SIRT2 promotes base excision repair by transcriptionally activating OGG1 in an ATM/ATR-dependent manner.

Sirtuin 2 (SIRT2) regulates the maintenance of genome integrity by targeting pathways of DNA damage response and homologous recombination repair. However, whether and how SIRT2 promotes base excision repair (BER) remain to be determined. Here, we found that independent of its catalytic activity SIRT2 interacted with the critical glycosylase OGG1 to promote OGG1 recruitment to its own promoter upon oxidative stress, thereby enhancing OGG1 promoter activity and increasing BER efficiency. Further studies revealed that SIRT2 was phosphorylated on S46 and S53 by ATM/ATR upon oxidative stress, and SIRT2 phosphorylation enhanced the SIRT2-OGG1 interaction and mediated the stimulatory effect of SIRT2 on OGG1 promoter activity. We also characterized 37 cancer-derived SIRT2 mutants and found that 5 exhibited the loss of the stimulatory effects on OGG1 transcription. Together, our data reveal that SIRT2 acts as a tumor suppressor by promoting OGG1 transcription and increasing BER efficiency in an ATM/ATR-dependent manner.

The application potential of mepiquat chloride in soybean: improvement of yield characteristics and drought resistance.

BACKGROUND: Drought can result in yield losses, the application of plant growth regulators is an effective measure to improve drought resistance and yield. The objective of the study was to explore the application potential of mepiquat chloride (MC) in regulating soybean yield and drought resistance. METHODS: In this study, a three-year field experiment was designed and combined with drought experiments to measure the yield of popularized varieties during 2021-2022 and drought-resistant and drought-sensitive varieties were selected, and planted in the field in 2023. RESULTS: MC increased the yield of HN84 and HN87 for two consecutive years from 2021 to 2022 and improved their physiological characteristics under field conditions. Under M200 treatment, the yield of HN84 increased by 6.93% and 9.46%, and HN87 increased by 11.11% and 15.72%. Different concentrations of MC have different effects on soybeans. The maximum increase of SOD, POD and proline in HN84 under M400 treatment reached 71.92%, 63.26% and 71.54%, respectively; the maximum increase of SOD, POD and proline in HN87 under M200 treatment reached 21.96%, 93.49% and 40.45%, respectively. In 2023, the foliar application of MC improved the physiological characteristics of HN44 and HN65 under drought-stress conditions. On the eighth day of drought treatment, compared to the drought treatment, the leaf and root dry weight of HN44 under M100 treatment increased by 17.91% and 32.76%, respectively; the dry weight of leaves and roots of HN65 increased by 20.74% and 29.29% under M200 treatment, respectively. MC also reduced malondialdehyde (MDA) content, decreased antioxidant enzyme activity and proline content. In addition, different concentrations of MC increased the chlorophyll fluorescence parameters (Fs, Fv/Fm, YII, and SPAD). In the field, the plant height of the two varieties decreased significantly, the yield increased, the number of two-grain and three-grain pods increased, and the stem length at the bottom and middle decreased with MC induction. CONCLUSIONS: The application of 100-200 mg/L MC effectively improved drought resistance and increased yield. This study provided support for the rational application of MC in soybean production.

Biochemical characterization and metabolic reprogramming of amino acids in Soybean roots under drought stress.

Amino acids play important roles in stress resistance, plant growth, development, and quality, with roots serving as the primary organs for drought response. We conducted biochemical and multi-omics analyses to investigate the metabolic processes of root amino acids in drought-resistant (HN44) and drought-sensitive (HN65) soybean (Glycine max) varieties. Our analysis revealed an increase in total amino acid content in both varieties, with phenylalanine, proline, and methionine accumulating in both. Additionally, several amino acids exhibited significant decreases in HN65 but slight increases in HN44. Multi-omics association analysis identified 13 amino acid-related pathways. We thoroughly examined the changes in genes and metabolites involved in various amino acid metabolism/synthesis and determined core genes and metabolites through correlation networks. The phenylalanine, tyrosine, and tryptophan metabolic pathways and proline, glutamic acid and sulfur-containing amino acid pathways were particularly important for drought resistance. Some candidate genes, such as ProDH and P4HA family genes, and metabolites, such as O-acetyl-L-serine, directly affected up- and downstream metabolism to induce drought resistance. This study provided a basis for soybean drought resistance breeding.

Deep learning-based pathology signature could reveal lymph node status and act as a novel prognostic marker across multiple cancer types.

BACKGROUND: Identifying lymph node metastasis (LNM) relies mainly on indirect radiology. Current studies omitted the quantified associations with traits beyond cancer types, failing to provide generalisation performance across various tumour types. METHODS: 4400 whole slide images across 11 cancer types were collected for training, cross-verification, and external validation of the pan-cancer lymph node metastasis (PC-LNM) model. We proposed an attention-based weakly supervised neural network based on self-supervised cancer-invariant features for the prediction task. RESULTS: PC-LNM achieved a test area under the curve (AUC) of 0.732 (95% confidence interval: 0.717-0.746, P < 0.0001) in fivefold cross-validation of multiple cancer types, which also demonstrated good generalisation in the external validation cohort with AUC of 0.699 (95% confidence interval: 0.658-0.737, P < 0.0001). The interpretability results derived from PC-LNM revealed that the regions with the highest attention scores identified by the model generally correspond to tumours with poorly differentiated morphologies. PC-LNM achieved superior performance over previously reported methods and could also act as an independent prognostic factor for patients across multiple tumour types. DISCUSSION: We presented an automated pan-cancer model for predicting the LNM status from primary tumour histology, which could act as a novel prognostic marker across multiple cancer types.

Graph model for multiple scattering in lithium niobate on insulator integrated photonic networks.

We present a graph-based model for multiple scattering of light in integrated lithium niobate on insulator (LNOI) networks, which describes an open network of single-mode integrated waveguides with tunable scattering at the network nodes. We first validate the model at small scale with experimental LNOI resonator devices and show consistent agreement between simulated and measured spectral data. Then, the model is used to demonstrate a novel platform for on-chip multiple scattering in large-scale optical networks up to few hundred nodes, with tunable scattering behaviour and tailored disorder. Combining our simple graph-based model with material properties of LNOI, this platform creates new opportunities to control randomness in large optical networks.

Reintroduction of highly pathogenic avian influenza A H7N9 virus in southwestern China.

Highly pathogenic (HP) avian influenza A H7N9 virus has emerged in China since 2016. In recent years, it has been most prevalent in northern China. However, several strains of HP H7N9 reappeared in southwestern China (Yunnan Province) in 2021. As a result, we are wondering if these viruses have re-emerged in situ or been reintroduced. Here, we present phylogenetic evidence that the HP H7N9 viruses isolated in Yunnan emigrated from northern to southwestern China in 2020. The northern subregion of China has become a novel epicenter in HP H7N9 dissemination. Meanwhile, a cleavage motif re-emerged due to the T341I mutation, implying a parallel evolution. This cross-region transmission, which originated in non-adjacent provinces and traveled a great geographic distance in an unknown way, indicates that HP H7N9 dissemination did not halt in 2020, even under the shadow of the COVID-19 pandemic. Additional surveillance studies in poultry are required to determine the HP H7N9 virus's geographic distribution and spread.

Development of chlorine dioxide sustained-release device using carboxymethyl cellulose-polyvinyl alcohol-ß-cyclodextrin ternary hydrogel and a new sustained-release kinetic model.

Owing to unique physiochemical and biological properties as well as the ability to be combined with a wide variety of materials for both biocompatibility and hydrophilia, carboxymethyl cellulose (CMC) is an excellent choice as a carrier. Loading Chlorine dioxide (ClO2) into biodegradable carrier for its good disinfection performance and high safety factors has attracted significantattention. Therefore, in this study, we used ClO2 as a model drug, and a sustained-ClO2-gas-release gel was developed from degradable materials, such as carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), and ß-cyclodextrin (ßCD), through a simple and benign crosslinking strategy. Notably, the gel had sustained-release property in a wide temperature range of 4-35 â„ƒ and released ClO2 gas effectively for more than 30 days. Furthermore, a loss factor was proposed based on the incomplete release of the drug in the sustained release process to a chieve a good fit with the gas diffusion process. A new diffusion model was designed based on the Korsmeyer-Peppas model, and an excellent fit was obtained. This sustained-ClO2-gas-release gel provides theoretical and technical guidance for the development of sustained-disinfectant-release agents for use in space and offers new insights into the sustained release model of skeleton-soluble hydrogels. Supplementary Information: The online version contains supplementary material available at 10.1007/s10570-023-05070-6.

Lysine Deprivation Suppresses Adipogenesis in 3T3-L1 Cells: A Transcriptome Analysis.

Growing evidence proves that amino acid restriction can reverse obesity by reducing adipose tissue mass. Amino acids are not only the building blocks of proteins but also serve as signaling molecules in multiple biological pathways. The study of adipocytes' response to amino acid level changes is crucial. It has been reported that a low concentration of lysine suppresses lipid accumulation and transcription of several adipogenic genes in 3T3-L1 preadipocytes. However, the detailed lysine-deprivation-induced cellular transcriptomic changes and the altered pathways have yet to be fully studied. Here, using 3T3-L1 cells, we performed RNA sequencing on undifferentiated and differentiated cells, and differentiated cells under a lysine-free environment, and the data were subjected to KEGG enrichment. We found that the differentiation process of 3T3-L1 cells to adipocytes required the large-scale upregulation of metabolic pathways, mainly on the mitochondrial TCA cycle, oxidative phosphorylation, and downregulation of the lysosomal pathway. Single amino acid lysine depletion suppressed differentiation dose dependently. It disrupted the metabolism of cellular amino acids, which could be partially reflected in the changes in amino acid levels in the culture medium. It inhibited the mitochondria respiratory chain and upregulated the lysosomal pathway, which are essential for adipocyte differentiation. We also noticed that cellular interleukin 6 (IL6) expression and medium IL6 level were dramatically increased, which was one of the targets for suppressing adipogenesis induced by lysine depletion. Moreover, we showed that the depletion of some essential amino acids such as methionine and cystine could induce similar phenomena. This suggests that individual amino acid deprivation may share some common pathways. This descriptive study dissects the pathways for adipogenesis and how the cellular transcriptome was altered under lysine depletion.

[Optimization of ethanol reflux extraction process of Ziziphi Spinosae Semen- Schisandrae Sphenantherae Fructus based on network pharmacology combined with response surface methodology].

The present study optimized the ethanol extraction process of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus drug pair by network pharmacology and Box-Behnken method. Network pharmacology and molecular docking were used to screen out and verify the potential active components of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus, and the process evaluation indexes were determined in light of the components of the content determination under Ziziphi Spinosae Semen and Schisandrae Sphenantherae Fructus in the Chinese Pharmacopoeia(2020 edition). The analytic hierarchy process(AHP) was used to determine the weight coefficient of each component, and the comprehensive score was calculated as the process evaluation index. The ethanol extraction process of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus was optimized by the Box-Behnken method. The core components of the Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus drug pair were screened out as spinosin, jujuboside A, jujuboside B, schisandrin, schisandrol, schisandrin A, and schisandrin B. The optimal extraction conditions obtained by using the Box-Behnken method were listed below: extraction time of 90 min, ethanol volume fraction of 85%, and two times of extraction. Through network pharmacology and molecular docking, the process evaluation indexes were determined, and the optimized process was stable, which could provide an experimental basis for the production of preparations containing Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus.

A dominant internal gene cassette of high pathogenicity avian influenza H7N9 virus raised since 2018.

The zoonotic H7N9 avian influenza virus emerged with the H9N2-origin internal gene cassette. Previous studies have reported that genetic reassortments with H9N2 were common in the first five human H7N9 epidemic waves. However, our latest work found that the circulating high pathogenicity H7N9 virus has established a dominant internal gene cassette and has decreased the frequency of reassortment with H9N2 since 2018. This dominant cassette of H7N9 was distinct from the cocirculating H9N2, although they shared a common ancestor. As a result, we suppose that this dominant cassette may benefit the viral population fitness and promote its continuous circulation in chickens.

Transcriptomic and Metabolomic Analysis of Seedling-Stage Soybean Responses to PEG-Simulated Drought Stress.

Soybean is an important crop grown worldwide, and drought stress seriously affects the yield and quality of soybean. Therefore, it is necessary to elucidate the molecular mechanisms underlying soybean resistance to drought stress. In this study, RNA-seq technology and ultra-performance liquid chromatography-tandem mass spectrometry were used to analyze the transcriptome and metabolome changes in soybean leaves at the seedling stage under drought stress. The results showed that there were 4790 and 3483 DEGs (differentially expressed genes) and 156 and 124 DAMs (differentially expressed metabolites), respectively, in the HN65CK vs. HN65S0 and HN44CK vs. HN44S0 comparison groups. Comprehensive analysis of transcriptomic and metabolomic data reveals metabolic regulation of seedling soybean in response to drought stress. Some candidate genes such as LOC100802571, LOC100814585, LOC100777350 and LOC100787920, LOC100800547, and LOC100785313 showed different expression trends between the two cultivars, which may cause differences in drought resistance. Secondly, a large number of flavonoids were identified, and the expression of Monohydroxy-trimethoxyflavone-O-(6″-malonyl)glucoside was upregulated between the two varieties. Finally, several key candidate genes and metabolites involved in isoflavone biosynthesis and the TCA cycle were identified, suggesting that these metabolic pathways play important roles in soybean response to drought. Our study deepens the understanding of soybean drought resistance mechanisms and provides references for soybean drought resistance breeding.

Chemical formation and source apportionment of PM2.5 at an urban site at the southern foot of the Taihang mountains.

The region along the Taihang Mountains in the North China Plain (NCP) is characterized by serious fine particle pollution. To clarify the formation mechanism and controlling factors, an observational study was conducted to investigate the physical and chemical properties of the fine particulate matter in Jiaozuo city, China. Mass concentrations of the water-soluble ions (WSIs) in PM2.5 and gaseous pollutant precursors were measured on an hourly basis from December 1, 2017, to February 27, 2018. The positive matrix factorization (PMF) method and the FLEXible PARTicle (FLEXPART) model were employed to identify the sources of PM2.5. The results showed that the average mass concentration of PM2.5 was 111 µg/m3 during the observation period. Among the major WSIs, sulfate, nitrate, and ammonium (SNA) constituted 62% of the total PM2.5 mass, and NO3- ranked the highest with an average contribution of 24.6%. NH4+ was abundant in most cases in Jiaozuo. According to chemical balance analysis, SO42-, NO3-, and Cl- might be present in the form of (NH4)2SO4, NH4NO3, NH4Cl, and KCl. The liquid-phase oxidation of SO2 and NO2 was severe during the haze period. The relative humidity and pH were the key factors influencing SO42- formation. We found that NO3- mainly stemmed from homogeneous gas-phase reactions in the daytime and originated from the hydrolysis of N2O5 in the nighttime, which was inconsistent with previous studies. The PMF model identified five sources of PM2.5: secondary origin (37.8%), vehicular emissions (34.7%), biomass burning (11.5%), coal combustion (9.4%), and crustal dust (6.6%).

Synthesis of 3D magnetic porous carbon derived from a metal-organic framework for the extraction of clenbuterol and ractopamine from mutton samples.

3D magnetic porous carbon (MPCK) was prepared using the metal-organic framework (MOF) MIL-100(Fe) as the carbon precursor by carbonisation and KOH activation strategies. Carbonisation and activation ensured that MPCK possessed excellent structural and thermal stability, strong magnetic responsiveness and high surface area. MPCK was used as an adsorbent for the magnetic solid-phase extraction of clenbuterol and ractopamine from mutton samples. The concentration levels of analytes were determined by ultra-high performance liquid chromatography-mass spectrometry. Under optimised extraction conditions, the peak area responded linearly to analytes over the concentration range from 0.05 µg L-1 to 40 µg L-1 (r ≥ 0.9972). The detection limits of clenbuterol and ractopamine were found to be 0.130 µg kg-1 and 0.150 µg kg-1, respectively. The satisfactory recoveries in mutton samples ranging from 95.64% to 114.65% indicated that 3D porous carbon is a promising adsorption material for the extraction of clenbuterol and ractopamine from complex biological matrixes.

Acoustic analysis and detection of pharyngeal fricative in cleft palate speech using correlation of signals in independent frequency bands and octave spectrum prominent peak.

BACKGROUND: Pharyngeal fricative is one typical compensatory articulation error of cleft palate speech. It passively influences daily communication for people who suffer from it. The automatic detection of pharyngeal fricatives in cleft palate speech can provide information for clinical doctors and speech-language pathologists to aid in diagnosis. RESULTS: This paper proposes two features (CSIFs: correlation of signals in independent frequency bands; OSPP: octave spectrum prominent peak) to detect pharyngeal fricative speech. CSIFs feature is proposed to detect the distribution characteristics of frequency components in pharyngeal fricative speech caused by the changed place of articulation and movement of articulators. While OSPP is presented to reflect the concentration degree of prominent peak which is closely related to the place of articulation in pharyngeal fricative, both features are investigated to relate to the altered production process of pharyngeal fricative. To evaluate the capability of these two features to detect pharyngeal fricative, we collected a speech database covering all the types of initial consonants in which pharyngeal fricatives occur. In this detection task, the classifier used to discriminate pharyngeal fricative speech and normal speech is based on ensemble learning. CONCLUSION: The detection accuracy obtained with CSIFs and OSPP features ranges from 83.5 to 84.5% and from 85 to 87%, respectively. When these two features are combined, the detection accuracy for pharyngeal fricative speech ranges from 88 to 89%, with an AUC (area under the receiver operating characteristic curve) value of 93%.

miR-25-3p promotes proliferation and inhibits autophagy of renal cells in polycystic kidney mice by regulating ATG14-Beclin 1.

MicroRNAs are involved in the regulation of the autophagy and proliferation in several diseases. This study aims to verify the role of miR-25-3p in the proliferation and autophagy of renal cells in polycystic kidney disease (PKD). We found that kidney to body weight and blood urea content were increased in PKD mice. Cystic dilations were increased in kidney tissue from PKD mice, and autophagy-related protein ULK1 and the ratio of LC3-II/LC3-I were decreased, indicating autophagy was inhibited in PKD mice. In addition, miR-25-3p was upregulated in PKD mice, and inhibition of miR-25-3p decreased cystic dilations in kidney tissues, increased ULK1 expression and the ratio of LC3-II/LC3-I, indicating inhibition of miR-25-3p enhanced the autophagy in PKD. Besides, inhibition of miR-25-3p suppressed the proliferation of renal cells and downregulated E2F-1 and PCNA expressions. Importantly, miR-25-3p targetedly suppressed ATG14 expression in PKD cells. Finally, silencing ATG14 abolished the inhibition effect of miR-25-3p inhibitor on renal cell proliferation, and reversed the inhibition effect of miR-25-3p inhibitor on E2F-1 and PCNA expressions in in vitro and in vivo experiments, which suggested that ATG14 was involved in the regulation of miR-25-3p-mediated kidney cell proliferation. Therefore, inhibition of miR-25-3p promoted cell autophagy and suppressed cell proliferation in PKD mice through regulating ATG14.

Mercury in Municipal Sewage and Sewage Sludge.

Wide occurrence of mercury species, including the highly toxic and readily bioaccumulative methylmercury (MeHg), in municipal sewage (MS) and sewage sludge (SS) has been evidenced in recent studies. Considering that vast amounts of MS and SS are produced globally each year and the majority of MS is discharged into aqueous environments, i.e., the main sites for MeHg bioaccumulation, special attention should be paid on the source and environmental behaviors of sewage-borne and sludge-borne mercury species. This review aims to summarize the findings on the occurrence of mercury species in MS and SS, their transport and transformation in MS treatment plants, as well as their fate and environmental implications.

Does economic development improve urban greening? Evidence from 289 cities in China using spatial regression models.

Significant differences in urban greening have occurred in Chinese cities, accompanied by China's rapid urbanization. However, there are relatively few studies on the spatial differentiation of urban greening in China at the city level. In addition, there is no unanimous conclusion on the main factors influencing the spatial differentiation of urban greening. Based on 2014 emission inventory data from 289 cities, the spatial differentiation pattern and spatial correlation characteristics of the urban green space ratio, urban green coverage rate, and public green area per capita were calculated and analyzed using global and local Moran's I. We then used ordinary least squares, spatial error model, spatial autoregression, and geographically weighted regression to quantify the impact and spatial variations of China's economy on urban greening. The results showed (1) a significant spatial dependence and heterogeneity existed in urban greening values, and the patterns showed influences of both the stage of economic development and spatial agglomeration; (2) regression models revealed per capita GDP had a positive effect on the urban green space ratio and public green area per capita while the urbanization rate, secondary industry, urban land, and population density had opposite effects on these two greening indexes; and (3) geographically weighted regression revealed per capita GDP had a greater influence on urban greening in the northwestern region than in the southeastern region. The study could constitute a valuable reference for mid-to-long-term green space planning policy in diverse parts of China and could further assist in coordinating the development of urban greening and economic growth.

Analysis of microcystins using high-performance liquid chromatography and magnetic solid-phase extraction with silica-coated magnetite with cetylpyridinium chloride.

Microcystins (MCs), produced by freshwater cyanobacteria, can be serious water pollutants, so it is important to monitor their concentration in drinking water. We have developed a method for rapid and accurate determination of microcystin levels in environmental water, using magnetic solid-phase extraction and high-performance liquid chromatography with UV detection. The magnetic composite material, which was combined with cetylpyridinium chloride, was prepared by hydrothermal synthesis. The optimal extraction of microcystins in water sample was achieved by optimizing the amount of adsorbent, time of adsorption, ratio of eluting solvent, and volume of eluent. Under the optimal conditions, the limit of detection of MC-LR was 0.001 µg/L, and the limit of quantification was 0.0028 µg/L. The limit of detection of MC-RR was 0.001 µg/L, and the limit of quantification was 0.003 µg/L. These values are far lower than those established by the International Health Organization for the maximum concentration of microcystins in drinking water. The magnetic solid-phase extraction adsorbent used in this method has the advantages of simple preparation, low price, and easy solid-liquid separation, and it can be used for the rapid and sensitive monitoring of trace microcystins in environmental water samples.

Beyond cones: an improved model of whisker bending based on measured mechanics and tapering.

The sense of touch is represented by neural activity patterns evoked by mechanosensory input forces. The rodent whisker system is exceptional for studying the neurophysiology of touch in part because these forces can be precisely computed from video of whisker deformation. We evaluate the accuracy of a standard model of whisker bending, which assumes quasi-static dynamics and a linearly tapered conical profile, using controlled whisker deflections. We find significant discrepancies between model and experiment: real whiskers bend more than predicted upon contact at locations in the middle of the whisker and less at distal locations. Thus whiskers behave as if their stiffness near the base and near the tip is larger than expected for a homogeneous cone. We assess whether contact direction, friction, inhomogeneous elasticity, whisker orientation, or nonconical shape could explain these deviations. We show that a thin-middle taper of mouse whisker shape accounts for the majority of this behavior. This taper is conserved across rows and columns of the whisker array. The taper has a large effect on the touch-evoked forces and the ease with which whiskers slip past objects, which are key drivers of neural activity in tactile object localization and identification. This holds for orientations with intrinsic whisker curvature pointed toward, away from, or down from objects, validating two-dimensional models of simple whisker-object interactions. The precision of computational models relating sensory input forces to neural activity patterns can be quantitatively enhanced by taking thin-middle taper into account with a simple corrective function that we provide.

Preparation and application of a coated-fiber needle extraction device.

In this study, a needle-trap device with fibers coated with a molecularly imprinted polymer was developed for separation. A number of heat-resistant Zylon filaments were longitudinally packed into a glass capillary, followed by coating with a molecularly imprinted polymer. Then, the molecularly imprinted polymer coating was copolymerized and anchored onto the surface of the fibers. The bundle of synthetic fibers coated with the molecularly imprinted polymer was packed into a 21G stainless-steel needle and served as an extraction medium. The coated-fiber needle extraction device was used to extract volatile organic compounds from paints and gasoline effectively. Subsequently, the extracted volatile organic compounds were analyzed by gas chromatography. Calibration curves of gaseous benzene, toluene, ethylbenzene, and o-xylene in the concentration range of 1-250 µg/L were obtained to evaluate the method, acceptable linearity was attended with correlation coefficients above 0.998. The limit of detection of benzene, toluene, ethylbenzene, and o-xylene was 11-20 ng/L using the coated-fiber needle-trap device. The relative standard deviation of needle-to-needle repeatability was less than 8% with an extraction time of 20 min. The loss rates after storage for 3 and 7 days at room temperature were less than 30%.