Salmonella-mediated methionine deprivation drives immune activation and enhances immune checkpoint blockade therapy in melanoma.

BACKGROUND: Although immune checkpoint inhibitor (ICI)-based therapy is advantageous for patients with advanced melanoma, resistance and relapse are frequent. Thus, it is crucial to identify effective drug combinations and develop new therapies for the treatment of melanoma. SGN1, a genetically modified Salmonella typhimurium species that causes the targeted deprivation of methionine in tumor tissues, is currently under investigation in clinical trials. However, the inhibitory effect of SGN1 on melanoma and the benefits of SGN1 in combination with ICIs remain largely unexplored. Therefore, this study aims to investigate the antitumor potential of SGN1, and its ability to enhance the efficacy of antibody-based programmed cell death-ligand 1 (PD-L1) inhibitors in the treatment of murine melanoma. METHODS: The antitumor activity of SGN1 and the effect of SGN1 on the efficacy of PD-L1 inhibitors was studied through murine melanoma models. Further, The Cancer Genome Atlas-melanoma cohort was clustered using ConsensusClusterPlus based on the methionine deprivation-related genes, and immune characterization was performed using xCell, Microenvironment Cell Populations-counter, Estimation of Stromal and Immune cells in MAlignant Tumor tissues using Expression data, and immunophenoscore (IPS) analyses. The messenger RNA data on programmed death-1 (PD-1) immunotherapy response were obtained from the Gene Expression Omnibus database. Gene Set Enrichment Analysis of methionine deprivation-up gene set was performed to determine the differences between pretreatment responders and non-responders. RESULTS: This study showed that both, the intratumoral and the intravenous administration of SGN1 in subcutaneous B16-F10 melanomas, suppress tumor growth, which was associated with an activated CD8+T-cell response in the tumor microenvironment. Combination therapy of SGN1 with systemic anti-PD-L1 therapy resulted in better antitumor activity than the individual monotherapies, respectively, and the high therapeutic efficacy of the combination was associated with an increase in the systemic level of tumor-specific CD8+ T cells. Two clusters consisting of methionine deprivation-related genes were identified. Patients in cluster 2 had higher expression of methionine_deprivation_up genes, better clinical outcomes, and higher immune infiltration levels compared with patients in cluster 1. Western blot, IPS analysis, and immunotherapy cohort study revealed that methionine deficiency may show a better response to ICI therapy CONCLUSIONS：: This study reports Salmonella-based SGN1 as a potent anticancer agent against melanoma, and lays the groundwork for the potential synergistic effect of ICIs and SGN1 brought about by improving the immune microenvironment in melanomas.

[Determination of 14 aniline and benzidine compounds in soil by gas chromatography-mass spectrometry].

The complex matrix of soil samples and low extraction efficiency of aniline compounds limit many methods developed for detecting aniline and benzidine compounds in soil. In this study, a rapid and sensitive method based on gas chromatography-mass spectrometry was developed for the simultaneous determination of 14 aniline and benzidine compounds in soil. The collected soil samples were sealed with 5% sodium sulfite solution and refrigerated to inhibit the oxidation of the target compounds for up to 7 d. The extraction efficiencies of accelerated solvent extraction and oscillating dispersion extraction were compared, and the recovery of accelerated solvent extraction was found to be unsuitable. Hence, three-phase oscillating dispersion extraction was adopted. A certain amount of alkaline aqueous solution was added to the test system during extraction to improve the extraction efficiency because aniline and benzidine compounds are weakly alkaline substances. When the pH of the extracted water phase was greater than 12, relatively good recoveries were obtained. Next, a mixed solvent of ethyl acetate-methylene chloride (1∶4, v/v) was added to extract the target compounds via oscillation for 20 min. The solid phase was discarded via centrifugation, and the aqueous and organic phases were transferred to a liquid separation funnel for further separation. Finally, the organic phase was retained. This pretreatment process prevents the co-extraction of acidic compounds or other impurities, thereby enhancing the purification ability of the method. Solid phase extraction (SPE) is generally recommended for soil extraction and purification. A preliminary test showed that compared with other columns, the Florisil SPE column could better retain the target substances and exhibited higher elution efficiency. After purification, the organic phase was concentrated to 1 mL using a nitrogen blower. The analytes were analyzed by gas chromatography-mass spectrometry using a capillary column (DB-35MS, 30 m×0.25 mm×0.25 µm). The temperature program was optimized to separate the target compounds at the baseline. Specifically, the initial oven temperature was set to 60 ℃, held for 2 min, increased to 130 ℃ at a rate of 5 ℃/min, increased to 300 ℃ at a rate of 30 ℃/min, and held for 4 min. The injector and ion source temperatures were 250 and 300 ℃, respectively. Aniline-d5 and acenaphthene-d10 were used as the internal standards for quantification. The effects of antioxidant addition, extraction solvent type, salting out, and other factors on extraction efficiency were investigated. The results showed that the method performed well under the optimized experimental conditions when actual soils were used as real sample matrices. The accuracy and precision of the proposed method were verified. A total of 14 aniline and benzidine compounds demonstrated good linearities in the range of 0.5-100 mg/L. The method detection limits (MDLs) ranged from 0.02 to 0.07 mg/kg, and the limits of quantification (LOQs) ranged from 0.08 to 0.28 mg/kg. The target compounds were spiked at contents of 1 and 10 mg/kg. The spiked recoveries of the 14 targets in actual soils were 62.9%-101%, and the relative standard deviations (RSDs) of six precision tests were 3.8%-10.3%. The proposed method effectively inhibited the oxidation of aniline and benzidine compounds during extraction, and the target compounds exhibited high recoveries and good stabilities in the presence of three phases. Moreover, the operating procedure was simple and easy to implement. The proposed method was applied to the soil collected from an industrial enterprise in Jiangsu province that was suspected to be contaminated with aniline, and two aniline compounds were detected. The developed method requires a small sample size, and the preservation step is simple and effective. In addition, it can be applied to various types of actual soils. The method meets the requirements of current soil pollution risk control standards for aniline and benzidine compounds in soils.

[Determination of polycyclic aromatic hydrocarbons in airborne particles by microwave extraction-high performance liquid chromatography].

A method by using microwave extraction and high performance liquid chromatography was developed to determine polycyclic aromatic hydrocarbons (PAHs) in airborne particles. The glass/quartz fiber filter, on which particles adsorbed, was extracted with a mixed solvent n-hexane/acetone (1:1, v/v) using microwave, and the subsequent purification of the extract was carried out by a florisil cartridge. The prepared sample was determined by high performance liquid chromatography coupled with photodiode array (PDA) and fluorescence dual-detector. The target compounds showed good linearity between the mass concentration of 20.0 µg/L and 500 µg/L, with the correlation coefficients not less than 0.9960. The spiked recoveries of target compounds in blank filter ranged from 56.3%-101%. The current method has been applied to investigate the distribution of PAHs in airborne particles in Nanjing City.

[Determination of trace and ultra-trace level bromate in water by large volume sample injection with enrichment column for on-line preconcentration coupled with ion chromatography].

A method for the determination of trace and ultra-trace level bromate in water by ion chromatography with large volume sample injection for on-line preconcentration was established. A high capacity Dionex IonPac AG23 guard column was simply used as the enrichment column instead of the loop for the preconcentration of bromate. High purity KOH solution used as eluent for gradient elution was on-line produced by an eluent generator automatically. The results showed that a good linear relationship of bromate was exhibited in the range of 0.05-51.2 µg/L (r ≥ 0.999 5), and the method detection limit was 0.01 µg/L. Compared with conventional sample injection, the injection volume was up to 5 mL, and the enrichment factor of this method was about 240 times. This method was successfully applied for several real samples of pure water which were purchased in the supermarket, and the recoveries of bromate were between 90%-100% with the RSDs (n = 6) of 2.1%-6.4% at two spiked levels. This method without pretreatment is simple, and of high accuracy and precision. The preconcentration can be achieved by large volume sample injection. It is suitable for the analysis of trace and ultra-trace level bromate.

[Determination of 21 phenolic compounds in soil by ultrasonic extraction-gas chromatography].

An effective method was developed to determine phenolic compounds in soil by ultrasonic extraction-gas chromatography (GC). The soil sample was extracted with methylene dichloride-hexane (2:1, v/v) for 3 min with the extraction temperature lower than 50 degrees C for 3 times. The extract was purified by alkaline aqueous solution (pH > 12), and the phenolic compounds were dissolved in aqueous solution. After the organic solution was discarded, the aqueous solution was adjusted to pH <3 and subsequently extracted with methylene dichloride-ethyl acetate (4:1, v/v) twice. The analytes were determined by GC-FID and quantified by external standard method. The limits of quantification for all tested phenolic compounds were 0.01-0.06 mg/kg in about 10 g soil samples. The recoveries were between 62.9% and 111.4% with the relative standard deviations (RSDs) in the range of 4.3%-24.0% (n = 6). The results showed that this method is sensitive, accurate, and suitable for the determination of the 21 phenolic compounds in soil.

[Determination of trace phenol compounds using gas chromatography-mass spectrometry coupled with Tenax adsorption tube for enrichment of air samples].

A novel determination method for trace of seven phenol compounds in air samples has been established. They were collected with Tenax adsorption tube (180 mm x 60 mm glass tube packed with 150 mg Tenax (40 - 60 mesh)) and desorbed with methanol. Five microlitres of naphthalene-D8 (internal standard) solution was added to the eluate. One microlitre of the mixture solution was injected into an HP-5MS capillary column (30 m x 0.25 mm x 0.25 microm) and determined by gas chromatography-mass spectrometry with selected ion monitoring (GC-MS/SIM). The selected ions were m/z 94, 95, 66, 108, 107, 77, 90, 122, 121, 107 and 136. The quantitative ions, m/z 94 for phenol, m/z 108 for cresol, m/z 122 for xylenol and m/z 136 for internal standard, were selected. The average recoveries of phenol compounds (spiked at the levels of 0.25, 1.00, 5.00 microg) ranged from 92.4% to 102% and the relative standard deviations were less than 4.8%. When the air sample volume was 10 L, the detection limits were less than 0.001 mg/m3. Good linearities were observed in the range from 0.05 to 20.0 mg/L. The method is simple, fast, sensitive and accurate for the determination of phenol compounds in air samples.

[Determination of seven nitrobenzene compounds in water samples by single-drop microextraction with capillary gas chromatography].

A method suitable for the extraction of nitrobenzene, nitrotoluenes and nitrochlorobenzenes from water samples using single-drop microextraction with capillary gas chromatography is presented. Parameters, such as organic solvents, drop volume of organics, agitation, immerge depth of needle point, aqueous sample volume, exposure time and temperature were controlled and optimized. The developed protocol was found to yield a linear calibration curve in a concentration range from 0.8 to 32 microg/L for nitrobenzene and nitrotoluenes and a range from 0.04 to 3.2 microg/L for nitrochlorobenzenes (r2 > 0.999). The limits of detection were found to be in the range between 0.01 and 0.3 microg/L. The relative standard deviations (RSDs) and relative recoveries (n = 5) from tap water fortified samples were 3.1% -7.9%, and 101% - 105%, and from waste water fortified samples were 3.3% - 7.9%, and 92.5% - 97.0%, respectively. The single-drop microextraction is proved to be a environmental friendly, sensitive, fast and simple tool for the preconcentration of trace nitrobenzene, nitrotoluenes and nitrochlorobenzenes from water samples.