight in blueberry fruits. The R treatment attained the maximum activities of superoxide dismutase (SOD) and peroxidase (POD), and the Y treatment displayed the maximum contents of ascorbic acid (AsA), glutathione (GSH) and total phenol in fruits, thus improving blueberry-fruit antioxidant capacity. Interestingly, there were differences in the solidity-acid ratio of fruit under different light-wavelength treatments. Moreover, blue light could significantly improve the expression levels of anthocyanin biosynthesis genes and anthocyanin content in fruits. Correlation and principal component analysis showed that total acid content and antioxidant enzymes were significantly negatively correlated with anthocyanin content in blueberry fruits. These results provide new insights for the application of light wavelength to improve blueberry fruit quality and anthocyanin content.

Physiological and Morphological Responses of Blackberry Seedlings to Different Nitrogen Forms.

Blackberries are an emerging third-generation fruit that are popular in Europe, and specific nitrogen (N) supply is an important factor affecting their growth and development. To study the optimal N fertilizer for blackberry seedlings, no N (CK), nitrate (NO3-)-N, ammonium (NH4+)-N and urea were applied to one-year-old 'Ningzhi 4' blackberry plants at a key growth period (from May to August) to explore the effects of different N forms on the physiological characteristics. Correlation and principal component analysis were used to determine the relationships between various indexes. Ammonium (NH4+) or urea-fed plants had a better growth state, showed a greater plant height, biomass, SPAD values and enhanced antioxidant enzyme activities and photosynthesis. In addition, NH4+ was beneficial to the accumulation of sugars and amino acids in leaves and roots, and promoted the transport of auxin and cytokinin to leaves. NO3- significantly inhibited root growth and increased the contents of active oxygen, malondialdehyde and antioxidants in roots. Correlation and principal component analysis showed that growth and dry matter accumulation were closely related to the antioxidant system, photosynthetic characteristics, amino acids and hormone content. Our study provides a new idea for N regulation mechanism of blackberry and proposes a scientific fertilization strategy.

Correlation between the Human Development Index and the Incidence and Mortality of Non-Hodgkin Lymphoma.

OBJECTIVE: This study was to examine the relationship between socioeconomic status and the incidence and mortality of non-Hodgkin lymphoma (NHL). METHODS: We compared the age-standardized incidence rate (ASIR), age-standardized mortality rate (ASMR), and the ASMR to ASIR ratio (MIR) at national and regional levels and studied the correlation between the MIR and the human development index (HDI) in 2012 and 2018. RESULTS: The highest ASIR was in North America in 2012 and in Australia in 2018, and the lowest ASIR was in Central and South Asia in both 2012 and 2018. The highest ASMR was in North Africa in both 2012 and 2018, and the lowest ASMR was in Eastern Asia and South-Central Asia in 2012 and in South-Central Asia in 2018. The lowest MIR was in Australia in both 2012 and 2018, and the highest MIR was in Western Africa in both 2012 and 2018. HDI was strongly negatively correlated with MIR (r: -0.8810, P<0.0001, 2012; r: -0.8895, P<0.0001, 2018). Compared to the 2012 data, the MIR in the intermediate HDI countries significantly deceased and the HDI in low and high HDI countries significantly increased in 2018. CONCLUSION: The MIR is negatively correlated with HDI. Increasing the HDI in low and intermediate HDI countries may reduce the MIR and increase the survival of patients with NHL.

A Target-Triggered Emission Enhancement Strategy Based on a Y-Shaped DNA Fluorescent Nanoprobe with Aggregation-Induced Emission Characteristic for microRNA Imaging in Living Cells.

DNA self-assembled fluorescent nanoprobes have been developed for bio-imaging owing to their high resistance to enzyme degradation and great cellular uptake capacity. In this work, we designed a new Y-shaped DNA fluorescent nanoprobe (YFNP) with aggregation-induced emission (AIE) characteristic for microRNA imaging in living cells. With the modification of the AIE dye, the constructed YFNP had a relatively low background fluorescence. However, the YFNP could emit a strong fluorescence due to the generation of microRNA-triggered AIE effect in the presence of target microRNA. Based on the proposed target-triggered emission enhancement strategy, microRNA-21 was detected sensitively and specifically with a detection limit of 122.8 pM. The designed YFNP showed higher bio-stability and cell uptake than the single-stranded DNA fluorescent probe, which has been successfully applied for microRNA imaging in living cells. More importantly, the microRNA-triggered dendrimer structure could be formed after the recognition of target microRNA, achieving a reliable microRNA imaging with a high spatiotemporal resolution. We expect that the proposed YFNP will become a promising candidate for bio-sensing and bio-imaging.

HPLC-MS/MS determination and the postmortem distribution or postmortem redistribution of paraquat and its metabolites in four fatal intoxication cases.

HPLC-MS/MS analysis and postmortem distribution or postmortem redistribution of paraquat and its two metabolites in poisoning death cases were reported. Paraquat, monoquat, and paraquat monopyridone were extracted from the sample with acetonitrile or methanol, respectively, detected by ZORBAX HILIC Plus (4.6 × 100 mm, 3.5 µm) chromatographic column, with 0.1 % formic acid aqueous solution - 0.1 % formic acid acetonitrile solution (v/v) as mobile phase. Paraquat, monoquat, and paraquat monopyridone had a good linear relationship within the range of 10-1000, 1-400, and 1-1000 ng/mL (or g), the correlation coefficient (r) were all ≥ 0.9996. Their detection limits were lower than 1 ng/mL (or g). The detection accuracy was 91.25∼113.44 %. The intra-day and inter-day precision were 1.51-3.99 % and 1.92-4.93 %, respectively. This method was used to detect and analyze four rare paraquat poisoning cases. The distribution of paraquat, monoquat, and paraquat monopyridone is uneven, which is relatively high in the heart, blood, lung, and kidney. Heart blood/Peripheral blood ratio of paraquat, monoquat, paraquat monopyridone concentration in two poisoned cases were 1.4, 2.0, 1.5 and 1.9, 1.3, 1.2, which showed a location dependent postmortem redistribution. This is the first time that HPLC-MS/MS and the postmortem distribution or postmortem redistribution of paraquat metabolites in poisoned death cases have been reported. This research provides scientific basis for forensic identification of paraquat poisoning cases and extraction of biological specimen.

Recent advances in metabolic engineering of microorganisms for the production of monomeric C3 and C4 chemical compounds.

Bio-based C3 and C4 bi-functional chemicals are useful monomers in biopolymer production. This review describes recent progresses in the biosynthesis of four such monomers as a hydroxy-carboxylic acid (3-hydroxypropionic acid), a dicarboxylic acid (succinic acid), and two diols (1,3-propanediol and 1,4-butanediol). The use of cheap carbon sources and the development of strains and processes for better product titer, rate and yield are presented. Challenges and future perspectives for (more) economical commercial production of these chemicals are also briefly discussed.

Simulating dynamic interaction between diazepam and ethanol targeting the GABAA receptor via in silico model.

Combined diazepam-ethanol poisoning is common in forensic toxicology. Both diazepam and ethanol can inhibit the central nervous system via the γ-aminobutyric acid (GABA) ligand gated chloride ion channel, GABAA Receptor (GABAAR). As the common target of diazepam and ethanol, whether GABAAR is the key target of their combined action remains unclear. This study aimed to explore their interaction based on the synergistic mechanisms between diazepam and ethanol targeting the GABAAR. Four models were built in silico based on the crystal structure of GABAAR. Molecular dynamic processes of the four models were simulated by the GPU-accelerated pmemd.cuda program in the Amber18 package. Results showed that ethanol inclined to combine the adjacent GABA or diazepam sites, minimized fluctuations of the root-mean-square deviation (RMSD) in the molecular dynamic process of GABA or diazepam binding the GABAAR, and increased the release of binding energy of GABA or diazepam binding the GABAAR. Results also showed that diazepam had less effect on the RMSD fluctuation or the binding energy release of GABA binding GABAAR. The formation of complex of diazepam and GABAAR could minimize the RMSD fluctuation and increase binding energy release of ethanol binding GABAAR. Thus, ethanol, bridging GABA and diazepam, could strengthen the complex of GABA binding the GABAAR, as well as the complex of diazepam binding the GABAAR. However, whether diazepam binds GABAAR or not, it cannot affect GABA binding the GABAAR; and yet the complex of diazepam and GABAAR can stabilize the complex of ethanol and GABAAR.

A comparative study of postmortem distribution and postmortem diffusion of tramadol in rabbits.

In recent years, the cases of tramadol intoxication have become more frequent in many countries. However, most of the previous studies have been based on cases of tramadol intoxication, and the detailed information on the differences between postmortem distribution and diffusion of tramadol remains unclear. To investigate this issue systematically, we established a postmortem distribution model and two postmortem diffusion models. Then, gas chromatography-mass spectrometry (GC/MS) was used to measure the concentrations of tramadol in various biological specimens of fluids and tissues. In postmortem distribution, the results showed an uneven distribution of tramadol in various biological specimens, and the concentrations of tramadol in urine were significantly higher than those in other fluids. In postmortem diffusion, the results showed a dosage-dependent increase of tramadol concentration in most specimens; at all time points from 0.25 to 6 h after postmortem administration, the concentrations of tramadol in fluids were not significantly different from those in tissues, and the concentrations of tramadol in urine were lower than those in both tissues and other fluids in most time points. We recommend a quantitative examination of the specimens of both fluids and tissues to provide more evidence for the forensic identification, and the realization that there is a correlation between the concentrations of fluids and tissues is important for determining antemortem and postmortem administration of tramadol. This information can serve as ancillary data in inferring the contribution of a drug to death in cases of suspected tramadol poisoning.

Transcriptomic and Metabolomic Profiling Reveals the Variations in Carbohydrate Metabolism between Two Blueberry Cultivars.

Blueberry is a high-quality fruit tree with significant nutritional and economic value, but the intricate mechanism of sugar accumulation in its fruit remains unclear. In this study, the ripe fruits of blueberry cultivars 'Anna' and 'Misty' were utilized as experimental materials, and physiological and multi-omics methodologies were applied to analyze the regulatory mechanisms of the difference in sugar content between them. The results demonstrated that the 'Anna' fruit was smaller and had less hardness than the 'Misty' fruit, as well as higher sugar content, antioxidant capability, and lower active substance content. A total of 7067 differentially expressed genes (DEGs) (3674 up-regulated and 3393 down-regulated) and 140 differentially abundant metabolites (DAMs) (82 up-regulated and 58 down-regulated) were identified between the fruits of the two cultivars. According to KEGG analysis, DEGs were primarily abundant in phenylpropanoid synthesis and hormone signal transduction pathways, whereas DAMs were primarily enriched in ascorbate and aldarate metabolism, phenylpropanoid biosynthesis, and the pentose phosphate pathway. A combined multi-omics study showed that 116 DEGs and 3 DAMs in starch and sucrose metabolism (48 DEGs and 1 DAM), glycolysis and gluconeogenesis (54 DEGs and 1 DAM), and the pentose phosphate pathway (14 DEGs and 1 DAM) were significantly enriched. These findings suggest that blueberries predominantly increase sugar accumulation by activating carbon metabolism network pathways. Moreover, we identified critical transcription factors linked to the sugar response. This study presents new understandings regarding the molecular mechanisms underlying blueberry sugar accumulation and will be helpful in improving blueberry fruit quality through breeding.

Integrated Physiological and Metabolomic Analyses Reveal the Differences in the Fruit Quality of the Blueberry Cultivated in Three Soilless Substrates.

With improving living standards, traditional blueberry planting modes cannot meet commercial demands, and blueberry cultivation with soilless substrate has become a popular solution in the blueberry industry. In this study, different soilless substrate treatments were found to markedly influence fruit appearance and intrinsic quality. The fruit in the 50:50 peat/pine bark (v/v) (FPB) treatment group had the maximum single fruit weight, largest vertical diameter, and brightest color, as well as the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) value, solid-acid ratio and anthocyanin content. The fruit in the 50:50 pine bark/rice husk (v/v) (FBR) treatment group had the highest total phenol and flavonoid levels, largest drip loss value, and lowest total pectin content and firmness value. Metabolomic analysis showed that flavonoid, carbohydrate, and carbohydrate conjugate, and amino acid, peptide, and analog levels were significantly different between groups. Fruit in the FPB group had the highest sucrose, D-fructose 1,6-bisphosphate, salidroside, tectorigenin, naringenin chalcone, trifolirhizin, and galangin contents. The increase in the relative expression of phenylalanine (Phe) promoted the synthesis of fruit polyphenols in the FBR group. Our results provide new insights into the effects of different substrates on the quality of blueberries and a reference for the soilless substrate cultivation of blueberries.

Simultaneous measurement of six biomarkers of dichlorvos in blood by ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry.

A protocol was developed, via a data-dependent high-resolution tandem mass spectrometry (ddHRMS/MS), to detect six biomarkers of dichlorvos in its metabolites and blood adducts of butyrylcholinesterase and albumin without using standard synthetic peptides. Firstly, the adducts of dimethoxy phosphonate (DMP-BChE) and the aged adducts of methoxy phosphonate (MxP-BChE) were isoloated by immunomagnetic separation (IMS), and then digested to DMP-nonapeptide and MxP-nonapeptide by pepsin. The dichlorvos and its metabolites (Trimethyl phosphate, Dimethyl phosphate) were analyzed in the supernatant of IMS treatment after protein precipitation. The precipitate was digested by pronase to phosphorylated tyrosine (DMP-Tyr), which were quantified by ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry (UPLC-MS). The linearity of detector response of all biomarkers was studied in their respective ranges, and the correlation coefficients (R2) were all greater than 0.9981. The limits of detection (LOD) and limits of quantification (LOQ) were 0.2-10 ng/mL and 0.5-20 ng/mL, respectively. The recoveries of the six biomarkers were 78.6-110.0 %, matrix effect were 64.1-106.8 %. Inter- and intra-assay precision had coefficients of variation of ≤13.2 % and ≤9.7 %, respectively. In addition, MxP- BChE, DMP-Tyr, TMP, DMP and DDVP were detected in the blood of three cases who died from DDVP poisoning, but DMP- BChE was not detected in all of them, which may be caused by the instability of DMP- BChE and its easy aging to form MxP-BCHE.