An unsupervised learning model based on CT radiomics features accurately predicts axillary lymph node metastasis in breast cancer patients: diagnostic study.

BACKGROUND: The accuracy of traditional clinical methods for assessing the metastatic status of axillary lymph nodes (ALNs) is unsatisfactory. In this study, the authors propose the use of radiomic technology and three-dimensional (3D) visualization technology to develop an unsupervised learning model for predicting axillary lymph node metastasis in patients with breast cancer (BC), aiming to provide a new method for clinical axillary lymph node assessment in patients with this disease. METHODS: In this study, we retrospectively analyzed the data of 350 patients with invasive BC who underwent lung-enhanced computed tomography (CT) and axillary lymph node dissection surgery at the Department of Breast Surgery of the Second Xiangya Hospital of Central South University. The authors used 3D visualization technology to create a 3D atlas of ALNs and identified the region of interest for the lymph nodes. Radiomic features were subsequently extracted and selected, and a prediction model for ALNs was constructed using the K-means unsupervised algorithm. To validate the model, the authors prospectively collected data from 128 BC patients who were clinically evaluated as negative at our center. RESULTS: Using 3D visualization technology, we extracted and selected a total of 36 CT radiomics features. The unsupervised learning model categorized 1737 unlabeled lymph nodes into two groups, and the analysis of the radiomic features between these groups indicated potential differences in lymph node status. Further validation with 1397 labeled lymph nodes demonstrated that the model had good predictive ability for axillary lymph node status, with an area under the curve of 0.847 (0.825-0.869). Additionally, the model's excellent predictive performance was confirmed in the 128 axillary clinical assessment negative cohort (cN0) and the 350 clinical assessment positive (cN+) cohort, for which the correct classification rates were 86.72 and 87.43%, respectively, which were significantly greater than those of clinical assessment methods. CONCLUSIONS: The authors created an unsupervised learning model that accurately predicts the status of ALNs. This approach offers a novel solution for the precise assessment of ALNs in patients with BC.

Prognostic value and immunological characteristics of a novel cuproptosis-related long noncoding RNAs risk signature in kidney renal clear cell carcinoma.

Background: Cuproptosis has been found as a novel cell death mode significantly associated with mitochondrial metabolism, which may be significantly associated with the occurrence and growth of tumors. LncRNAs take on critical significance in regulating the development of kidney renal clear cell carcinoma (KIRC), whereas the correlation between cuproptosis-related LncRNAs (CRLs) and KIRC is not clear at present. Therefore, this study built a prognosis signature based on CRLs, which can achieve accurate prediction of the outcome of KIRC patients. Methods: The TCGA database provided the expression profile information and relevant clinical information of KIRC patients. Univariate Cox, Lasso, and multivariate Cox were employed for building a risk signature based on CRLs. Kaplan-Meier (K-M) survival analysis and time-dependent receiver operating characteristic (ROC) curve were employed for the verification and evaluation of the reliability and accuracy of risk signature. Then, qRT-PCR analysis of risk LncRNAs was conducted. Finally, the possible effect of the developed risk signature on the microenvironment for tumor immunization was speculated in accordance with ssGSEA and ESTIMATE algorithms. Results: A prognosis signature composed of APCDD1L-DT, MINCR, AL161782.1, and AC026401.3 was built based on CRLs. As revealed by the results of the K-M survival study, the OS rate and progression-free survival rate of highrisk KIRC patients were lower than those of lowrisk KIRC patients, and the areas under ROC curves of 1, 3, and 5 years were 0.828, 0.780, and 0.794, separately. The results of the immune analysis showed that there were significant differences in the status of immunization and the microenvironment of tumor between groups at low-risk and at high-risk. The qRT-PCR results showed that the relative expression level of MINCR and APCDD1L-DT were higher in 786-O and 769-P tumor cells than in HK-2 cells, which were normal renal tubular epithelial cells. Conclusion: The developed risk signature takes on critical significance in the prediction of the prognosis of patients with KIRC, and it can bring a novel direction for immunotherapy and clinical drug treatment of KIRC. In addition, 4 identified risk LncRNAs (especially APCDD1L-DT and MINCR) can be novel targets for immunotherapy of KIRC patients.

A novel defined risk signature based on pyroptosis-related genes can predict the prognosis of prostate cancer.

BACKGROUND: Pyroptosis can not only inhibit the occurrence and development of tumors but also develop a microenvironment conducive to cancer growth. However, pyroptosis research in prostate cancer (PCa) has rarely been reported. METHODS: The expression profile and corresponding clinical data were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Patients were divided into different clusters using consensus clustering analysis, and differential genes were obtained. We developed and validated a prognostic biomarker for biochemical recurrence (BCR) of PCa using univariate Cox analysis, Lasso-Cox analysis, Kaplan-Meier (K-M) survival analysis, and time-dependent receiver operating characteristics (ROC) curves. RESULTS: The expression levels of most pyroptosis-related genes (PRGs) are different not only between normal and tumor tissues but also between different clusters. Cluster 2 patients have a better prognosis than cluster 1 patients, and there are significant differences in immune cell content and biological pathway between them. Based on the classification of different clusters, we constructed an eight genes signature that can independently predict the progression-free survival (PFS) rate of a patient, and this signature was validated using a GEO data set (GSE70769). Finally, we established a nomogram model with good accuracy. CONCLUSIONS: In this study, PRGs were used as the starting point and based on the expression profile and clinical data, a prognostic signature with a high predictive value for biochemical recurrence (BCR) following radical prostatectomy (RP) was finally constructed, and the relationship between pyroptosis, immune microenvironment, and PCa was explored, providing important clues for future research on pyroptosis and immunity.

Preparation and characterization of calcium carbonate microspheres and their potential application as drug carriers.

The influence of lecithin from egg yolk (LE) on calcium carbonate (CaCO3) biomineralization was investigated. In the present study, spherical CaCO3 particles were synthesized via coprecipitation in the presence of LE. LE multilamellar liposomes were first tuned by sonication to provide better control over the nucleation of CaCO3. Subsequently, monodisperse microspheres ~2 µm in size were generated by controlling the aggregation and growth of CaCO3 under appropriate concentrations of LE. In contrast to unstable vaterite, the microspheres generated in aqueous solution remained stable for at least 10 days without transforming into calcite, due to the strong interaction between the LE and calcium ions. The microspheres as drug carriers of doxorubicin (DOX) were assessed and were observed to have a good encapsulation efficiency, sustained drug release without a burst release and notable pH sensitivity. In addition, in vivo tumor inhibition examination demonstrated that DOX­loaded CaCO3 microspheres formulation had more superior efficacy to significantly restrain tumor growth. These novel LE/CaCO3 hybrids may provide novel options for various biomedical applications.

Metabonomic study on the plasma of streptozotocin-induced diabetic rats treated with Ge Gen Qin Lian Decoction by ultra high performance liquid chromatography-mass spectrometry.

Changes in endogenous metabolites in the plasma of streptozotocin (STZ)-induced diabetic rats treated with Ge Gen Qin Lian Decoction (GGQLD) were studied. The endogenous compounds in plasma were detected using ultra high performance liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS). Rats were divided into three groups: control, model, and administration (4.95g crude drug/kg body weight). After the final administration, plasma samples from the three groups were analyzed using metabonomics. The three sample groups could be clearly distinguished. The administration group exhibited a distinct return to the levels of phytosphingosine and dihydrosphingosine of the control group according to the principal component analysis score, and the corresponding biomarkers were defined. Significant changes in endogenous metabolites, such as dihydrosphingosine, phytosphingosine, cholylglycine, and pantothenic acid, were identified in STZ-induced diabetic rats. These biochemical changes are associated with the metabolism of sphingolipids, fats, and acetyl coenzyme-A, which could be useful to further investigate the characteristics of STZ-induced diabetes mellitus and the therapeutic mechanism of action of GGQLD. This metabonomic analysis could provide a useful starting point toelucidate the therapeutic effects and mechanism of action of GGQLD in diabetes mellitus.

Cytotoxicity and mutagenicity of sidestream cigarette smoke particulate matter of different particle sizes.

Sidestream cigarette smoke, the major component of environmental tobacco smoke, is a complex and reactive aerosol. The particulate matter (PM) in sidestream smoke is one of the carriers of chemical constituents. However, particle size-dependent toxicological effects of PM are poorly understood. In this study, we evaluated the relationship between the PM size and in vitro cytotoxicity and mutagenicity of sidestream cigarette smoke. A NanoMoudi-II(™) 125A was used to collect PM samples ranging from 10 nm to 10 µm. The in vitro toxicity of PM was evaluated using a neutral red cytotoxicity assay and Salmonella mutagenicity assay. The results showed that the cytotoxicity and mutagenicity of PM larger than 1 µm was significantly lower than PM sized 10 nm-1 µm. Furthermore, there was a noticeable trend that the smaller the size of the PM of sidestream cigarette smoke, the greater the toxicity. This study suggests that the toxicity of PM in sidestream cigarette smoke is size-dependent.

Boron-chelating fluorescent probe (BOPB) in the red region combined with CE-LIF for the detection of NO in mice liver.

Precise measurement of nitric oxide (NO) is of great importance to understand the function of NO in liver and the mechanism of liver injury. 8-(3',4'-Diamino phenyl)-3,5-(2-hydroxyphenyl)-dimethylene pyrrole (BOPB), a fluorescent probe in the red region (>600 nm) newly developed in our group, has good photostability and excitation/emission wavelength of 622/643 nm matching well with commercial 635 nm semiconductor laser of CE-LIF detection. Therefore, BOPB was used in CE-LIF for the determination of NO in mice liver. Both derivatization and separation conditions were optimized. Derivatization reaction of BOPB and NO was carried out in pH 7.4 PBS buffer at 35°C for 12 min and the separation of NO derivative of BOPB (BOPB-T) was achieved within 7.0 min in pH 9.0 running buffer containing 15 mM H3 BO3 -NaOH and 15 mM SDS. Good linearity was found in the range of 1.0 × 10(-9) -5.0 × 10(-7) M with the LOD of 0.02 nM. The proposed method was applied to the analysis of NO in real samples, and NO concentration was obviously increased in acute liver injury of mice. Compared to existing derivatization-based CE-LIF methods for NO, this method has lower LOD and less background interference owing to detection wavelength of BOPB in the red region.

One step physically adsorbed coating of silica capillary with excellent stability for the separation of basic proteins by capillary zone electrophoresis.

The coating of capillary inner surface is considered to be an effective approach to suppress the adsorption of proteins on capillary inner surface in CE. However, most of coating materials reported are water-soluble, which may dissolve in BGE during the procedure of electrophoresis. In this study, a novel strategy for selection of physically coating materials has been illustrated to get coating layer with excellent stability using materials having poor solubility in commonly used solvents. Taking natural chitin as example (not hydrolyzed water soluble chitosan), a simple one step coating method using chitin solution in hexafluoroisopropanol was adopted within only 21 min with good coating reproducibility (RSDs of EOF for within-batch coated capillaries of 1.55% and between-batch coated capillaries of 2.31%), and a separation of four basic proteins on a chitin coated capillary was performed to evaluate the coating efficacy. Using chitin coating, the adsorption of proteins on capillary inner surface was successfully suppressed with reversed and stable EOF, and four basic proteins including lysozyme, cytochrome c, ribonuclease A and α-chymotrypsinogen A were baseline separated within 16 min with satisfied separation efficiency using 20 mM pH 2.0 H3PO4-Na2HPO4 as back ground electrolyte and 20 kV as separation voltage. What is more important, the chitin coating layer could be stable for more than two months during this study, which demonstrates that chitin is an ideal material for preparing semi-permanent coating on bare fused silica capillary inner wall and has hopeful potential in routine separation of proteins with CE.

The natural degradation of benzophenone at low concentration in aquatic environments.

The natural degradation caused by sun irradiation and microbes in aquatic environments is of major significance in the elimination process of benzophenone (BP). In this study, the fate of BP in surface water at a low concentration of 10 µg/L was investigated, including both photodegradation and microbial degradation. The result showed that the photodegradation rate of BP was affected by several parameters such as the initial concentration, continuous input, and the presence of the analogue, ions and small molecules. Meanwhile, the rate of microbial degradation of BP was mainly influenced by the kind and amount of microbes in the environmental water.

Capillary electrophoresis strategy to monitor the released and remaining nitric oxide from the same single cell using a specially designed water-soluble fluorescent probe.

Gasotransmitters including nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) have attracted more and more attention in the past decades due to their unique signaling and functions. However, as a fundamental issue in the investigations of gasotransmitters, the cell membrane permeability and release behavior of them is controversial in reports because of the lack of an efficient approach to determine gasotransmitters released out of and remaining in the same cells simultaneously. To solve such problem, taking NO as representative, a robust and facile strategy has been reported based on a completely water-soluble fluorescent probe and a commercially available capillary electrophoresis system. A specially designed boron-dipyrromethene (BODIPY)-based fluorescent probe with two sulfonate groups, disodium 2,6-disulfonate-1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl) difluoroboradiaza-s-indance (TMDSDAB), has been developed. As a turn-on fluorescent probe, TMDSDAB can react with NO promptly in aqueous media, and 540-fold enhancement of fluorescence is obtained. Using TMDSDAB, the trapping and quantification of NO released out of and remaining in the same single cell was achieved by capillary electrophoresis with laser-induced fluorescence detection. The limit of detection is 0.5 nM for NO. The proposed method has been applied to estimate the release behavior of single macrophages, and the results indicated that the cell membrane should be a barrier to NO diffusion.

Efficient reactivation of p53 in cancer cells by a dual MdmX/Mdm2 inhibitor.

The aberrant interaction between p53 and Mdm2/MdmX is an attractive target for cancer drug discovery because the overexpression of Mdm2 and/or MdmX ultimately impairs the function of p53 in approximately half of all human cancers. Recent studies have shown that inhibition of both Mdm2 and MdmX is more efficient than that of a single target in promoting cellular apoptosis in cancers. In this study, we demonstrate that a dual small-molecule antagonist of Mdm2/MdmX can efficiently reactivate the p53 pathway in model cancer cells overexpressing MdmX and/or Mdm2. The dual antagonist was rationally designed based on segmental mutational analysis of the N-terminal domain of MdmX and the crystal structure of the N-terminal domain of Mdm2 in complex with nutlin-3a (an Mdm2-specific inhibitor). The current work establishes a small molecule therapeutic candidate that targets cancers overexpressing Mdm2 and/or MdmX.

The combined toxicological effects of titanium dioxide nanoparticles and bisphenol A on zebrafish embryos.

Environmental pollutants co-exist and exhibit interaction effects that are different from those associated with a single pollutant. As one of the more commonly manufactured nanomaterials, titanium dioxide nanoparticles (TiO2-NPs) are most likely to bind to other contaminants in water. In this paper, we aimed to study the combined toxicological effects of TiO2-NPs and bisphenol A (BPA) on organism. First, in vitro adsorption experiments were conducted to determine the adsorptive interaction between TiO2-NPs and BPA. Second, zebrafish embryo toxicity tests were performed to monitor for changes in the toxicological effects associated with the two chemicals. The study results demonstrated that adsorptive interactions exist between the two chemicals and increased toxicity effects which included an advanced toxicological effect time, decreased survival, increased morphological abnormalities, and delayed embryo hatching. Also, we suggest that the mode of combined action has a synergistic effect. Based on this, we postulate that concomitant exposure to TiO2-NPs and BPA increased BPA bioavailability and uptake into cells and organisms. Further studies are required to understand the mechanisms of interactions of this mixture.

Simultaneous determination of primary and secondary phenethylamines in biological samples by high-performance liquid chromatographic method with fluorescence detection.

Phenylalanine is an essential amino acid and its metabolites relate to various physiological and immune functions of living organisms. To monitor the alteration of concentration of primary and secondary phenethylamines including N-methyltyramine, octopamine, tyramine, tyrosine and phenylalanine in the metabolic pathway of phenylalanine, a sensitive and selective reversed-phase high-performance liquid chromatographic method has been developed in this study. The identification and quantification of phenethylamines were performed by fluorescent detection after pre-column derivatization with 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene, an excellent fluorescent probe which could react with both primary and secondary amino groups simultaneously. The derivatization was carried out at 25 °C for 25 min, and the separation was performed on a C18 column within 20 min. The linear ranges were from 2.0 to 100 nM for phenylalanine and tyramine to 5.0 to 250 for tyrosine and octopamine, with the detection limits of 0.1 nM for octopamine, tyramine, tyrosine and phenylalanine and 0.2 nM for N-methyltyramine (signal-to-noise ratio=3), which allowed for the sure determination of phenethylamines at trace levels in the real samples without complex pretreatment or enrichment during multitudinous samples analysis. The proposed method has been validated by the analysis of the five target compounds in biological samples with spiked recoveries of 96.4-104.4% and the relative standard deviation of 1.0 and 4.4%.

Comparative proteomic analysis of ovary for Chinese rare minnow (Gobiocypris rarus) exposed to chlorophenol chemicals.

Pentachlorophenol (PCP) and 2,4,6-trichlorophenol (TCP) are suspected of disrupting the endocrine system and thus affecting human and wildlife reproduction, but the potential common mechanisms and biomarkers of chlorophenols (CPs) in the ovary are not fully elucidated. In the present study, the female rare minnow (Gobiocypris rarus) was exposed to PCP (0.5, 5.0, and 50 µg/L), TCP (1.0, 10, and 100 µg/L) and 17ß-estradiol (as a positive control) for 28 days, and the matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF/TOF) mass spectrometry analysis was employed to investigate the alteration of protein expression in the ovary. After comparison of the protein profiles from treated and control groups, 22 protein spots were observed to be altered in abundance (>2-fold) from female treated groups, and 14 protein spots were identified successfully. These proteins were related to molecular response patterns, endocrine effects, metabolic pathways, and even the possible carcinogens in response to CP exposure. The seven differentially expressed mRNA encoding proteins were measured by quantitative real-time PCR (QRT-PCR) and histopathology was also measured. Our data demonstrate that alterations of multiple pathways may be associated with the toxic effects of CPs on ovaries. BIOLOGICAL SIGNIFICANCE: Although numerous studies have shown the affection of the endocrine system with exposure to chlorophenols (CPs), there is little report on the alterations of protein expression in the ovaries from rare minnows following exposure to PCP or TCP. In the present study, a comparative proteomic approach using two dimensional gel electrophoresis and mass spectrometry (MALDI-TOF/TOF MS) has been developed to identify certain proteins in the ovaries of Chinese rare minnow, whose abundance changes during exposure to CPs. After comparison of the protein profiles from treated and control groups, 22 protein spots were observed to be altered in abundance (>2-fold) from female treated groups, and 14 protein spots were identified successfully. These proteins were related to molecular response patterns, endocrine effects, metabolic pathways, and even the possible carcinogens in response to CP exposure. Because the mechanism often involves changes in the expression of multiple proteins rather than a single protein, a global analysis of the protein alterations can result in valuable information to understand the CP action mechanism. All the above results demonstrate that the Vtg, SUMO, Lec-3 and PIMT protein are potential biomarkers and involved in the toxicity pathway of CP exposure in aquatic animals, which should be the primary focus of studies on the CP ovary toxicity mechanism in the future.

A new BODIPY-based long-wavelength fluorescent probe for chromatographic analysis of low-molecular-weight thiols.

A new long-wavelength fluorescent probe, 1,7-dimethyl-3,5-distyryl-8-phenyl-(4'-iodoacetamido)difluoroboradiaza-s-indacene (DMDSPAB-I), was designed and synthesized for thiol labeling in high-performance liquid chromatography (HPLC). The excitation and emission wavelengths of DMDSPAB-I are 620 and 630 nm, respectively, with a high fluorescence quantum yield of 0.557, which is advantageous in preventing interference of intrinsic fluorescence from complex biological matrices and enabling high sensitivity HPLC. Based on DMDSPAB-I, a reversed-phase HPLC method was developed for measuring low-molecular-weight thiols including glutathione, cysteine, homocysteine, N-acetylcysteine, cysteinylglycine, and penicillamine. After the specific reaction of DMDSPAB-I with thiols, baseline separation of all six stable derivatives was achieved through isocratic elution on a C18 column within 25 min, with the limits of detection (signal-to-noise ratio = 3) from 0.24 nmol L(-1) for glutathione to 0.72 nmol L(-1) for penicillamine. The proposed method was validated in part by measuring thiols in blood samples from mice, with recoveries of 95.3-104.3%.

Rapid and sensitive determination of free thiols by capillary zone electrophoresis with near-infrared laser-induced fluorescence detection using a new BODIPY-based probe as labeling reagent.

A CZE with near-infrared (NIR) LIF detection method has been developed for the analysis of six low molecular weight thiols including glutathione, homocysteine, cysteine, γ-glutamylcysteine, cysteinylglycine, and N-acetylcysteine. For this purpose, a new NIR fluorescent probe, 1,7-dimethyl-3,5-distyryl-8-phenyl-(4'-iodoacetamido)difluoroboradiaza-s-indacene was utilized as the labeling reagent, whose excitation wavelength matches the commercially available NIR laser line of 635 nm. The optimum procedure included a derivatization step of the free thiols at 45°C for 25 min and CZE analysis conducted within 14 min in the running buffer containing 16 mmol/L pH 7.0 sodium citrate and 60% v/v ACN. The LODs (S/N = 3) ranged from 0.11 nmol/L for N-acetylcysteine to 0.31 nmol/L for γ-glutamylcysteine, which are better than or comparable to those reported with other derivatization-based CE-LIF methods. As the first trial of NIR CE-LIF method for thiol determination, the practical application of the proposed method has been validated by detecting thiols in cucumber and tomato samples with recoveries of 96.5-104.3%.

Dual labeling for simultaneous determination of nitric oxide, glutathione and cysteine in macrophage RAW264.7 cells by microchip electrophoresis with fluorescence detection.

A simple, rapid and efficient method based on microchip electrophoresis coupled with fluorescence detection (MCE-FLD) was developed for simultaneous determination of nitric oxide (NO), glutathione (GSH) and cysteine (Cys) using dual labeling strategy. Two highly reactive fluorogenic probes, 1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacene (DAMBO) and 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene (TMPAB-o-M), were used for labeling NO and thiols, respectively, under physiological conditions. The rapid separation and sensitive detection of the derivatives were achieved on a glass microchip within 70s in a running buffer of 20mM H3Cit-Na2HPO4 solution (pH 7.4) containing 15% (v/v) acetonitrile at a separation voltage of 2400V. The limits of detection (S/N=3) for NO, GSH and Cys were 7.0, 3.0 and 2.0nM, respectively. The proposed method was validated by measuring intracellular levels of NO and biothiols in macrophage RAW264.7 cells.

Toxicity of TDCPP and TCEP on PC12 cell: changes in CAMKII, GAP43, tubulin and NF-H gene and protein levels.

TDCPP and TCEP are two major types of organophosphorus flame retardants (OPFRs) that are bioaccumulative and persistent in the environment. The toxicity effects of TDCPP and TCEP on PC12 cell are not well understood. In the present study, we investigated morphology, viability and apoptosis in cultured PC12 cells in response to TDCPP and TCEP. The mRNA and protein expression levels of CAMKII, GAP43, tubulin and NF-H were quantified in PC12 cells treated with varying concentrations of the two agents. Results indicate that, upon treatment with the two OPFRs, cell growth decreased, apoptosis increased, morphology was altered and significant changes were found in the gene and protein levels. Treatment with TDCPP caused a reduction in the levels of each of the six proteins studied and in the gene levels of GAP43, NF-H and the two tubulins, but it resulted in an increase in CAMKII gene levels. Treatment with TCEP resulted in similar changes in gene levels to TDCPP and led to decreases in the protein levels of GAP43 and the tubulins while increasing the CAMKII and NF-H protein levels. These results suggest that changes in the gene and protein levels of the regulatory proteins (CAMKII, GAP43) and the structural proteins (tubulin, NF-H) are due to different mechanisms of the toxins, and these proteins may be useful biomarkers for the cytotoxicity and neurotoxicity.

Comparison of extraction methods for analysis of citrinin in red fermented rice.

There is no standard method for the extraction and analysis of citrinin in red fermented rice (RFR). In the study, five extraction methods were compared for their efficiency to analyse citrinin in RFR by HPLC-FLD (reversed phase high performance liquid chromatography with fluorescence detection), including, (1) ultrasonic extraction with EW solution (ethanol:water, 7:3, v/v); (2) ultrasonic extraction with TEF solvent mixtures (toluene:ethyl acetate:formic acid, 7:3:1, v/v); (3) shaking extraction with EW; (4) shaking extraction with EF solvent mixtures (ethyl acetate:formic acid, 1:1, v/v); (5) shaking combined with ultrasonic extraction in EW. Comparison of chromatograms of citrinin by HPLC-FLD with different extraction methods revealed that EW was the best extraction solvent. It was also found that shaking combined with ultrasonic extraction in EW was the most efficient extraction method to extract citrinin from RFR for qualitative and quantitative analysis.

Development of a potential method based on microchip electrophoresis with fluorescence detection for the sensitive determination of intracellular thiols in RAW264.7 cells.

This paper, for the first time, reported the development of a simple, rapid, and reliable method for the separation and sensitive determination of four thiol compounds including homocysteine, cysteine, glutathione, and N-acetylcysteine based on glass MCE with fluorescence detection using a highly reactive fluorogenic probe, 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene (TMPAB-o-M), as the labeling reagent. TMPAB-o-M reacted selectively with thiols to produce highly fluorescent derivatives and the highest derivatization efficiency was achieved within 6 min in physiological conditions. After the optimization of separation conditions, a baseline separation of the four thiol compounds was achieved with the detection limits ranging from 2 nM for glutathione to 4 nM for cysteine (S/N = 3) and RSDs (n = 5) in the range of 3.2-3.8%. The proposed method was significantly sensitive compared to those using electrochemical or even LIF detection in MCE-based setup reported previously, and applied to the determination of intracellular thiols in macrophage RAW264.7 cells.