Physiologic and Nanoscale Distinctions Define Glutamatergic Synapses in Tonic vs Phasic Neurons.

Neurons exhibit a striking degree of functional diversity, each one tuned to the needs of the circuitry in which it is embedded. A fundamental functional dichotomy occurs in activity patterns, with some neurons firing at a relatively constant "tonic" rate, while others fire in bursts, a "phasic" pattern. Synapses formed by tonic versus phasic neurons are also functionally differentiated, yet the bases of their distinctive properties remain enigmatic. A major challenge toward illuminating the synaptic differences between tonic and phasic neurons is the difficulty in isolating their physiological properties. At the Drosophila neuromuscular junction, most muscle fibers are coinnervated by two motor neurons: the tonic "MN-Ib" and phasic "MN-Is." Here, we used selective expression of a newly developed botulinum neurotoxin transgene to silence tonic or phasic motor neurons in Drosophila larvae of either sex. This approach highlighted major differences in their neurotransmitter release properties, including probability, short-term plasticity, and vesicle pools. Furthermore, Ca2+ imaging demonstrated ∼2-fold greater Ca2+ influx at phasic neuron release sites relative to tonic, along with an enhanced synaptic vesicle coupling. Finally, confocal and super-resolution imaging revealed that phasic neuron release sites are organized in a more compact arrangement, with enhanced stoichiometry of voltage-gated Ca2+ channels relative to other active zone scaffolds. These data suggest that distinctions in active zone nano-architecture and Ca2+ influx collaborate to differentially tune glutamate release at tonic versus phasic synaptic subtypes.SIGNIFICANCE STATEMENT "Tonic" and "phasic" neuronal subtypes, based on differential firing properties, are common across many nervous systems. Using a recently developed approach to selectively silence transmission from one of these two neurons, we reveal specialized synaptic functional and structural properties that distinguish these specialized neurons. This study provides important insights into how input-specific synaptic diversity is achieved, which could have implications for neurologic disorders that involve changes in synaptic function.

Retrospective cohort study of neonatal blood transfusion in China.

BACKGROUND: Blood transfusion therapy is extremely important for certain neonatal diseases, but the threshold for neonatal blood transfusion is not the same in different countries. Until now, clinical studies to determine the suitable threshold for newborns in China are lacking. Therefore, it is of high importance to establish a multi-center cohort study to explore appropriate transfusion thresholds for newborns in China. METHODS: This retrospective cohort study investigated neonatal blood transfusion therapy administered from January 1, 2017 to June 30, 2018, with the aim of evaluating the effect of restricted and nonrestricted blood transfusion on neonatal health. The subjects were enrolled in 46 hospitals in China. A total of 5669 neonatal cases were included in the study. Clinical diagnosis and transfusion treatment of these neonates were collected and the data were retrospectively analyzed. The neonates were followed up 1 week and 1 month after leaving the hospital. The newborns' and their mothers' data were collected containing 280 variables in the database. The primary outcome of the study was mortality, and the secondary outcomes were complications, hospital stays, NICU hospital stays and hospital costs. RESULTS: Results from the < 1500 g group showed that there was a higher mortality rate in the restricted transfusion group (11.41%) when compared with the non-restricted transfusion group (5.12%) (P = 0.000). Among the secondary outcomes, the restricted transfusion group had fewer costs. Results from the 1500-2500 g group showed that the mortality rates of the restricted and non-restricted transfusion groups were 3.53% and 4.71%, respectively, however there was no statistical significance between the two groups (P = 0.345). Among the secondary outcomes, the restricted transfusion group had fewer hospital stays, NICU hospital stays and hospital costs. The incidence of necrotizing enterocolitis was lower in the restricted transfusion group (OR, 2.626; 95% confidence interval [CI], 1.445 to 4.773; P = 0.003). The results from the ≥ 2500 g restricted transfusion group suggested that the mortality rate of (3.02%) was significantly lower than that of non-restricted transfusion group (9.55%) (P = 0.000). Among the secondary outcomes, the restricted transfusion group had fewer hospital stays and hospital costs. The incidence of retinopathy of prematurity was lower in the restricted transfusion group (OR, 4.624; 95% confidence interval [CI], 2.32 to 9.216; P = 0.000). CONCLUSIONS: Current transfusion protocols for newborns weighing less than 1500 g may be inappropriate and lead to higher mortality. The current transfusion threshold performed better for the other two weight groups.

The auxiliary glutamate receptor subunit dSol-1 promotes presynaptic neurotransmitter release and homeostatic potentiation.

Presynaptic glutamate receptors (GluRs) modulate neurotransmitter release and are physiological targets for regulation during various forms of plasticity. Although much is known about the auxiliary subunits associated with postsynaptic GluRs, far less is understood about presynaptic auxiliary GluR subunits and their functions. At the Drosophila neuromuscular junction, a presynaptic GluR, DKaiR1D, localizes near active zones and operates as an autoreceptor to tune baseline transmission and enhance presynaptic neurotransmitter release in response to diminished postsynaptic GluR functionality, a process referred to as presynaptic homeostatic potentiation (PHP). Here, we identify an auxiliary subunit that collaborates with DKaiR1D to promote these synaptic functions. This subunit, dSol-1, is the homolog of the Caenorhabditis elegans CUB (Complement C1r/C1s, Uegf, Bmp1) domain protein Sol-1. We find that dSol-1 functions in neurons to facilitate baseline neurotransmission and to enable PHP expression, properties shared with DKaiR1D Intriguingly, presynaptic overexpression of dSol-1 is sufficient to enhance neurotransmitter release through a DKaiR1D-dependent mechanism. Furthermore, dSol-1 is necessary to rapidly increase the abundance of DKaiR1D receptors near active zones during homeostatic signaling. Together with recent work showing the CUB domain protein Neto2 is necessary for the homeostatic modulation of postsynaptic GluRs in mammals, our data demonstrate that dSol-1 is required for the homeostatic regulation of presynaptic GluRs. Thus, we propose that CUB domain proteins are fundamental homeostatic modulators of GluRs on both sides of the synapse.

Knockdown of long non-coding RNA TUG1 depresses apoptosis of hippocampal neurons in Alzheimer's disease by elevating microRNA-15a and repressing ROCK1 expression.

OBJECTIVE: Many studies have already suggested the role of long non-coding RNAs (lncRNAs) in Alzheimer's disease (AD), but the functions of lncRNA Taurine Upregulated Gene 1 (TUG1) in AD have been scarcely discussed. This study aims to verify how TUG1 affects hippocampal neurons in AD through modulation of microRNA-15a (miR-15a)/Rho-associated protein kinase 1 (ROCK1). METHOD: AD mice was modeled through injection of ß-amyloid 25-35 (Aß25-35) into the lateral ventricle. After modeling, the mice were injected with altered TUG1 and/or miR-15a agomir lentiviruses. The spatial learning ability and memory ability of mice were detected through Morris water maze test. Hippocampal neuronal apoptosis and oxidative stress indicators in AD mice were then detected. The hippocampal neuron AD model was induced by Aß25-35. Next, the neurons were, respectively, transfected with altered TUG1 vector and/or miR-15a mimics to determine the proliferation inhibition and apoptosis of hippocampal neurons. The interactions between TUG1 and miR-15a, and between miR-15a and ROCK1 were assessed using bioinformatic prediction, dual luciferase reporter gene assay and RNA-pull-down assay. RESULTS: In the animal models, Aß25-35-induced mice exhibited decreased spatial learning and memory ability, obvious pathological injury, promoted hippocampal neuronal apoptosis and decreased antioxidant ability. TUG1 silencing and miR-15a elevation improved spatial learning ability and memory ability, ameliorated pathological injury, depressed neuronal apoptosis, and strengthened antioxidant ability of hippocampal neurons in AD mice. In cellular models, Aß25-35-treated hippocampal neurons presented inhibited neuronal viability and promoted neuronal apoptosis. TUG1 silencing and miR-15a elevation increased viability and limited apoptosis of Aß25-35-treated hippocampal neurons. TUG1 specifically bound to miR-15a, and miR-15a targeted ROCK1. CONCLUSION: Collectively, this study reveals that TUG1 knockdown restricts apoptosis of hippocampal neurons in AD by elevating miR-15a and suppressing ROCK1 expression, and provides a new therapeutic target for AD treatment.

The Drosophila Postsynaptic DEG/ENaC Channel ppk29 Contributes to Excitatory Neurotransmission.

The protein family of degenerin/epithelial sodium channels (DEG/ENaCs) is composed of diverse animal-specific, non-voltage-gated ion channels that play important roles in regulating cationic gradients across epithelial barriers. Some family members are also enriched in neural tissues in both vertebrates and invertebrates. However, the specific neurophysiological functions of most DEG/ENaC-encoding genes remain poorly understood. The fruit fly Drosophila melanogaster is an excellent model for deciphering the functions of DEG/ENaC genes because its genome encodes an exceptionally large number of DEG/ENaC subunits termed pickpocket (ppk) 1-31 Here we demonstrate that ppk29 contributes specifically to the postsynaptic modulation of excitatory synaptic transmission at the larval neuromuscular junction. Electrophysiological data indicate that the function of ppk29 in muscle is necessary for normal postsynaptic responsivity to neurotransmitter release and for normal coordinated larval movement. The ppk29 mutation does not affect gross synaptic morphology and ultrastructure, which indicates that the observed phenotypes are likely due to defects in glutamate receptor function. Together, our data indicate that DEG/ENaC ion channels play a fundamental role in the postsynaptic regulation of excitatory neurotransmission.SIGNIFICANCE STATEMENT Members of the degenerin/epithelial sodium channel (DEG/ENaC) family are broadly expressed in epithelial and neuronal tissues. To date, the neurophysiological functions of most family members remain unknown. Here, by using the power of Drosophila genetics in combination with electrophysiological and behavioral approaches, we demonstrate that the DEG/ENaC-encoding gene pickpocket 29 contributes to baseline neurotransmission, possibly via the modulation of postsynaptic glutamate receptor functionality.

Association between maternal nonresponsive feeding practice and child's eating behavior and weight status: children aged 1 to 6 years.

The purposes of this study are to investigate the prevalence of nonresponsive feeding practice (NRFP) and child's eating behavior (CEB) and to explore the hypothetical association between child's weight status, NRFP and CEB for 1- to 6-year-old children. In this study, 2423 caregivers of 1- to 6-year-old children are from the Nanjing Maternal and Child Health Hospital who completed the self-report questionnaires about their NRFP and CEB as well as their children's sociodemographic data. Chi-square test and multiple regression analyses were used to examine the correlation between child's weight status and NRFP and CEB. The total prevalence of overweight and obesity was 15.2 and 7.3%, respectively. High prevalence of CEB problems and NRFP was detected at 2- and 5-year-old children. Moreover, maternal NRFP was significantly positively associated with CEB. The regression and correlation analysis revealed CEB and maternal NRFP are closely associated with BMI. For instance, refusing new food (OR = 3.57, 95%CI, 1.37-9.33, 1.5-year-old) and restriction (OR = 3.01, 95%CI, 1.34-6.76) are likely to be associated with underweight. Preferring junk food (OR = 4.892, 95%CI, 1.71-14.01, 1-year-old) and inattention (OR = 2.24, 95%CI, 1.16-4.35, 1-year-old) are prone to be overweight and obese, and pressure (OR = 0.23, 95%CI, 0.06-0.91, 1-year-old) is less likely to be associated with underweight. CONCLUSION: The findings provide strong evidence for the correlation between NRFR and CEB, and this indicates that prevention and intervention of unhealthy weight should start in early life. However, further research is necessary to gain an understanding of the impact of NRFP on CEB and weight. What is known: • Responsive feeding practice is crucial to the formation of eating behavior, and poor practice is associated with the current epidemics of childhood obesity and underweight. What is new: • The findings provide a strong evidence for the correlation between NRFR and CEB. • This finding indicates that NRFR and CEB are associated with child's unhealthy weight.

Uric acid quantification in fingernail of gout patients and healthy volunteers using HPLC-UV.

The presence of elevated uric acid (UA) levels is a sign of gout, that is, hyperuricemia. In this study the monitoring of the UA levels in less-invasive biological samples, such as the human fingernail, is suggested for the diagnosis and therapy of gout. Twenty-six healthy volunteers (HV) and 22 gout patients (GP) were studied. The UA was extracted from human fingernail samples, then separated on an Inertsil ODS-3 column (250 × 4.6 mm i.d., 4.0 µm, GL Sciences) by isocratic elution using methanol-74 mm phosphate buffer (pH 2.2) 2:98 (v/v). A UV detector was used to monitor the samples at 284 nm. Using the developed method, different UA concentrations were found in the GP and HV. When comparing the concentrations from GP with those from HV, a statistically significant correlation was observed between the UA (p < 0.01). In this study, the UA was confirmed as a potential biomarker for the diagnosis and therapy of gout. We have developed a novel sensitive, and simple method for the determination of UA in the fingernails of GP and HV. The human fingernail may serve as a noninvasive biosample for the diagnosis of gout. Copyright © 2016 John Wiley & Sons, Ltd.

Levels of polybrominated diphenyl ethers in settled house dust from urban dwellings with resident preschool-aged children in Nanjing, China.

We investigated the levels and possible determinants of polybrominated diphenyl ethers (PBDEs) in the settled house-dust (SHD) of urban dwellings with resident preschool-aged children in Nanjing, China. The possible neurodevelopmental effects of house-dust PBDEs were also explored. SHD was collected from 216 urban houses. Levels of 8 PBDEs were measured by gas chromatography-negative chemical ionization mass spectrometry. The Child Behavior Checklist and the Gesell Development Inventory were used to evaluate the child's development. BDE47, BDE99, BDE153, BDE18, and BDE209 were detected in the SHD of >90 % of houses, of which BDE209 predominated. Most PBDEs were found at significantly greater levels in indoor than in outdoor dust (P < 0.05). Levels of BDE28 and BDE154 in houses with solid-wood floors were significantly greater than those in houses with plywood floors (P < 0.05). BDE154 levels in houses with wallpaper were significantly greater than those without wallpaper (P < 0.05). Greater BDE47 concentrations were found in houses with less natural ventilation time (linear trend P < 0.05). After dichotomization at the geometric mean concentration, BDE209 and total BDEs showed significant risks for depressed behavior problems and lower personal social developmental quotients (DQs); BDE99 and BDE153 indicated a risk for lower personal social DQs. In conclusion, PBDEs (especially BDE209) are ubiquitous in urban SHD in Nanjing residences. Natural ventilation and floor materials potentially influence PBDE levels in SHD. The potential adverse effect of postnatal exposure to PBDEs on the behavior and neurodevelopment of preschool-age children requires follow-up in larger studies.

Synthesis and photocatalytic properties of CuO nanostructures.

CuO nanostructures were grown by decomposition of a mixture of Cu(CH3COO)2 x H2O and NaCl at different temperatures. The nanostructure properties were studied by X-ray diffractometer, scanning electron microscope and Raman spectroscope. Photodegradation activity of the nanostructures towards methyl orange was also examined. CuO spheres and hollow spheres composed of nanoparticles were obtained. CuO nanoparticle size increases with an increase in the growth temperature. More specifically, it increases slowly when the temperature was lower than 280 degrees C and increases dramatically in a higher temperature range. The degradation activity is sensitive to the nanostructure growth temperatures, but the degradation activity varies with the growth temperatures or the size of nanoparticles composing of nanospheres non-monotonously. The hollow spheres composed of nanoparticles grown at 280 degrees C show superior photocatalytic activity towards the degradation of methyl orange than that grown at lower and higher temperatures.

A dried sweat spot paper (DSSP) method based on novel mass spectrometry probe labeling for detection and resolution of DL-lactate enantiomers as potential biomarkers for diabetes mellitus.

BACKGROUND: Human sweat can be collected non-invasively with low infectivity; however, its application as a determination method has been challenged due to the presence of trace amounts of chiral metabolites. Moreover, its application as a biological fluid for disease diagnosis has not been previously reported. In this study, the human dried sweat spot paper (DSSP) method was proposed for the derivatization of a novel mass spectrometric chiral probe, N-[1-Oxo-5-(triphenylphosphonium) pentyl]-(S)-3-aminopyrrolidine (OTPA), determination and resolution of DL-lactic acid (DL-LA) enantiomers in human elbow sweat. RESULTS: The methodological validation revealed the resolution (Rs) as 1.78, the limit of detection (S/N = 3) as 20.83 fmol, good linearity (R2 ≥ 0.9996), and the intra-day and intra-day stability with RSD ranging from 0.53 to 10.85 %, while the average recovery rate of D-LA and L-LA were 104.00 % ± 4.68 % and 107.41 % ± 8.34 %, respectively, with high accuracy. In addition, the method was applied for the determination of DL-LA in the sweat on elbow of 10 healthy volunteers and 30 diabetic patients. The results demonstrated that the D/L ratio and L/D ratio were significantly different (p < 0.0001). In addition, a moderate positive linear correlation between the D/L-LA ratio in human sweat and fasting blood glucose level (r = 0.7744, p < 0.0001) was observed, thereby suggesting that the D/L ratio of lactate in human sweat correlate the glucose level in human fasting blood. SIGNIFICANCE AND NOVELTY: The D/L lactate ratio in human sweat could be used as a potential biomarker for diabetes screening. The method can be used to screen for diabetes by providing a dry sweat paper to test equipment and has the potential to be a non-invasive early-warning diagnostic tool for diabetes.

Relative quantitation of chiral thiol compounds labeled based on isotope novel mass spectrometry probes: Monitoring of the dynamic changes of chiral thiol compounds in human urine during normal, exercise, and rest recovery states.

Monitoring changes in the content of chiral thiol compounds in the human body is crucial for the early diagnosis of oxidative stress-related diseases and the exploration of their pathogenesis. To address this, we synthesized a novel isotope mass spectrometry (MS) probe, denoted as (R)-(5-(3-isothiocyanato (13C) pyrrolidin-1-yl)-5-oxopentyl) triphenylphosphonium (N13CS-OTPP), with triphenylphosphine as its parent structure. In this study, we established a new ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLCHRMS) relative quantitative method to monitor chiral thiol compounds in human urine under varying oxidative stress conditions. This method relies on the ratio of 12C/13C isotope-labeled peak areas. To assess the chiral separation efficiency of N13CS-OTPP, we employed three types of thiol compounds (D/L-GSH, D/L-Cys, and D/L-Hcy) and observed separation degrees (Rs) ranging from 1.82 to 1.89. We further validated the accuracy and feasibility of our relative quantitative methods using D/L-Cys-as a model compound. N12C/13CS-OTPP-Cys-exhibited excellent linearity (R2 = 0.9993-0.9994) across different molar ratios (D/L-Cys = 10:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:10) and achieved a low limit of detection (LOD) of 2.5 fmol. Additionally, we monitored the dynamic changes in urine D/L-Cys-and D/L-Hcy ratios in 12 healthy volunteers (six males and six females) under various oxidative stress states. We generated fitting curves and investigated the trends in chiral thiol compounds in vivo. This study introduces a novel method for the relative quantitative monitoring of chiral thiol compounds in different oxidative stress states within the human body. It also presents a new strategy for understanding the pathogenesis of related diseases resulting from the abnormal metabolism of thiol compounds.

Simultaneous determination of free DL-amino acids in human hair with a novel DBD-M-Pro derivatization by UHPLC-HRMS: An application in diabetes patients.

Human hair is a non-invasive biological sample that is easy to collect and store and can reflect long-term body health. However, the correlation between DL-amino acids and metabolic diseases in hair samples has not been studied. Therefore, we propose a novel UHPLC-HRMS method for analyzing seven free chiral amino acids (DL-Thr, DL-Glu, DL-Ala, DL-Val, DL-Pro, DL-Leu, and DL-Phe) simultaneously in hair samples by derivatization of chiral probe 4-(N,N-dmethylaminosulfonyl)-2,1,3-benzoxadiazole-trans-2-methyl-L-proline (DBD-M-Pro) labeled with targeted amino functional groups. Gradient elution was carried out using an ACQUITYTM BEH C18 (100×2.1 mm,1.7 µm) column with a mobile phase of 0.15 % formic acid (FA) in 10 mM ammonium acetate (CH3-COONH4) and 0.2 % FA in acetonitrile. The labelled DL-amino acid diastereoisomers could be completely separated, with a resolution (Rs) of 1.59-11.44. These amino acids show a strong linear correlation within the range of 3.1-99.2 pmol (R2 ≥ 0.9990). Intraday and interday precision was 1.87 %-14.87 %. The average recovery was 96.12 %-105.33 %. The limit of detection (LOD) ranged from 0.29 to 2.11 pmol. We then employed the method to determine the concentration of free chiral amino acids in hair samples from 30 healthy volunteers (HVs) and 30 diabetes patients (DPs). Male diabetes patients had significantly higher levels of L-Thr, L-Val, L-Leu (p < 0.05), and D-Ala (p < 0.01) in their hair samples than male healthy volunteers and female diabetes patients had significantly higher levels of D-Ala (p < 0.05) in their hair samples than female healthy volunteers. This is the first study to confirm the feasibility of using free DL-amino acids in human hair as potential biomarkers for diabetes.

Simultaneous analysis of natural and artificial sweeteners in sugar-free drinks and urine samples by column-switching UHPLC-charged aerosol detection method.

Sweeteners are considered an alternative to high-calorie foods or drinks and have been widely used globally. However, the simultaneous separation and detection of high-polarity natural and artificial sweeteners are challenging owing to their broad-spectrum physical and chemical properties. Herein, we developed a column-switching UHPLCCAD method and used it for detecting and quantitating 12 sweeteners, including natural sweeteners (erythritol, mannitol, xylitol, sorbitol and stevioside) and artificial sweeteners (acesulfame potassium, saccharin sodium salt, sodium cyclamate, sucralose, aspartame, alitame and neotame). The LOD and LOQ were 0.932-6.25 µg/mL and 3.10-20.83 µg/mL, respectively, and the method demonstrated excellent linearity (R² ≥ 0.9990), good precision (intraday and interday precision was 0.59-6.88 %), and high recovery (average recoveries were 85.16-108.64 %). This method was applied to determine the sweeteners in 15 sugar-free drinks purchased from the local Chinese supermarkets. What's more, natural sweetener erythritol and artificial sweetener acesulfame potassium were suspected over addition in sugar-free drinks. Meanwhile the method was applied to the sweeteners in various sugar-free drinks and the dynamic monitoring of transit and excretion in vivo after drinking. Those prove that the method can be used to the detection of sugar free drinks and quality control of the sweeteners. The study highlights the potential of UHPLC-charged aerosol detection technology in detection of multiple components in food industry.

Acoustic spin rotation in heavy-metal-ferromagnet bilayers.

Through pumping a spin current from ferromagnet into heavy metal (HM) via magnetization precession, parts of the injected spins are in-plane rotated by the lattice vibration, namely acoustic spin rotation (ASR), which manifests itself as an inverse spin Hall voltage in HM with an additional 90° difference in angular dependency. When reversing the stacking order of bilayer with a counter-propagating spin current or using HMs with an opposite spin Hall angle, such ASR voltage shows the same sign, strongly suggesting that ASR changes the rotation direction due to interface spin-orbit interaction. With the drift-diffusion model of spin transport, we quantify the efficiency of ASR up to 30%. The finding of ASR endows the acoustic device with an ability to manipulate spin, and further reveals a new spin-orbit coupling between spin current and lattice vibration.

Development of a diphenyl sulfide structure derivatization reagent for amino acid enantiomers analysis: Application of dynamic monitoring in human urine after drinking wine.

We developed a novel chiral mass spectrometry derivatization reagent (S)-(3-(4-carboxythiazolidin-3-yl)-3-oxopropyl) diphenylsulfonium (CTOD) with a positively charged sulfur-containing structure for high-sensitivity detection of the chiral resolution of amino acid enantiomers. CTOD reacted with DL-amino acids at 60oC for 60 min to generate the corresponding diastereomers, fifteen chiral amino acid-derived products were separated. Resolution (Rs) values were of the range 1.54-4.36, except Asn 1.07, achieving good separation. A highly sensitive and selective UHPLC-MS/MS method for the simultaneous determination and chiral separation of five chiral amino acids (Pro, Ala, Glu, Asp, and Phe) based on CTOD derivatization was established and applied to the detection of chiral amino acids in different wines. The diastereomeric resolution of the five amino acids was 1.71-5.42, and an excellent linear relationship was obtained in the range of 0.25-500 pmol (R2 ≥0.9993). The detection limit was 0.05-0.25 pmol. The intra- and inter-day precisions were 0.51-5.76% and 0.78-5.18%, respectively, and the average recovery was 90.03-99.99%. In addition, the metabolic concentration of chiral amino acids was monitored after drinking red wine and white wine, and the fitting curve of metabolic concentration was drawn.

Development and evaluation of a novel fluorescent chiral derivatization reagent DBD-S-M-Pro: first observation of four chiral amino acids in human hair.

This study reports a novel fluorescent chiral derivatization reagent, 4-(N,N-dmethylaminosulfonyl)-2,1,3-benzoxadiazole-(2-succinimidoxy)-trans-2-methyl-L-proline (DBD-S-M-Pro), with a benzoxadiazole structure containing an N-hydroxysuccinimide activation group. DBD-S-M-Pro targets chiral amino-functional compounds under alkaline conditions without a condensation agent. Gradient elution was performed on a BEH C18 (100 × 2.1 mm, 1.7 µm) column with a mobile phase of 0.05% formic acid (FA) in 10 mM ammonium acetate (CH3COONH4) and 0.1% FA in acetonitrile or methanol. The efficiency of the chiral resolution was evaluated under excitation and emission wavelengths of 450 nm and 560 nm, respectively. The 19 chiral amino acids were separated in the range of 1.45-14.84. The resolutions of almost all DL-amino acids exceeded 1.5; the exceptions were serine (Ser) and lysine (Lys), with resolutions of 1.45 and 1.46, respectively. In addition, a new approach was devised for the simultaneous analysis of four chiral amino acids (DL-Glu, DL-Ala, DL-Val, and DL-Phe) in human hair. These amino acids were analyzed in the range of 12.5-400 pmol, with R2 ≥ 0.9990, limits of detection (S/N = 3) of 4-10 pmol, and intraday and interday precisions of 0.57-6.23%. The average spikes in the hair recoveries were 89.76-111.54%, and the matrix effects were 92.47-102.40%. Next, the contents of free chiral amino acids in the hair samples of 10 healthy volunteers (five males and five females) were analyzed with this method, and the differences were compared. The developed DBD-S-M-Pro provides a novel strategy for the sensitive determination of free chiral amino acids in living organisms.

A novel UHPLC-MS/MS method for the determination of four α-dicarbonyl compounds in wine and dynamic monitoring in human urine after drinking.

α-dicarbonyl compounds (α-DCs) serve as potential biomarkers for oxidative stress-related diseases but are difficult to detect.To study the metabolism of carbonyl compounds, we developed a new mass spectrometry probe, 3-benzyl-2-oxo-4λ3-thiazolidine-4-carbohydrazide (BOTC), containing hydrazyl groups for the targeted detection of carbonyl functional groups.In a novel approach, we used BOTC pre-column derivatization with ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to simultaneously detect four kinds of α-DCs in red wine as well as in urine after drinking. The α-DCs were completely separated (R2 ≥ 0.9995), detection was sensitive (detection limit was 12.5-50 fmol), consistent (intraday and interday precision was 0.1-5.7 %), and efficient (average recoveries were 103.3-110.2 %). The method was applied to the analysis of α-DCs in different wines and the dynamic monitoring of transit and excretion in vivo after drinking. Our novel method provides a new strategy for the detection of α-dicarbonyl compounds in red wine and dicarbonyl compounds produced in oxidative stress-related diseases.

Stable isotope labeling differential glycans discovery in the serum of acute myocardial infarction by ultrahigh-performance liquid chromatography-quadrupole-Orbitrap high resolution mass spectrometry.

Acute myocardial infarction (AMI) poses a grave threat to human life. However, most clinical biomarkers have limitations of low sensitivity and specificity. Therefore, screening novel glycan biomarkers with high sensitivity and specificity is crucial for the prevention and treatment of AMI. The novel method of ultrahigh-performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) with d0/d5-BOTC probe labeling for relative quantification of glycans based on Pronase E digestion was established to screen novel glycan biomarkers in the serum of 34 AMI patients relative to healthy volunteers. The monosaccharide model D-glucosamine was used to investigate the effectiveness of the derivatization; the limit of detection (S/N = 3) was 10 amol. The accuracy was verified based on the consistency of different theoretical molar ratios (d0/d5 = 1:2, 2:1) and intensity ratios following digestion of glycoprotein ribonuclease B. Expressions of H4N4F3SA, H4N6F2, H4N6SA, H4N6F3 and H5N4FSA in the serum were significantly different (p < 0.0005) between AMI patients and healthy volunteers. The area under the receiver operating characteristic curve (AUC) for H4N6SA, H5N4FSA, and H4N6F2 was greater than 0.9039. Based on the proposed method, H4N6SA, H5N4FSA, and H4N6F2 in human serum showed high accuracy and specificity and may serve as potential glycan biomarkers, crucial for the diagnosis and treatment monitoring of AMI.

Knockdown of NYGGF4 increases glucose transport in C2C12 mice skeletal myocytes by activation IRS-1/PI3K/AKT insulin pathway.

NYGGF4, an obesity-related gene, is proposed to be involved in the development of insulin resistance. Skeletal muscle is a primary target organ for insulin and NYGGF4 showed a relatively high expression level in skeletal muscle. Therefore, this study aimed to explore the effect of NYGGF4 on insulin sensitivity of skeletal muscle cells. RNA interference (RNAi) was adopted to silence NYGGF4 expression in mice C2C12 skeletal myocytes. A remarkably increased insulin-stimulated glucose uptake and GLUT4 translocation was observed in NYGGF4 silencing C2C12 cells. Importantly, the enhanced glucose uptake induced by NYGGF4 silencing could be abrogated by the PI3K inhibitor LY294002. In addition, the crucial molecules involved in PI3K insulin signaling pathway were detected by western blotting. The results showed that NYGGF4 knockdown dramatically activate the insulin-stimulated phosphorylation of IRS-1 and AKT. Taken together, these data demonstrate that NYGGF4 knockdown increases glucose transport in myocytes by activation of the IRS-1/PI3K/AKT insulin pathway.