Objective To establish and validate a method for simultaneous analysis of fluconazole,linezolid,voriconazole and cont-ezolid by liquid chromatography-tandem mass spectrometry,and conduct preliminary assessment its value of application value in clinical therapeutic drug monitoring.Methods Using an isotopic internal standard method,the serum samples were pretreated with protein precipitation.The supernatant was diluted after centrifugation,and detected by liquid chromatography-tandem mass spectrometer.Refer-ring to the Recommendations for Clinical Application of Liquid Chromatography-Mass Spectrometry(LC-MS)and Clinical and Labora-tory Standards Instituhe(CLSI)C62A,the performance of the LC-MS method was verified,including quantitation limits,linearity,trueness,precision,matrix effect,carry-over,interference,dilution consistency and stability.The blood samples from patients who were treated with fluconazole,linezolid,voriconazole,and contezolid were collected and measured for trough or peak concentrations.Results The quantitation limits of fluconazole,linezolid,voriconazole and contezolid by this method were 1 μg/mL,0.25 μg/mL,0.25 μg/mL and 0.1 μg/mL,respectively.The linear ranges were 1-100 μg/mL,0.25-25 μg/mL,0.25-25 μg/mL,and 0.1-10μg/mL,respectively.The recovery rates were 103.0％-105.7％,103.1％-108％,102.4％-106.2％and 101.0％-109.9％,respectively.The precisions,expressed as coefficient of variation(CV),were 1.7％-3.4％,2.1％-4.8％,1.9％-3.1％,and 3.1％-6.8％,respective-ly.No obvious matrix effect,carry-over contamination and interference were found.The dilution consistency and stability were satisfac-tory.The concentrations of fluconazole,linezolid and voriconazole within the reference interval accounted for 49.1％,52.5％and 80.7％of the total samples,respectively.The peak concentration of contezolamide was(14.02±4.94)μg/mL(n=4),and the trough concen-tration was(0.34±0.20)μg/mL(n=5).Conclusion In this study,a method for simultaneous analysis of the concentrations of flu-conazole,linezolid,voriconazole and contezolid was successfully established and verified by liquid chromatography-tandem mass spec-trometry.This method is simple,rapid,and suitable for therapeutic drug monitoring,and providing a basis for the optimization of drug regimens.

Objective In this study,2,4-dinitrochlorobenzene(DNCB)was used to induce the establishment of an atopic dermatitis(AD)model in BALB/c homozygous mice to simulate the skin inflammatory complications in patients with clinical malignancies.Methods BALB/c mice were divided into different groups:negative control group(NC group),model group(MODEL group),atopic dermatitis group(AD group),and dexamethasone group(DEX group).After the mice in MODEL group and DEX group were inoculated with S-180 tumor cells in the axilla,MODEL group,AD group and DEX group were stimulated with DNCB on the dorsal skin and the ear to establish an animal model of atopic dermatitis in tumor-bearing mice.Changes in body weight were observed and recorded,the dorsal skin condition of mice was assessed after the last administration of the drug,the spleen was taken to calculate the spleen coefficient,the difference in the mass of mouse ear slices was determined to calculate the degree of auricular swelling and the rate of inhibition of swelling,and histopathological tests were performed on the dorsal skin tissues to detect the levels of IgE,TNF-α,IL-4,and IL-17 in the serum using an ELISA assay.Results Compared with the NC group,the skin of mice in the MODEL and AD groups showed erythematous,papular,scaly and mossy changes,accompanied by weight loss,and a significant increase in splenic coefficient and auricular swelling.Pathologic findings showed an incomplete skin structure,a significant increase in skin thickness,a large infiltration of inflammatory cells,and an increase in the number of mast cells.Serum levels of IgE,TNF-α,IL-4 and IL-17 were increased.Compared with the MODEL group,the DEX group showed an improvement in all the assays.Conclusions DNCB excitation can successfully establish an animal model of AD in hormonal mice,which is drug-controllable,which provides a useful scientific tool for conducting scientific research related to malignant tumors and skin inflammation.

BACKGROUND:Non-alcoholic fatty liver disease is one of the common chronic liver diseases in the world.Aerobic exercise is considered to be an important means for the treatment of non-alcoholic fatty liver disease.However,the mechanism of exercise to improve non-alcoholic fatty liver disease has not been fully clarified.OBJECTIVE:To investigate the effects of aerobic exercise on the protein kinase B/glycogen synthase kinase-3β pathway mediated by Canopy FGF signaling regulator 2(CNPY2)in the liver canopy of non-alcoholic fatty liver disease mice and its mechanism.METHODS:Thirty male CNPY2 knockout mice(ko)and thirty their litters of wild-type mice(wt)were fed adaptively for one week and randomly divided into control group,model group,and model exercise group,with 10 mice in each group.The control group was fed with ordinary diet.The model group and the model exercise group were fed with high-fat diet for 17 weeks.The model exercise group received continuous aerobic exercise intervention from week 10 until the end of the experiment at week 18.Liver histopathology was observed by hematoxylin-eosin and oil red O staining.The levels of serum lipids and liver function were detected by automatic biochemical analyzer.The expression levels of CNPY2,protein kinase B/glycogen synthase kinase-3β pathway,and Caspase-3 protein in liver tissues were detected by Western Blotting.The apoptosis rate of hepatocytes was detected by TUNEL staining.RESULTS AND CONCLUSION:(1)Compared with wt control group,CNPY2 expression in liver tissues of wt model group was increased(P＜0.05),while CNPY2 expression in wt model exercise group was decreased compared with wt model group(P＜0.05).Compared with control group,wt mice and ko mice in model group showed steatosis,increased lipid droplets,abnormal blood lipids and liver function,decreased protein kinase B/glycogen synthase kinase-3β expression(P＜0.05)and increased Caspase-3 expression(P＜0.05),and increased hepatocyte apoptosis rate in liver tissue(P＜0.05).(2)Compared with the model group,wt mice and ko mice showed improvement in the above indexes in model exercise group.(3)Compared with wt mice,the above indexes of ko mice were improved.(4)These findings indicate that CNPY2 gene deletion and aerobic exercise can effectively improve non-alcoholic fatty liver disease.The mechanism may be related to aerobic exercise reducing CNPY2 expression,activating protein kinase B/glycogen synthase kinase-3β signaling pathway,and thus inhibiting hepatocyte apoptosis.

Objective:? To summarize the achievements in improving the consistency of clinical liquid chromatography-tandem mass spectrometry (LC-MS/MS) testing results.Methods:? From 2021 to 2024, Zhongshan Hospital Affiliated to Fudan University recruited laboratories voluntarily participating in the MSHP (Clinical LC-MS/MS Testing Consistency Program). As of Batch 202404, a total of 76 laboratories had enrolled, including 60 medical institutions (all tertiary hospitals) and 16 third-party laboratories. Test items were established, and comparative samples were distributed regularly-each item′s samples covered three concentrations (high, medium, and low). Samples were shipped via cold chain and tested within one week. Our laboratory′s measurements served as the target, with participating labs′ results within ±25% of the target deemed qualified. Passing-Bablok regression and Bland-Altman analysis were used to assess consistency.Results:Taking 3-MT (3-methoxytyramine) as an example, the coefficients of variation (CVs) for the project′s three concentration levels improved from 17.00%, 47.18%, and 4.88% in the first comparative batch to 9.59%, 9.59%, and 6.1% in Batch 202404. Passing-Bablok regression results for the 5 units participating in 3-MT testing showed that Laboratory A had proportional bias but no systematic bias (regression slope [95% CI]: 0.903 [0.862-0.952]; intercept [95% CI]: 0.912 [-1.921-6.073]). The remaining laboratories exhibited no proportional or systematic bias with the target (Laboratory B: slope 1.031 [0.961-1.147], intercept-0.733 [-4.641-8.272]; Laboratory C: slope 0.982 [0.940-1.009], intercept-0.576 [-2.675-1.891]; Laboratory D: slope 0.973 [0.939-1.066], intercept-1.168 [-6.108-1.649]; Laboratory E: slope 0.999 [0.905-1.051], intercept-1.876 [-6.111-3.508]). Bland-Altman analysis indicated that all 5 laboratories′ results generally showed good consistency with the target. Through quality feedback and optimizing sample preparation concentrations, result consistency was enhanced.? Conclusion:? Clinical LC-MS/MS testing consistency programs contribute to improving the comparability of test results.

Objective In this study,2,4-dinitrochlorobenzene(DNCB)was used to induce the establishment of an atopic dermatitis(AD)model in BALB/c homozygous mice to simulate the skin inflammatory complications in patients with clinical malignancies.Methods BALB/c mice were divided into different groups:negative control group(NC group),model group(MODEL group),atopic dermatitis group(AD group),and dexamethasone group(DEX group).After the mice in MODEL group and DEX group were inoculated with S-180 tumor cells in the axilla,MODEL group,AD group and DEX group were stimulated with DNCB on the dorsal skin and the ear to establish an animal model of atopic dermatitis in tumor-bearing mice.Changes in body weight were observed and recorded,the dorsal skin condition of mice was assessed after the last administration of the drug,the spleen was taken to calculate the spleen coefficient,the difference in the mass of mouse ear slices was determined to calculate the degree of auricular swelling and the rate of inhibition of swelling,and histopathological tests were performed on the dorsal skin tissues to detect the levels of IgE,TNF-α,IL-4,and IL-17 in the serum using an ELISA assay.Results Compared with the NC group,the skin of mice in the MODEL and AD groups showed erythematous,papular,scaly and mossy changes,accompanied by weight loss,and a significant increase in splenic coefficient and auricular swelling.Pathologic findings showed an incomplete skin structure,a significant increase in skin thickness,a large infiltration of inflammatory cells,and an increase in the number of mast cells.Serum levels of IgE,TNF-α,IL-4 and IL-17 were increased.Compared with the MODEL group,the DEX group showed an improvement in all the assays.Conclusions DNCB excitation can successfully establish an animal model of AD in hormonal mice,which is drug-controllable,which provides a useful scientific tool for conducting scientific research related to malignant tumors and skin inflammation.

BACKGROUND:Non-alcoholic fatty liver disease is one of the common chronic liver diseases in the world.Aerobic exercise is considered to be an important means for the treatment of non-alcoholic fatty liver disease.However,the mechanism of exercise to improve non-alcoholic fatty liver disease has not been fully clarified.OBJECTIVE:To investigate the effects of aerobic exercise on the protein kinase B/glycogen synthase kinase-3β pathway mediated by Canopy FGF signaling regulator 2(CNPY2)in the liver canopy of non-alcoholic fatty liver disease mice and its mechanism.METHODS:Thirty male CNPY2 knockout mice(ko)and thirty their litters of wild-type mice(wt)were fed adaptively for one week and randomly divided into control group,model group,and model exercise group,with 10 mice in each group.The control group was fed with ordinary diet.The model group and the model exercise group were fed with high-fat diet for 17 weeks.The model exercise group received continuous aerobic exercise intervention from week 10 until the end of the experiment at week 18.Liver histopathology was observed by hematoxylin-eosin and oil red O staining.The levels of serum lipids and liver function were detected by automatic biochemical analyzer.The expression levels of CNPY2,protein kinase B/glycogen synthase kinase-3β pathway,and Caspase-3 protein in liver tissues were detected by Western Blotting.The apoptosis rate of hepatocytes was detected by TUNEL staining.RESULTS AND CONCLUSION:(1)Compared with wt control group,CNPY2 expression in liver tissues of wt model group was increased(P＜0.05),while CNPY2 expression in wt model exercise group was decreased compared with wt model group(P＜0.05).Compared with control group,wt mice and ko mice in model group showed steatosis,increased lipid droplets,abnormal blood lipids and liver function,decreased protein kinase B/glycogen synthase kinase-3β expression(P＜0.05)and increased Caspase-3 expression(P＜0.05),and increased hepatocyte apoptosis rate in liver tissue(P＜0.05).(2)Compared with the model group,wt mice and ko mice showed improvement in the above indexes in model exercise group.(3)Compared with wt mice,the above indexes of ko mice were improved.(4)These findings indicate that CNPY2 gene deletion and aerobic exercise can effectively improve non-alcoholic fatty liver disease.The mechanism may be related to aerobic exercise reducing CNPY2 expression,activating protein kinase B/glycogen synthase kinase-3β signaling pathway,and thus inhibiting hepatocyte apoptosis.

Objective To establish and validate a method for simultaneous analysis of fluconazole,linezolid,voriconazole and cont-ezolid by liquid chromatography-tandem mass spectrometry,and conduct preliminary assessment its value of application value in clinical therapeutic drug monitoring.Methods Using an isotopic internal standard method,the serum samples were pretreated with protein precipitation.The supernatant was diluted after centrifugation,and detected by liquid chromatography-tandem mass spectrometer.Refer-ring to the Recommendations for Clinical Application of Liquid Chromatography-Mass Spectrometry(LC-MS)and Clinical and Labora-tory Standards Instituhe(CLSI)C62A,the performance of the LC-MS method was verified,including quantitation limits,linearity,trueness,precision,matrix effect,carry-over,interference,dilution consistency and stability.The blood samples from patients who were treated with fluconazole,linezolid,voriconazole,and contezolid were collected and measured for trough or peak concentrations.Results The quantitation limits of fluconazole,linezolid,voriconazole and contezolid by this method were 1 μg/mL,0.25 μg/mL,0.25 μg/mL and 0.1 μg/mL,respectively.The linear ranges were 1-100 μg/mL,0.25-25 μg/mL,0.25-25 μg/mL,and 0.1-10μg/mL,respectively.The recovery rates were 103.0％-105.7％,103.1％-108％,102.4％-106.2％and 101.0％-109.9％,respectively.The precisions,expressed as coefficient of variation(CV),were 1.7％-3.4％,2.1％-4.8％,1.9％-3.1％,and 3.1％-6.8％,respective-ly.No obvious matrix effect,carry-over contamination and interference were found.The dilution consistency and stability were satisfac-tory.The concentrations of fluconazole,linezolid and voriconazole within the reference interval accounted for 49.1％,52.5％and 80.7％of the total samples,respectively.The peak concentration of contezolamide was(14.02±4.94)μg/mL(n=4),and the trough concen-tration was(0.34±0.20)μg/mL(n=5).Conclusion In this study,a method for simultaneous analysis of the concentrations of flu-conazole,linezolid,voriconazole and contezolid was successfully established and verified by liquid chromatography-tandem mass spec-trometry.This method is simple,rapid,and suitable for therapeutic drug monitoring,and providing a basis for the optimization of drug regimens.

Objective:? To summarize the achievements in improving the consistency of clinical liquid chromatography-tandem mass spectrometry (LC-MS/MS) testing results.Methods:? From 2021 to 2024, Zhongshan Hospital Affiliated to Fudan University recruited laboratories voluntarily participating in the MSHP (Clinical LC-MS/MS Testing Consistency Program). As of Batch 202404, a total of 76 laboratories had enrolled, including 60 medical institutions (all tertiary hospitals) and 16 third-party laboratories. Test items were established, and comparative samples were distributed regularly-each item′s samples covered three concentrations (high, medium, and low). Samples were shipped via cold chain and tested within one week. Our laboratory′s measurements served as the target, with participating labs′ results within ±25% of the target deemed qualified. Passing-Bablok regression and Bland-Altman analysis were used to assess consistency.Results:Taking 3-MT (3-methoxytyramine) as an example, the coefficients of variation (CVs) for the project′s three concentration levels improved from 17.00%, 47.18%, and 4.88% in the first comparative batch to 9.59%, 9.59%, and 6.1% in Batch 202404. Passing-Bablok regression results for the 5 units participating in 3-MT testing showed that Laboratory A had proportional bias but no systematic bias (regression slope [95% CI]: 0.903 [0.862-0.952]; intercept [95% CI]: 0.912 [-1.921-6.073]). The remaining laboratories exhibited no proportional or systematic bias with the target (Laboratory B: slope 1.031 [0.961-1.147], intercept-0.733 [-4.641-8.272]; Laboratory C: slope 0.982 [0.940-1.009], intercept-0.576 [-2.675-1.891]; Laboratory D: slope 0.973 [0.939-1.066], intercept-1.168 [-6.108-1.649]; Laboratory E: slope 0.999 [0.905-1.051], intercept-1.876 [-6.111-3.508]). Bland-Altman analysis indicated that all 5 laboratories′ results generally showed good consistency with the target. Through quality feedback and optimizing sample preparation concentrations, result consistency was enhanced.? Conclusion:? Clinical LC-MS/MS testing consistency programs contribute to improving the comparability of test results.

Objective:The aim of this study was to develop and validate a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of melatonin in human serum.Methods:We describe the performance and validation of melatonin by LC-MS/MS. 182 serum samples from the patients diagnosed with Sleep disturbance who visited the Department of Psychiatry at Zhongshan Hospital affiliated to Fudan University from February 2022 to March 2023(56 males,162 females,mean age [45.51±16.31]years), as well as 182 healthy individuals were included(87 males,95 females,mean age [48.55±11.93]years). The two groups were used to assess the application of serum melatonin levels as a diagnostic indicator for sleep disorders (SDs). The liquid chromatography mass spectrometry (LC-MS) system with an chromatography column (2.1×100 mm, 1.8 μm) was used for separation. The column temperature was set at 35 ℃, as well as the mobile phase consisting of a 0.1% formic acid aqueous solution and pure acetonitrile. The flowing rate was set at 0.4 ml/min for gradient elution. The LC-MS/MS method was validated according to guidance documents, including the following parameters: specificity, selectivity, matrix effect, carryover contamination and reproducibility, lower limit of measuring interval, linearity, precision, recovery rate, dilution consistency, and serum sample stability. Then, it was subsequently employed to profile melatonin changes in Sleep disturbance.Results:The lower limit of quantification for melatonin was 1 pg/ml, and the linear range of detection was 1 pg/ml to 500 pg/ml ( r=0.999). The intra-day and intra-batch precision, expressed as the coefficient of variation ( CV), was within the range from 3.07% to 6.86%, which met the requirement of less than 15%. The recovery rate of the spiked samples ranged from 105.91% to 116.30%. The level of serum melatonin in the sleep disturbance group was significantly lower than that in the healthy control group ([2.00(1.00,3.28)] vs [8.35(4.28,14.80)] pg/ml, P<0.001). Conclusions:The LC-MS/MS method we developed for the quantification of melatonin is clinical practicable.

TSCI have dyskinesia and sensory disturbance that can cause various life-threaten complications. The patients with traumatic spinal cord injury (TSCI), seriously affecting the quality of life of patients. Based on the epidemiology of TSCI and domestic and foreign literatures as well as expert investigations, this expert consensus reviews the definition, injury classification, rehabilitation assessment, rehabilitation strategies and rehabilitation measures of TSCI so as to provide early standardized rehabilitation treatment methods for TSCI.