Deubiquitinase cylindromatosis (CYLD) regulates antibacterial immunity and apoptosis in Chinese mitten crab (Eriocheir sinensis).

Ubiquitination and deubiquitination of target proteins is an important mechanism for cells to rapidly respond to changes in the external environment. The deubiquitinase, cylindromatosis (CYLD), is a tumor suppressor protein. CYLD from Drosophila melanogaster participates in the antimicrobial immune response. In vertebrates, CYLD also regulates bacterial-induced apoptosis. However, whether CYLD can regulate the bacterial-induced innate immune response in crustaceans is unknown. In the present study, we reported the identification and cloning of CYLD in Chinese mitten crab, Eriocheir sinensis. Quantitative real-time reverse transcription polymerase chain reaction analysis showed that EsCYLD was widely expressed in all the examined tissues and was upregulated in the hemolymph after Vibrio parahaemolyticus challenge. Knockdown of EsCYLD in hemocytes promoted the cytoplasm-to-nucleus translocation of transcription factor Relish under V. parahaemolyticus stimulation and increased the expression of corresponding antimicrobial peptides. In vivo, silencing of EsCYLD promoted the removal of bacteria from the crabs and enhanced their survival. In addition, interfering with EsCYLD expression inhibited apoptosis of crab hemocytes caused by V. parahaemolyticus stimulation. In summary, our findings revealed that EsCYLD negatively regulates the nuclear translocation of Relish to affect the expression of corresponding antimicrobial peptides and regulates the apoptosis of crab hemocytes, thus indirectly participating in the innate immunity of E. sinensis.

Integrinß1/FAK/ERK signalling pathway is essential for Chinese mitten crab Eriocheir sinensis hemocyte survival.

Integrins are cellular adhesion molecules that mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions. Integrins can stimulate various signaling pathways by binding to different ligands, thereby exerting immunological functions. While integrins have been found to primarily play a role in bacterial agglutination, phagocytosis, and inhibition of apoptosis in invertebrates, the specific signaling pathway and mechanism of action remain unclear. In vertebrates, ß1 integrin and extracellular matrix interactions can associate with focal adhesion kinase (FAK) to initiate MAPK/ERK signaling and regulate cell survival; however, in invertebrates (e.g., Chinese mitten crab), the mechanisms of integrins are poorly understood. The purpose of this study was to investigate whether integrinß1/FAK activation of the MAPK/ERK pathway regulates hemocyte survival and the associated mechanism. Treatment with an integrinß1 inhibitor RGD (a conserved tripeptide Arg-Gly-Asp), decreased the levels of FAK and ERK expression and phosphorylation, followed by an intensification of apoptosis. Similar results were obtained following siRNA knockdown of integrinß1 expression. We further found that the attenuation of ERK phosphorylation enhanced the level of Caspase-3 expression. Together, these findings suggest that integrinß1 activates the FAK/ERK signaling cascade and is involved in the survival of Chinese mitten crab hemocytes.

Effects of the tumor necrosis factor on hemocyte proliferation and bacterial infection in Chinese mitten crab (Eriocheir sinensis).

Tumor necrosis factor (TNF) is an important cytokine that can regulate a variety of cellular responses by binding tumor necrosis factor receptor (TNFR). We studied whether the TNF of Eriocheir sinensis can regulate hemocyte proliferation. The results showed that the EsTNF and EsTNFR were constitutively expressed in all tested tissues, including the heart, hepatopancreas, muscles, gills, stomachs, intestines, and hemocytes. We found that low levels of EsTNF and EsTNFR transcripts were present in hemocytes. The gene expression levels were significantly increased in the hemocytes after being stimulated by Staphylococcus aureus or Vibrio parahaemolyticus. We also found some genes related to cell proliferation were expressed at a higher level in pulsing rTNF-stimulated hemocytes compared with the control group. We also knocked down the EsTNFR gene with RNAi technology. The results showed that the expression level of these genes related to cell proliferation was significantly down-regulated compared with the control group when the TNF does not bind TNFR. We used Edu technology to repeat the above experiments and the results were similar. Compared with the control group, the hemocytes stimulated by rTNF showed more significant proliferation, and the proliferation rate was significantly down-regulated after knocking down the EsTNFR gene. Therefore, we indicate that TNF binding TNFR can affect the proliferation of E. sinensis hemocytes, which might be manifested by affecting the expression of some proliferation-related genes.

Immune functions of the Dscam extracellular variable region in Chinese mitten crab.

In arthropods, there is only a single copy of Down Syndrome Cell Adhesion Molecule (Dscam) in the genome, but it can exist as numerous splice variants. There are three hypervariable exons in the extracellular domain and one hypervariable exon in the transmembrane domain. In Chinese mitten crab (Eriocheir sinensis), exons 4, 6 and 14 can produce 25, 34 and 18 alternative splice variants, respectively. In this study, through Illumina sequencing, we identified additional splice variants for exons 6 and 14, hence there may be > 50,000 Dscam protein variants. Sequencing of exons 4, 6 and 14 showed that alternative splicing was altered after bacterial stimulation. Therefore, we expressed and purified the extracellular variable region of Dscam (EsDscam-Ig1-Ig7). Exons 4.3, 6.46 and 14.18, three variable exons of the recombinant protein, were randomly selected. The functions of EsDscam-Ig1-Ig7 in immune defences of E. sinensis were subsequently explored. EsDscam-Ig1-Ig7 was discovered to bind to both Gram-positive Staphylococcus aureus and Gram-negative Vibrio parahaemolyticus, but it did not exhibit antibacterial activity. By promoting hemocyte phagocytosis and bacterial removal, EsDscam-Ig1-Ig7 can also shield the host from bacterial infection. The findings highlight the immunological activities of Dscam alternative splicing and reveal the potential for many more Dscam isoforms than were previously predicted in E. sinensis.

Genome-wide identification and analysis of the MAPKK gene family in Chinese mitten crab (Eriocheir sinensis) and its response to bacterial challenge.

Protein kinases of the MAPK cascade family (MAPKKK-MAPKK-MAPK) play an important role in the growth and development of organisms and their response to environmental stress. The MAPKK gene families in the Chinese mitten crab Eriocheir sinensis have never been systematically analyzed. We identified four MAPKK family genes, EsMEK, EsMAPKK4, EsMAPKK6, and EsMAPKK7, in E. sinensis and analyzed their molecular features and expression patterns. All four MAPKK genes are composed of multiple exons and introns, all have a conserved domain, and all have 10 conserved motifs (except EsMEK and EsMAPKK7 which are missing motif 10). The four MAPKK genes are on four different chromosomes and have no gene duplications, and the results of phylogenetic tree analysis indicate that the ESMAPKK gene family is highly conserved evolutionarily. The EsMAPKK genes were widely expressed in all the examined tissues with higher expression in hemocytes, hepatopancreas, and gills. Notably, EsMAPKK6 was also highly expressed in the ovary. Vibrio parahaemolyticus infection significantly increased the mRNA levels of the EsMAPKK genes in hemocytes. Further disruption of the EsMAPKK gene family expression affects the expression levels of multiple antimicrobial peptides in hemocytes. Our experimental results provide a starting point for a more in-depth study of the innate immunity functional roles of members of the MAPKK gene families in E. sinensis.

Improving aptamer performance: key factors and strategies.

Aptamers are functional single-stranded oligonucleotide fragments isolated from randomized libraries by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), exhibiting excellent affinity and specificity toward targets. Compared with traditional antibody reagents, aptamers display many desirable properties, such as low variation and high flexibility, and they are suitable for artificial and large-scale synthesis. These advantages make aptamers have a broad application potential ranging from biosensors, bioimaging to therapeutics and other areas of application. However, the overall performance of aptamer pre-selected by SELEX screening is far from being satisfactory. To improve aptamer performance and applicability, various post-SELEX optimization methods have been developed in the last decade. In this review, we first discuss the key factors that influence the performance or properties of aptamers, and then we summarize the key strategies of post-SELEX optimization which have been successfully used to improve aptamer performance, such as truncation, extension, mutagenesis and modification, splitting, and multivalent integration. This review shall provide a comprehensive summary and discussion of post-SELEX optimization methods developed in recent years. Moreover, by discussing the mechanism of each approach, we highlight the importance of choosing the proper method to perform post-SELEX optimization.

A novel Dicistro-like virus discovered in Procambarus clarkii with "Black May" disease.

The peak period of morbidity and death in cultured Procambarus clarkii is around May each year and is called the "Black May" disease. The pathogen causing "Black May" disease is believed to be a white spot syndrome virus (WSSV). In 2018, a significant number of P. clarkii died in the pond culture of Xinglong Township, Xuyi County. Two sampling tests on the affected pond showed that, in addition to WSSV, a novel Dicistro-like virus (PcDV) was present. Genomic sequence analysis indicated that this new virus belongs to the Dicistroviridae family, Picornaviridaes order. A high number of spherical particles were detected in gill tissues of P. clarkii with "Black May" disease by electron microscopy, a finding consistent with viruses from the Picornaviridaes order. From October 2018 to September 2019, we took monthly samples from Hubei, Jiangsu and Anhui provinces, and tested for the presence of PcDV and WSSV in P. clarkii. The detection rates of PcDV in P. clarkii peaked from April to June, consistent with the onset of the "Black May" disease. In conclusion, we believe that the discovery of PcDV will provide new research directions for investigating the pathogens causing "Black May" disease in P. clarkii.

Response Surface Optimization of an Extraction Method for the Simultaneous Detection of Sulfamethoxazole and 17ß-Estradiol in Soil.

Antibiotics and hormones widely exist in fertilizers and manures, which are excessively used in agriculture and animal husbandry. Considering their potential harm to the environment and human health, the detection of antibiotics and hormones has become a necessity. However, current methods find it difficult to simultaneously extract and detect antibiotics and hormones in soil and to maintain a high level of accuracy and a low cost. In this study, a straightforward, convenient, and simultaneous extraction and detection method of a representative antibiotic (sulfamethoxazole, SMZ) and hormone (17ß-Estradiol, E2) in soil has been established. Ultrasound-assisted extraction (UAE) was used in the pretreatment process and high-performance liquid chromatography with the ultraviolet detector (HPLC-UV) method was then chosen in the detection process. By means of single factors and response surface experiments, optimal extraction conditions were a 41-mL buffer solution (pH 4.27) mixed with 1 g of soil sample, an ultrasonication time of 36 min, an ultrasonication temperature of 25 °C, and two extraction cycles. The detection limits of 0.3-10 µg/kg and quantification limits of 1-30 µg/kg have been obtained. Finally, the optimized simultaneous extraction and detection method was validated by three different real soil samples with recoveries ranging from 79.49% to 86.47%.

Fluorometric determination for ofloxacin by using an aptamer and SYBR Green I.

A fluorometric method is described for the determination of ofloxacin (OFL). It is based on the use of the fluorescent intercalator SYBR Green I (SG-I). The OFL-aptamer has G-quadruplex structures and can be recognized by SG-I. It results in strong fluorescence of SG-I. If OFL is present, OFL will bind to its aptamer to form stable complexes. This induces the despiralization of partial dsDNA regions, leads to changes in the structure of the aptamer. Thus, SG-I is released from the OFL-aptamer into solution. Hence, the fluorescence of SG-I drops. Fluorescence decreases linearly in the 1.1 to 200 nM OFL concentration range, and the limit of detection is 0.34 nM. The method shows good selectivity to much interference including analogues, hormones, pesticides. It is also effortless and fast with the times of measurement of <40 min. In addition, good recoveries of 91.3-119.0% were found for tap water, river water and artificial urine spiked with OFL with relative standard deviation (RSD) of ≤11.6%. Graphical abstract A sensitive fluorometric method is developed for ofloxacin (OFL) detection in aqueous samples based on the fluorescence intensity change of SYBR Green I (SG-I) with or without OFL.

Colorimetric determination of ofloxacin using unmodified aptamers and the aggregation of gold nanoparticles.

A colorimetric method is presented for the determination of the antibiotic ofloxacin (OFL) in aqueous solution. It is based on the use of an aptamer and gold nanoparticles (AuNPs). In the absence of OFL, the AuNPs are wrapped by the aptamer and maintain dispersed even at the high NaCl concentrations. The solution with colloidally dispersed AuNPs remains red and has an absorption peak at 520 nm. In the presence of OFL, it will bind to the aptamer which is then released from the AuNPs. Hence, AuNPs will aggregate in the salt solution, and color gradually turns to blue, with a new absorption peak at 650 nm. This convenient and specific colorimetric assay for OFL has a linear response in the 20 to 400 nM OFL concentration range and a 3.4 nM detection limit. The method has a large application potential for OFL detection in environmental and biological samples. Graphical abstract Schematic of a sensitive and simple colorimetric aptasensor for ofloxacin (OFL) detection in tap water and synthesic urine. The assay is based on the salt-induced aggregation of gold nanoparticles which results in a color change from red to purple.

Effects of biochar on the emissions, soil distribution, and nematode control of 1,3-dichloropropene.

Emissions of volatile soil fumigant 1,3-dichloropropene (1,3-D) from soil to air are a significant concern in relation to air quality, and cost-effective strategies to reduce such emissions are urgently required by growers to help them comply with increasingly stringent regulations. In this work, application of a rice husk-derived biochar to the surface of a sandy loam soil chamber reduced soil-air emissions of 1,3-D from 42% in a control (no biochar) to 8% due to adsorption onto the biochar. This adsorbed 1,3-D showed a potential for re-volatilization into air and solubilization into the soil-liquid phase. Biochar at the soil surface also reduced soil-gas concentrations in the upper soil; based on the determination of concentration-time values, this may limit 1,3-D-induced nematode control in the upper soil. In batch studies, the mixing of biochar into the soil severely limited nematode control; 1,3-D application rates around four times greater than the maximum permissible limit would be required to give nematode control under such conditions. Therefore, the use of biochar as a surface amendment, while showing an emission reduction benefit, may limit pest control during subsequent fumigations if, as seems probable, it is plowed into the soil.

Biochar Amendment to the Soil Surface Reduces Fumigant Emissions and Enhances Soil Microorganism Recovery.

During soil fumigation, it is ideal to mitigate soil fumigant emissions, ensure pest control efficacy, and speed up the recovery of the soil microorganism population established postapplication. However, no current fumigant emission reduction strategy can meet all these requirements. In the present study, replicated soil columns were used to study the effect of biochar derived from rice husk (BR) and green waste (BG) applied to the soil surface on 1,3-dichloropropene (1,3-D) and chloropicrin (CP) emissions and soil gas distribution, and on microorganism population re-establishment. Relative to fumigated bare soil (no emission reduction strategy), high-density polyethylene (HDPE), and ammonium thiosulfate (ATS) treatments, BR gave dramatic emission reductions for both fumigants with no obvious emission peak, whereas BG was very effective only for 1,3-D. With BR application, the concentration of fumigant in the soil gas was higher than in the bare soil and ATS treatment. After the soil column experiment, mixing the BR with the fumigated soil resulted in higher soil respiration rates than were observed for HDPE and ATS treatments. Therefore, biochar amendment to the soil surface may be an effective strategy for fumigant emission reduction and the recovery of soil microorganism populations established postapplication.

Kinetics and the mass transfer mechanism of hydrogen sulfide removal by biochar derived from rice hull.

UNLABELLED: The biochar derived from rice hull was evaluated for its abilities to remove hydrogen sulfide (H2S) from gas phase. The surface area and pH of the biochar were compared. The biochar derived from rice hull was evaluated for its abilities to remove hydrogen sulfide (H2S) from gas phase. The surface area and pH of the biochar were compared. The different pyrolysis temperature has great influence on the adsorption of H2S. At the different pyrolysis temperature, the H2S removal efficiency of rice hull-derived biochar was different. The adsorption capacities of biochar were 2.09 mg·g(-1), 2.65 mg·g(-1), 16.30 mg·g(-1), 20.80 mg·g(-1), and 382.70 mg·g(-1), which their pyrolysis temperatures were 100 °C, 200 °C, 300 °C, 400 °C and 500 °C respectively. Based on the Yoon-Nelson model, it analyzed the mass transfer mechanism of hydrogen sulfide adsorption by biochar. IMPLICATIONS: The paper focuses on the biochar derived from rice hull-removed hydrogen sulfide (H2S) from gas phase. The surface area and pH of the biochar were compared. The different pyrolysis temperatures have great influence on the adsorption of H2S. At the different pyrolysis temperatures, the H2S removal efficiency of rice hull-derived biohar was different. The adsorption capacities of biochar were 2.09, 2.65, 16.30, 20.80, and 382.70 mg·g(-1), and their pyrolysis temperatures were 100, 200, 300, 400, and 500 °C, respectively. Based on the Yoon-Nelson model, the mass transfer mechanism of hydrogen sulfide adsorption by biochar was analyzed.

Optimization of ultrasonic-assisted extraction for determination of polycyclic aromatic hydrocarbons in biochar-based fertilizer by gas chromatography-mass spectrometry.

Application of biochar-based fertilizers is increasingly being considered for its potential agronomic and environmental benefits. However, biochar may contain residues of polycyclic aromatic hydrocarbons (PAHs) as a result of its production by pyrolysis. The strong adsorption of PAHs to biochar makes extraction and analysis of biochar-based fertilizers difficult. This study optimizes the extraction of PAHs in biochar-based fertilizer samples by using an ultrasonic bath for quantification by gas chromatography-mass spectrometry. Among 12 solvents, acetone-cyclohexane (1:1) mixture was selected as the optimum solvent for extraction. Three variables affecting the extraction were studied by Box-Behnken design. The optimum conditions were 57 °C extraction temperature, 81 min extraction time, and two extraction cycles, which were validated by assessing the linearity of analysis, LOD, LOQ, recovery, and levels of PAHs in real biochar-based fertilizer samples. Results revealed that the 16 U.S. EPA PAHs had good linearity, with squared correlation coefficients greater than 0.99. LODs were low, ranging from 2.2 ng g(-1) (acenaphthene) to 23.55 ng g(-1) (indeno[1,2,3-cd]perylene), and LOQs varied from 7.51 ng g(-1) to 78.49 ng g(-1). The recoveries of 16 individual PAHs from the three biochar-based fertilizer samples were 81.8-109.4 %. Graphical Abstract Use of RSM to optimize UAE for extraction of the PAHs in biochar-based fertilizer.

Optimization and determination of polycyclic aromatic hydrocarbons in biochar-based fertilizers.

The agronomic benefit of biochar has attracted widespread attention to biochar-based fertilizers. However, the inevitable presence of polycyclic aromatic hydrocarbons in biochar is a matter of concern because of the health and ecological risks of these compounds. The strong adsorption of polycyclic aromatic hydrocarbons to biochar complicates their analysis and extraction from biochar-based fertilizers. In this study, we optimized and validated a method for determining the 16 priority polycyclic aromatic hydrocarbons in biochar-based fertilizers. Results showed that accelerated solvent extraction exhibited high extraction efficiency. Based on a Box-Behnken design with a triplicate central point, accelerated solvent extraction was used under the following optimal operational conditions: extraction temperature of 78°C, extraction time of 17 min, and two static cycles. The optimized method was validated by assessing the linearity of analysis, limit of detection, limit of quantification, recovery, and application to real samples. The results showed that the 16 polycyclic aromatic hydrocarbons exhibited good linearity, with a correlation coefficient of 0.996. The limits of detection varied between 0.001 (phenanthrene) and 0.021 mg/g (benzo[ghi]perylene), and the limits of quantification varied between 0.004 (phenanthrene) and 0.069 mg/g (benzo[ghi]perylene). The relative recoveries of the 16 polycyclic aromatic hydrocarbons were 70.26-102.99%.

Glutamine supplementation and immune function during heavy load training.

Athletes with heavy training loads are prone to infectious illnesses, suggesting that their training may suppress immune function. This study sought to determine whether supplementation with the amino acid glutamine, which supports immune health, alters immune function in athletes during heavy load training. 24 athletes were randomly assigned to either an experimental group (n = 12) or a control group (n = 12). Athletes exercised using heavy training loads for 6 weeks. Athletes in the experimental group took 10 g glutamine orally once a day beginning 3 weeks after initial testing, while athletes in the control group were given a placebo. Immune function was assessed by measuring the following immunity markers: CD4⁺ and CD8⁺ T cell counts, serum IgA, IgG, and IgM levels, and natural killer (NK) cell activity both before and after the completion of training. The percentages of circulating CD8⁺ T cells were significantly different before (39.13 ± 5.87%) and after (26.63 ± 3.95%) training in the experimental group (p < 0.05). Although CD8⁺ T cell percentages in the control group were similar before (38.57 ± 5.79%) and after (37.21 ± 5.58%) training, the post-training CD8⁺ T cell percentages were significantly different between the two groups (p < 0.05). The ratios of CD4⁺/CD8⁺ cells in the experimental group were significantly different before (0.91 ± 0.14) and after (1.39 ± 0.19) training (p < 0.05). The CD4⁺/CD8⁺ ratios in the control group were similar before (0.93  ± 0.15) and after (0.83 ± 0.11) training, but the post-training CD4⁺T/CD8⁺ T cell ratio was higher in the experimental group than in the control group (p < 0.05). NK cell activity was also significantly different between the two groups after training (experimental, 25.21 ± 3.12 vs. control, 20.21 ± 2.59; p < 0.05). However, no differences were observed in serum IgA, IgG, or IgM levels. Thus, glutamine supplementation may be able to restore immune function and reduce the immunosuppressive effects of heavy-load training.

Effect of biochar on nitrous oxide emission and its potential mechanisms.

Extensive use of biochar to mitigate nitrous oxide (N2O) emission is limited by the lack of understanding on the exact mechanisms altering N2O emission from biochar-amended soil. Biochars produced from rice straw and dairy manure at 350 and 500 degrees C by oxygen-limited pyrolysis were used to investigate their influence on N2O emission. A quadratic effect of biochar levels was observed on the N2O emissions. The potential mechanisms were explored by terminal restriction fragment length polymorphism (T-RFLP) and real-time polymerase chain reaction (qPCR). A lower relative abundance of bacteria, which included ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), was observed at 4% biochar application rate. Reduced copy numbers of the ammonia monooxygenase gene amoA and the nitrite reductase gene nirS coincided with decreased N2O emissions. Therefore, biochar may potentially alter N2O emission by affecting ammonia-oxidizing and denitrification bacteria, which is determined by the application rate of biochar in soil. Implications: Biochar research has received increased interest in recent years because of the potential beneficial effects of biochar on soil properties. Recent research shows that biochar can alter the rates of nitrogen cycling in soil systems by influencing nitrification and denitrification, which are key sources of the greenhouse gas nitrous oxide (N2O). However, there are still some controversial data. The purpose of this research was to (1) examine how applications of different dose of biochar to soil affect emission of N2O and (2) improve the understanding of the underlying mechanisms.

Real-Time Temperature Monitoring of Lithium Batteries Based on Ultrasonic Technology.

Electrochemical energy storage stations serve as an important means of load regulation, and their proportion has been increasing year by year. The temperature monitoring of lithium batteries necessitates heightened criteria. Ultrasonic thermometry, based on its noncontact measurement characteristics, is an ideal method for monitoring the internal temperature of lithium batteries. In this study, temperature and ultrasonic time delay measurement experiments were conducted on 18650 lithium batteries and laminated and wound lithium batteries to obtain the corresponding relationship between temperature and time delay and validate the temperature measurement for the same type of battery. The experimental results show that (1) the ultrasonic temperature measurement technique exhibits a relatively large error when used for 18650 Li-ion batteries under experimental conditions; (2) in the experiments on laminated and wound soft-pack lithium batteries, the relationship between temperature and time delay exhibits a nonlinear characteristic; and (3) under the experimental conditions, the ultrasonic temperature measurement errors were ±1.1 °C for stacked Li-ion batteries and ±1.4 °C for wound Li-ion batteries.

Review of Acoustic Agglomeration Technology Research.

Acoustic agglomeration is employed as a precursor technique that modifies the sound field of fine particles to increase their size, thereby facilitating more efficient emission control. This paper reviews progress in the field of acoustic agglomeration technology, clarifies the mechanisms at play within the acoustic agglomeration process, and outlines its applicability in both gas-liquid and gas-solid phases. Furthermore, it analyzes the factors impacting the efficacy of acoustic agglomeration, summarizes the numerical simulation research of acoustic agglomeration, and proposes directions for technological enhancement.

Combination of injection volume calibration by creatinine and MS signals' normalization to overcome urine variability in LC-MS-based metabolomics studies.

It is essential to choose one preprocessing method for liquid chromatography-mass spectrometry (LC-MS)-based metabolomics studies of urine samples in order to overcome their variability. However, the commonly used normalization methods do not substantially reduce the high variabilities arising from differences in urine concentration, especially for signal saturation (abundant metabolites exceed the dynamic range of the instrumentation) or missing values. Herein, a simple preacquisition strategy based on differential injection volumes calibrated by creatinine (to reduce the concentration differences between the samples), combined with normalization to "total useful MS signals" or "all MS signals", is proposed to overcome urine variabilities. This strategy was first systematically compared with other popular normalization methods by application to serially diluted urine samples. Then, the method has been verified using rat urine samples of pre- and postinoculation of Walker 256 carcinoma cells. The results showed that the calibration of injection volumes based on creatinine values could effectively eliminate intragroup differences caused by variations in the concentrations of urinary metabolites, thus giving better parallelism and clustering effects. In addition, peak area normalization could further eliminate intraclass differences. Therefore, the strategy of combining peak area normalization with calibration of injection volumes of urine samples based on their creatinine values is effective for solving problems associated with urinary metabolomics.