Quadrupole-Linear Ion Trap Tandem Mass Spectrometry System for Clinical Biomarker Analysis

The accurate and efficient measurement of small molecule disease markers for clinical diagnosis is of great importance.In this study,a quadrupole-linear ion trap(Q-LIT)tandem mass spectrometer was designed and built in our laboratory.Target precursor ions were first selected in the quadrupole,and then injected,trapped,and fragmented simultaneously in the linear ion trap(LIT)to reduce the space charge effect,enrich the target product ions,and promote sensitivity.The targeted analytes were measured with selected reaction monitoring using a positive scan mode with electrospray ionization(ESI).Ions with a mass-to-charge ratio(m/z)ranging from 195 to 2022 were demonstrated.When scanning at 1218amu.s^(-1),unit resolution and an accuracy of higher than m/z 0.28 was obtained for m/z up to 2000.The dimensionless Mathieu parameter(q)value used in this study was 0.40 for collision-induced dissociation(CID),which was activated by resonance excitation.And an overall CID efficiency of 64%was achieved(activation time,50 ms).Guanidinoacetic acid(GAA)and creatine(CRE)were used as model compounds for small molecule clinical biomarkers.The limits of quantification were 1.0 and 0.2 nmol.L^(-1)for GAA and CRE,respectively.A total of 77 actual samples were successfully analyzed by the home-built ESI-Q-LIT tandem mass spectrometry system.The developed method can reduce matrix interference,minimize space charge effects,and avoid the chromatographic separation of complex samples to simplify the pretreatment process.This novel Q-LIT system is expected to be a good candidate for the determination of biomarkers in clinical diagnosis and therapeutics.

NPY Genes Play an Essential Role in Root Gravitropic Responses in Arabidopsis

Plants can sense the direction of gravity and orient their growth to ensure that roots are anchored in soil and that shoots grow upward. Gravitropism has been studied extensively using Arabidopsis genetics, but the exact mecha- nisms for gravitropism are not fully understood. Here, we demonstrate that five NPY genes play a key role in Arabidopsis root gravitropism. NPYgenes were previously identified as regulators of auxin-mediated organogenesis in a genetic pathway with the AGC kinases PID, PID2, WAG1, and WAG2. We show that all five NPYgenes are highly expressed in primary root tips. The single npy mutants do not display obvious gravitropism defects, but the npyl npy2 npy3 npy4 npy5 quin- tuple mutants show dramatic gravitropic phenotypes. Systematic analysis of all the npy double, triple, and quadruple combinations demonstrates that the five NPY genes all contribute to gravitropism. Our work indicates that gravitropism, phototropism, and organogenesis use analogous mechanisms in which at least one AGC kinase, one NPH3/NPY gene, and one ARF are required.

Urinary analysis reveals high Alternaria mycotoxins exposure in the general population from Beijing, China

Alternaria mycotoxins are of concern due to its adverse health effect, they affect various cereal crops and grain-based food along with modified forms that contribute to overall exposure. This study aimed to determine the frequency and level of exposure to Alternaria mycotoxins(tenuazonic acid, TeA;alternariol, AOH;alternariol monomethyl ether, AME;tentoxin, TEN;and altenuene, ALT) in human urine from Beijing adults. A total of 2212 urine samples were collected and analyzed for five mycotoxins using LC–ESI–MS/MS. More than98% of the samples had at least one Alternaria mycotoxin detected. Among the mycotoxins,AME had the highest detection rate(96.0%), followed by TeA(70.5%). The calculated average daily intake values of AME(12.5 ng/kg b.w.) was 5 times the TTC value(2.5 ng/kg b.w.) set by the EFSA, indicating the potential health risks associated with mycotoxins. Immediate attention and subsequent actions should be taken to identify the sources of mycotoxins and the corresponding exposure pathways to humans in the investigated regions.

Two homologous INDOLE-3-ACETAIVHDE (IAM) HYDROLASE genes are required for the auxin effects of IAM in Arabidopsis

Indole-3-acetamide(IAM)is the first confirmed auxin biosynthetic intermediate in some plant pathogenic bacteria.Exogenously applied IAM or production of IAM by overexpressing the bacterial iaaM gene in Arabidopsis causes auxin overproduction phenotypes.However,it is still inconclusive whether plants use IAM as a key precursor for auxin biosynthesis.Herein,we reported the isolation IAM HYDROLASE 1(IAMH1)gene in Arabidopsis from a forward genetic screen for IAM-insensitive mutants that display normal auxin sensitivities.IAMH1 has a close homolog named IAMH2 that is located right next to IAMH1on chromosomeⅣin Arabidopsis.We generated iamh1 iamh2 double mutants using our CRISPR/Cas9gene editing technology.We showed that disruption of the IAMH genes rendered Arabidopsis plants resistant to IAM treatments and also suppressed the iaaM overexpression phenotypes,suggesting that IAMH1 and IAMH2 are the main enzymes responsible for converting IAM into indole-3-acetic acid(IAA)in Arabidopsis.The iamh double mutants did not display obvious developmental defects,indicating that IAM does not play a major role in auxin biosynthesis under normal growth conditions.Our findings provide a solid foundation for clarifying the roles of IAM in auxin biosynthesis and plant development.

Overexpression of the bacterial tryptophan oxidase RebO affects auxin biosynthesis and Arabidopsis development

Both tryptophan （Trp） and auxin are essential for plant growth and Trp is a precursor for auxin biosynthesis. Concentrations of Trp and auxin need to be tightly con- trolled to ensure optimal growth and development. It has been very difficult to study the homeostasis of these two essential and inter-dependent compounds. Auxin is mainly synthesized from Trp via a two-step pathway using indole- 3-pyruvate （IPA） as the intermediate. Here we used a bacterial Trp oxidase RebO, which does not exist in Ara- bidopsis and which converts Trp to the imine form of IPA, to modulate IPA levels in Arabidopsis. Our results demonstrate that Arabidopsis plants use two strategies to ensure that no excess IPA is made from Trp. IPA is made from Trp by the TAA family of aminotransferases, which we show catalyzes the reverse reaction when IPA level is high. Moreover, excess IPA is converted back to Trp by the YAS1 aminotransferase. We show that the VASl-cat- alyzed reaction is very important for Trp homeostasis. This work not only elucidates the intricate biochemical mech- anisms that control the homeostasis of Trp, IPA, and auxin, but also provides novel tools for further biochemical studies on Tip metabolism and auxin biosynthesis in plants.

ARTICLE OPEN Viral dynamics and antibody responses in people with asymptomatic SARS-CoV-2 infection

Over 40% of the coronavirus disease 2019(COVID-19)COVID-19 patients were asymptomatically infected with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and the immune responses of these asymptomatic individuals is a critical factor for developing the strategy to contain the COVID-19 pandemic.Here,we determined the viral dynamics and antibody responses among 143 asymptomatic individuals identified in a massive screening of more than 5 million people in eight districts of Wuhan in May 2020.Asymptomatic individuals were admitted to the government-designated centralized sites in accordance with policy.The incidence rate of asymptomatic infection is〜2.92/100,000.These individuals had low viral copy numbers(peaked at 315 copies/mL)and short-lived antibody responses with the estimated diminish time of 69 days.The antibody responses in individuals with persistent SARS-CoV-2 infection is much longer with the estimated diminish time of 257 days.These results imply that the immune responses in the asymptomatic individuals are not potent enough for preventing SARS-CoV-2 re-infection,which has recently been reported in recovered COVID-19 patients.This casts doubt on the efficacy of forming"herd-immunity"through natural SARS-CoV-2 infection and urges for the development of safe and effective vaccines.

A Preliminary Urinary Metabolomics Study of Sprague-Dawley Rats after Short‑term Ketamine Administration by Proton Nuclear Magnetic Resonance Spectroscopy

Drug abuse has become a global problem.The mass spectrometry‑based metabolic consequences of ketamine administration in anesthesia and therapy have been well studied,but to the best of our knowledge,metabolomic studies of ketamine abuse based on nuclear magnetic resonance(NMR)spectroscopy are still lacking.In this study,twenty Sprague–Dawley rats were randomly assigned into two groups:a control group(n=10)and a ketamine group(n=10).The animals in the ketamine group received intraperitoneal injections of ketamine twice daily at 12‑h intervals at progressively increasing doses over a period of 9 days,while the control group received an equal volume of saline.The urine samples were collected for 24 h at days 0,1,3,5,7,and 9 for the metabolomics study.The metabolic changes in urine after short‑term ketamine administration were analyzed by proton NMR coupled with multivariate statistical analysis.The results indicated that short‑term ketamine exposure led to significant alterations of the metabolites in the urine of the rats.Specifically,1,3,7‑trimethyluric acid,1,3‑dimethyluric acid,acetoacetic acid,acetylglycine,creatine,sarcosine,dimethylglycine,glycine,and theobromine were significantly increased in the urine.Significant changes were also found in metabolites related to antioxidant and energy metabolism,including acetoacetic acid,succinate,1,3,7‑trimethyluric acid,1,3‑dimethyluric acid,creatine,and taurine.Our findings indicated that short‑term ketamine administration leads to disorder of energy metabolism and oxidative stress.In addition,the modified metabolites identified could serve as the new biological markers and potential biological indices reflecting the underlying mechanism of ketamine abuse.

Allelic Analyses of the Arabidopsis YUC1 Locus Reveal Residues and Domains Essential for the Functions of YUC Family of Flavin Monooxygenases

Flavin monooxygenases （FMOs） play critical roles in plant growth and development by synthesizing auxin and other signaling molecules. However, the structure and function relationship within plant FMOs is not understood. Here we defined the important residues and domains of the Arabidopsis YUC1 FMO, a key enzyme in auxin biosynthesis. We previously showed that simultaneous inactivation of YUC1 and its homologue YUC4 caused severe defects in vascular and floral development. We mutagenized the yuc4 mutant and screened for mutants with phenotypes similar to those of yucl yuc4 double mutants. Among the isolated mutants, five of them contained mutations in the YUC1 gene. Interestingly, the mutations identified in the new yucl alleles were concentrated in the two GXGXXG motifs that are highly conserved among the plant FMOs. One such motif presumably binds to flavin adenine dinucleotide （FAD） cofactor and the other binds to nicotinamide adenine dinucleotide phosphate （NADPH）. We also identified the Ser139 to Phe conversion in yucl, a mutation that is located between the two nucleotide-binding sites. By analyzing a series of yucl mutants, we identified key residues and motifs essential for the functions of YUC1 FMO.

A Preliminary Gas Chromatography‑Mass Spectrometry‑Based Metabolomics Study of Rats Ingested Diazepam or Clonazepam

Drug-facilitated sexual assault(DFSA)is a sexual act in which the victim is unable to give or rescind consent due to alcohol or drug intoxication,which involved the abuse of benzodiazepines around the world.Conventional techniques used for the analysis of benzodiazepines have the limitation of short detection time window due to the rapid metabolism of these drugs in body.This study aimed to investigate the characteristic changes of metabolites in the blood of rats after ingesting diazepam/clonazepam through a gas chromatography-mass spectrometry-based metabolomics method,allowing the indirect reveal of the rats ingested diazepam/clonazepam.First,we found that diazepam and clonazepam in the blood of rats could not be detected by liquid chromatography-tandem mass spectrometry after 48 h of ingestion.Then,orthogonal partial least squares discrimination analysis regression models were,respectively,constructed to determine whether the rats ingested diazepam/clonazepam after 48 h.The results showed that 5 metabolites were found to be associated with diazepam exposure,and 7 metabolites were found to be associated with clonazepam exposure,which may be characterization for the evaluation of digestion of diazepam and clonazepam in rat.

A Gas Chromatography-Mass Spectrometry‑Based Metabonomic Study on Estimation of Toxicant in Rats

The aim of this study was to develop a gas chromatography‑mass spectrometry(GC‑MS)‑based metabolomics method to distinguish different kinds of poisons in the blood.We examined the changes in blood metabolites using GC‑MS following administration of four different poisons(paraquat,dichlorvos,aconitine,and sodium nitrite).The data were analyzed with orthogonal partial least squares.Then,total and single differential metabolite profiles were evaluated with support vector machine(SVM)models.The results showed that various metabolites(5‑ketone proline,1,5‑anhydrohexitol,lactic acid,glycine 2,2‑furoic acid,and 3‑hydroxybutyric acid)were differential between the experimental groups and the control groups.The accuracy rates of the SVM models established using total and single differential metabolites were 80%and 100%,respectively.In conclusion,we successfully developed a poison screening method.The established SVM models can distinguish four kinds of poisons and could be used to establish a complete poison metabonomic information database.Furthermore,some of the metabolites could be biomarkers of these poisons.Finally,both the models and potential biomarkers may reduce the time required for poison detection and provide direction for solving cases and auxiliary diagnosis.

Determination of Chlorpyrifos in Human Blood by Gas Chromatography-Mass Spectrometry

Gas chromatography-mass spectrometry method was developed for the qualitative and quantitative analyses of chlorpyrifos in human blood samples.The chlorpyrifos and parathion(internal standard)in human blood were extracted with a mixed solvent of hexane and acetonitrile.Chlorpyrifos was well separated from the internal standard.The linear range of chlorpyrifos was 0.01-2 μg/ml in blood.The limit of detection and limit of quantification were estimated at 0.002 and 0.01μg/ml,respectively.The inter-and intra-day precisions,accuracy,and recovery were assessed to verify this method.The results showed that the developed method is rapid,sensitive,and reliable.It is suitable for the determination of chlorpyrifos in forensic toxicological analysis and clinical diagnosis.