A Two-color Single-molecule Sequencing Platform and Its Clinical Applications.

DNA sequencers have become increasingly important research and diagnostic tools over the past 20 years. In this study, we developed a single-molecule desktop sequencer, GenoCare 1600 (GenoCare), which utilizes amplification-free library preparation and two-color sequencing-by-synthesis chemistry, making it more user-friendly compared with previous single-molecule sequencing platforms for clinical use. Using the GenoCare platform, we sequenced an Escherichia coli standard sample and achieved a consensus accuracy exceeding 99.99%. We also evaluated the sequencing performance of this platform in microbial mixtures and coronavirus disease 2019 (COVID-19) samples from throat swabs. Our findings indicate that the GenoCare platform allows for microbial quantitation, sensitive identification of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, and accurate detection of virus mutations, as confirmed by Sanger sequencing, demonstrating its remarkable potential in clinical application.

Occurrence and exposure risk assessment of pesticide residues in green tea samples cultivated in Hangzhou area, China.

The concentration of pesticide residues in 105 green tea samples grown in Hangzhou area were investigated. Of the 14 pesticides analysed using gas chromatography-tandem mass spectrometry and the 27 pesticides analysed using liquid chromatography-tandem mass spectrometry, only 18 were detected in the tea samples. The most frequently detected pesticide residues were imidacloprid (35.2%), acetamiprid (26.7%), carbendazim (21.0%), bifenthrin (21.0%), and cyhalothrin (19.1%). Carbofuran was the only pesticide which exceeded in one sample the maximum residue limit. The concentrations of the analytes in tea samples ranged from below the limit of detection (LOD) to 2.64 mg/kg. Their mean concentrations were all below the LOD, except for imidacloprid, acetamiprid, carbendazim, bifenthrin and cyhalothrin. Based on a preliminary long-term exposure assessment, the hazard quotient values of the detected pesticides varied in the range 0.47 × 10-3 - 1.1 × 10-3%, which indicates that these levels did not pose a risk to human health in Hangzhou area.

Monochloropropanediol in edible vegetable oils from Hangzhou market in China: occurrence and exposure risk assessment.

In this study, 241 vegetable-oil food samples were collected from the Hangzhou market in China and analysed for fatty acid esters of 3- and 2-monochloropropanediol (3-MCPD and 2-MCPD) using non-derivative gas chromatography tandem mass spectrometry (GC-MS/MS). Food consumption data were taken from a food consumption survey of urban and rural residents in Hangzhou city performed in 2010-2011. Levels of 3-MCPD esters in edible oil ranged from not detected to 7.98 mg/kg, and the highest mean levels were found in tea seed oil, with concentrations of 2.94 mg/kg. Esters of 2-MCPD levels ranged from not detected to 4.03 mg/kg, and the highest mean levels were also found in tea seed oil, containing 1.49 mg/kg. The range of mean dietary intake of 3-MCPD esters in different groups of edible oil was from 0.096 to 1.54 µg/kg body weight (bw) per day, which is lower than the tolerable daily intake (TDI) established by the European Food Safety Authority (EFSA) (2 µg/kg bw/day). For people aged above 6 years old, the dietary intake of 3-MCPD from edible oil was 0.42 µg/kg bw per day (mean) and 1.22 µg/kg bw per day (P97.5). The range of mean dietary intake of 2-MCPD esters in different groups of edible oil was from 0.025 to 0.79 µg/kg bw/day, and 2-MCPD esters intake was 0.20 µg/kg bw per day (mean) and 0.60 µg/kg bw per day (P97.5). In addition, the dietary intake exposure to 3-MCPD and 2-MCPD esters for urban residents was lower than that for rural residents. The findings indicate that the potential health risks caused by dietary 3-MCPD esters from edible oils were of low concern for most of the Hangzhou residents. However, the exposure risk for consumers with excessive consumption of certain kind of edible oil calls for attention.

Determination of phthalate esters in breast milk before and after frozen storage in milk storage bags.

This study describes the migration of 20 phthalates from breast milk storage bags to breast milk after storage frozen for 1 to 6 months. Analysis of the investigated phthalates was performed by GC-MS/MS. Nine phthalates were found in some of the 27 breast milk storage bags, six phthalates were detected in some of the 40 breast milk samples, but the concentrations were low, except for one sample, which contained dibutyl phthalate (DBP) at the level of 211.2 µg/kg. For most of the breast milk samples stored in breast milk storage bags, the migration of diisobutyl phthalate (DIBP), DBP and di-ethyl-hexyl phthalate (DEHP) increased with increasing storage time, but the migration of DBP and DEHP was lower than the migration limits of 300 µg/kg and 1500 µg/kg from the National Standards of the PRC even after storage for six months. The average daily intakes of DMP, DEP, DIBP, DBP and DEHP were 0.099, 0.39, 0.62, 2.89 and 1.13 µg/kg/d, respectively, and the hazard indices were far below 1.

Detection of 20 phthalate esters in breast milk by GC-MS/MS using QuEChERS extraction method.

A detection method for 20 different phthalate esters (PAEs) in breast milk analyzed by quick, easy, cheap, effective, rugged, and safe (QuEChERS) clean-up with five internal standards and quantitative GC-MS/MS was established. This method can effectively remove interfering substances such as lipids and fatty acids from breast milk using acetonitrile as the extraction solvent and PSA/C18 as clean-up materials. The 20 PAEs had a linear range of 5.0-500.0 µg/L and recoveries of 83.3-123.3% with RSDs of 0.2-7.6% (n = 6). The method detection limits (LODs) were 0.004-1.3 µg/kg. Dimethyl phthalate (DMP), Diethyl phthalate (DEP), Diisobutyl phthalate (DIBP), Di-n-butyl phthalate (DBP) and bis(2-ethylhexyl) phthalate (DEHP) were detected in the 35 breast milk samples, with median levels of 1.2, 6.8, 7.3, 32.9 and 13.6 µg/kg, respectively, and the concentrations of 20 PAEs ranged between 0.5 and 137.3 µg/kg. This is a fast, simple, sensitive and accurate method for the detection of 20 PAEs in breast milk or dairy products.

Mutation of a histidine-rich calcium-binding-protein gene in wheat confers resistance to Fusarium head blight.

Head or ear blight, mainly caused by Fusarium species, can devastate almost all staple cereal crops (particularly wheat), resulting in great economic loss and imposing health threats on both human beings and livestock1-3. However, achievement in breeding for highly resistant cultivars is still not satisfactory. Here, we isolated the major-effect wheat quantitative trait locus, Qfhs.njau-3B, which confers head blight resistance, and showed that it is the same as the previously designated Fhb1. Fhb1 results from a rare deletion involving the 3' exon of the histidine-rich calcium-binding-protein gene on chromosome 3BS. Both wheat and Arabidopsis transformed with the Fhb1 sequence showed enhanced resistance to Fusarium graminearum spread. The translation products of this gene's homologs among plants are well conserved and might be essential for plant growth and development. Fhb1 could be useful not only for curbing Fusarium head blight in grain crops but also for improving other plants vulnerable to Fusarium species.

Detection of 20 Phthalate Esters in Different Kinds of Food Packaging Materials by GC-MS/MS with Five Internal Standards.

>Background: Phthalate esters (PAEs) are a group of chemical compounds widely used as plasticizers to increase the flexibility of plastics that are used in the manufacturing of kitchen utensils and food containers. Objective: In this study, a simple, rapid, and sensitive method for the determination of 20 PAEs in different kinds of food packaging materials has been developed. Methods: Samples injected with five internal standards were extracted with acetonitrile saturated with n-hexane and then detected by GC-MS/MS without a purification step. Results: The standard calibration curves were linear for all analytes over the concentration range of 5-500 µg/L, and the correlation coefficients ranged from 0.9913 to 0.9999. The LODs and LOQs were in the ranges of 1.7-62.5 and 5.5-208.3 µg/kg, respectively. The accuracy of this method was evaluated by measuring the recovery from spiked samples. The recoveries of all 20 phthalates from samples spiked at three different concentrations were measured, and the recovery was in the range of 82.1-110.8% and the relative standard deviation range of recovery result (n = 6) was 0.3-9.7%. Conclusions: The method presented here is simple, rapid, and sensitive and can be applied to large-scale detection of PAEs in plastic materials. Highlights: Instead of only one solvent, acetonitrile saturated with n-hexane was used as the extraction solvent. Samples were pretreated without a purification step. Five internal standards were used for quantitative determination.

Determination of Sterigmatocystin in Infant Cereals from Hangzhou, China.

A simple method was developed for the determination of sterigmatocystin in infant cereals. The method consists of a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction approach and cleanup with an SPE HLB cartridge. After purification for separation and quantification of sterigmatocystin, HPLC with UV detection and diode-array detection (DAD) was used at sterigmatocystin levels >10 µg/kg, whereas ultra-performance LC with electrospray ionization (ESI) and tandem MS (MS/MS) was used at levels <10 µg/kg. The compound was determined by UV-DAD at 325 nm and by ESI-MS/MS in the positive ionization mode. Good r(2) values (≥0.99) were found with both UV-DAD and ESI-MS/MS, and satisfactory recoveries (84.8-96.2%) of sterigmatocystin in infant cereals were demonstrated, with RSD values ≤11.43%. The developed analytical method was used for the determination of sterigmatocystin in infant cereals from Hangzhou, China.

Rapid determination of nitrite in foods in acidic conditions by high-performance liquid chromatography with fluorescence detection.

In this study, a simple, rapid, and sensitive method for the determination of nitrite (NO2 (-) ) in food samples by high-performance liquid chromatography with fluorescence detection in acidic conditions had been developed. The derivatization of the nitrite with 2,3-diaminonaphthalene was performed in acidic conditions to yield the highly fluorescent 2,3-naphthotriazole, which was directly analyzed by high-performance liquid chromatography with fluorescence detection without adjusting the solution to alkaline. The analysis column was reversed-phase C8 column. A constant flow rate of 1.0 mL/min was employed using water/acetonitrile as the mobile phase in isocratic mode (70:30, v/v). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. The standard calibration curves were linear for nitrite in different matrixes in the concentration range of 0-100 µg/L, and the correlation coefficients ranged from 0.9978 to 0.9998. The limits of detection and quantification were in the ranges of 0.012-0.060 and 0.040-0.20 mg/kg, respectively. The recoveries of nitrite from samples spiked at three different concentrations were 74.0-113.2%, and the relative standard deviations of the recovery results (n = 6) were 1.67-10.8%. The proposed method has good repeatability and is very sensitive and simple. It has been successfully used to determine nitrite in foods.

Simultaneous determination of 20 phthalate esters in oil-free food by UPLC/MS/MS.

A quantitative method was established for the determination of 20 phthalate esters (PAEs) in oil-free food by ultra-performance LC/MS/MS and was used to evaluate the PAEs of 111 oil-free samples from supermarkets in Hangzhou City. By quantification with the internal standard D4-bis(2-ethylexyl) phthalate, linearity ranges of the 20 PAEs were observed with correlation coefficients of 0.9990-0.9999. For liquid and solid sample, the spiking recoveries were 65.5-129.9% with RSD of 2.7-9.7% and 70.9-126.9% with RSD of 1.6-9.8% (n = 6), and the method LODs were 0.05-7.4 microg/L and 0.6-14.4 microg/kg, respectively. Most of the 111 oil-free samples had detectable PAEs; the detection rate was 23.0-42.0%, and the concentration of PAEs was in the range of 1.0-38 610.9 microg/kg. The detection rate in the drink packaged in a glass bottle was the highest, next was laver, and the detection rate in the drink packaged in a plastic bottle was the lowest.

Ab initio calculations on halogen-bonded complexes and comparison with density functional methods.

A systematic theoretical investigation on a series of dimeric complexes formed between some halocarbon molecules and electron donors has been carried out by employing both ab initio and density functional methods. Full geometry optimizations are performed at the Moller-Plesset second-order perturbation (MP2) level of theory with the Dunning's correlation-consistent basis set, aug-cc-pVDZ. Binding energies are extrapolated to the complete basis set (CBS) limit by means of two most commonly used extrapolation methods and the aug-cc-pVXZ (X = D, T, Q) basis sets series. The coupled cluster with single, double, and noniterative triple excitations [CCSD(T)] correction term, determined as a difference between CCSD(T) and MP2 binding energies, is estimated with the aug-cc-pVDZ basis set. In general, the inclusion of higher-order electron correlation effects leads to a repulsive correction with respect to those predicted at the MP2 level. The calculations described herein have shown that the CCSD(T) CBS limits yield binding energies with a range of -0.89 to -4.38 kcal/mol for the halogen-bonded complexes under study. The performance of several density functional theory (DFT) methods has been evaluated comparing the results with those obtained from MP2 and CCSD(T). It is shown that PBEKCIS, B97-1, and MPWLYP functionals provide accuracies close to the computationally very expensive ab initio methods.

L-Proline as an efficient and reusable promoter for the synthesis of coumarins in ionic liquid.

The effect of L-proline as a promoter on the condensation reaction of salicylaldehyde or its derivatives with ethyl acetoacetate in neutral ionic liquid [emim]BF4 was studied. All reactions were carried out under mild reaction conditions and achieved high yields. Moreover, the ionic liquid containing L-proline could be recycled and reused for several times without noticeably decreasing in productivity. The results show that the L-proline-[emim]BF4 system has a potential in contribution to the development of environmentally friendly and inexpensive processes in organic syntheses.

Binding interactions of pefloxacin mesylate with bovine lactoferrin and human serum albumin.

The binding of pefloxacin mesylate (PFLX) to bovine lactoferrin (BLf) and human serum albumin (HSA) in dilute aqueous solution was studied using fluorescence spectra and absorbance spectra. The binding constant K and the binding sites n were obtained by fluorescence quenching method. The binding distance r and energy-transfer efficiency E between pefloxacin mesylate and bovine lactoferrin as well as human serum albumin were also obtained according to the mechanism of Förster-type dipole-dipole nonradiative energy-transfer. The effects of pefloxacin mesylate on the conformations of bovine lactoferrin and human serum albumin were also analyzed using synchronous fluorescence spectroscopy.

Fluorometric study on the interaction between lomefloxacin and bovine lactoferrin.

The binding of lomefloxacin to bovine lactoferrin (BLf) in a dilute aqueous solution was studied using fluorescence spectra. The binding constant (K) and the number of binding sites (n) were obtained by a fluorescence quenching method. The binding distance (r) and energy-transfer efficiency (E) between lomefloxacin and bovine lactoferrin were also obtained according to the mechanism of Foörster-type dipole-dipole nonradiative energy-transfer. The effect of lomefloxacin on the conformation of bovine lactoferrin was also analyzed by synchronous fluorescence spectroscopy. The interaction between lomefloxacin and bovine lactoferrin is strong. Lomefloxacin can affect the conformation of bovine lactoferrin to some degree.