Objective Piperazine derivatives are a group of emerging psychoactive substances with excitatory and hallucinogenic effects on the central nervous system.This study established a high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)screening method for the detection of 40 piperazine compounds in urine.Methods A 200 μL urine sample(spiked with an internal standard at 1 ng/mL)was subjected to liquid-liquid extraction with ethyl acetate.After nitrogen evaporation,the residue was redissolved in 200 μL methanol and injected for analysis.Separation was performed on a Waters Acquity UPLC? HSS T3 column(100 mm × 2.1 mm,1.8 μm).The mobile phase consisted of(A)20 mmol/L ammonium acetate buffer containing 0.1％formic acid and 5％acetonitrile,and(B)acetonitrile.Gradient elution was applied,and detection was carried out in multiple reaction monitoring(MRM)mode.Quantification was achieved using an internal standard calibration curve.Results The 40 piperazine substances demonstrated good linearity within the range of 1-50 ng/mL,with correlation coefficients of 0.995-0.998.The extraction recovery ranged from 51.51％to 104.1％.Intra-day precision was below 5％,while inter-day precision ranged from 1.61％to 10.17％.Accuracy was between-7.84％and 8.77％.The limits of detection were 0.2-1 ng/mL,and the limit of quantification was 1 ng/mL.Conclusion The proposed method requires only a small sample volume,exhibits high sensitivity,selectivity,and stability,and offers short run times.It is suitable for the qualitative and quantitative determination of piperazine derivatives in urine in forensic toxicology practice.

BACKGROUND: Diabetic foot is a complex condition with high incidence, recurrence, mortality, and disability rates. Current treatments for diabetic foot ulcers are often insufficient. This study was conducted to identify potential therapeutic targets for diabetic foot. METHODS: Datasets related to diabetic foot and diabetic skin were retrieved from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified using R software. Enrichment analysis was conducted to screen for critical gene functions and pathways. A protein interaction network was constructed to identify node genes corresponding to key proteins. The DEGs and node genes were overlapped to pinpoint target genes. Plasma and chronic ulcer samples from diabetic and non-diabetic individuals were collected. Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assays were performed to verify the S100 calcium binding protein A9 (S100A9), inflammatory cytokine, and related pathway protein levels. Hematoxylin and eosin staining was used to measure epidermal layer thickness. RESULTS: In total, 283 common DEGs and 42 node genes in diabetic foot ulcers were identified. Forty-three genes were differentially expressed in the skin of diabetic and non-diabetic individuals. The overlapping of the most significant DEGs and node genes led to the identification of S100A9 as a target gene. The S100A9 level was significantly higher in diabetic than in non-diabetic plasma (178.40 ± 44.65 ng/mL vs. 40.84 ± 18.86 ng/mL) and in chronic ulcers, and the wound healing time correlated positively with the plasma S100A9 level. The levels of inflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-1, and IL-6) and related pathway proteins (phospho-extracellular signal regulated kinase [ERK], phospho-p38, phospho-p65, and p-protein kinase B [Akt]) were also elevated. The epidermal layer was notably thinner in chronic diabetic ulcers than in non-diabetic skin (24.17 ± 25.60 μm vs. 412.00 ± 181.60 μm). CONCLUSIONS S100A9 was significantly upregulated in diabetic foot and was associated with prolonged wound healing. S100A9 may impair diabetic wound healing by disrupting local inflammatory responses and skin re-epithelialization.
Calgranulin B/therapeutic use* ; Diabetic Foot/metabolism* ; Humans ; Datasets as Topic ; Computational Biology ; Mice, Inbred C57BL ; Animals ; Mice ; Protein Interaction Maps ; Immunohistochemistry

Objective To observe the value of Cox proportional hazards regression model constructed based on high-resolution MRI for predicting the risk of esophageal squamous cell carcinoma(ESCC)progression after definitive chemoradiotherapy(dCRT).Methods Thirty ESCC patients who underwent dCRT were retrospectively enrolled.Quantitative and qualitative indicators of primary tumor and imaging-defined metastatic lymph nodes were analyzed based on pre-treatment high-resolution M RI.The progression-free survival(PFS)of patients were recorded.A Cox proportional hazards regression model was established to predict the risk of tumor progression based on MRI indices,and the risks of tumor progression were stratified into high and low according to the median prediction.PFS rates were compared between patients with high or low risk of tumor progression.Results Tumor thickness(HR[95％CI]=1.210[1.025,1.429],P=0.024),relationship between the tumor and aorta(HR[95％CI]=4.275[1.064,17.168],P=0.041)and lymph node signal change rate on delayed phase pre-treatment MRI(HR[95％CI]=0.049[0.007,0.362],P=0.003)were all independent factors for predicting PFS.Based on Cox proportional hazards regression model and its predicted value,PFS rate in high risk patients was lower than that in low risk patients(P＜0.05).Conclusion High-resolution MRI could be used to predict prognosis of ESCC after dCRT.

Objective Piperazine derivatives are a group of emerging psychoactive substances with excitatory and hallucinogenic effects on the central nervous system.This study established a high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)screening method for the detection of 40 piperazine compounds in urine.Methods A 200 μL urine sample(spiked with an internal standard at 1 ng/mL)was subjected to liquid-liquid extraction with ethyl acetate.After nitrogen evaporation,the residue was redissolved in 200 μL methanol and injected for analysis.Separation was performed on a Waters Acquity UPLC? HSS T3 column(100 mm × 2.1 mm,1.8 μm).The mobile phase consisted of(A)20 mmol/L ammonium acetate buffer containing 0.1％formic acid and 5％acetonitrile,and(B)acetonitrile.Gradient elution was applied,and detection was carried out in multiple reaction monitoring(MRM)mode.Quantification was achieved using an internal standard calibration curve.Results The 40 piperazine substances demonstrated good linearity within the range of 1-50 ng/mL,with correlation coefficients of 0.995-0.998.The extraction recovery ranged from 51.51％to 104.1％.Intra-day precision was below 5％,while inter-day precision ranged from 1.61％to 10.17％.Accuracy was between-7.84％and 8.77％.The limits of detection were 0.2-1 ng/mL,and the limit of quantification was 1 ng/mL.Conclusion The proposed method requires only a small sample volume,exhibits high sensitivity,selectivity,and stability,and offers short run times.It is suitable for the qualitative and quantitative determination of piperazine derivatives in urine in forensic toxicology practice.

Objective To observe the value of Cox proportional hazards regression model constructed based on high-resolution MRI for predicting the risk of esophageal squamous cell carcinoma(ESCC)progression after definitive chemoradiotherapy(dCRT).Methods Thirty ESCC patients who underwent dCRT were retrospectively enrolled.Quantitative and qualitative indicators of primary tumor and imaging-defined metastatic lymph nodes were analyzed based on pre-treatment high-resolution M RI.The progression-free survival(PFS)of patients were recorded.A Cox proportional hazards regression model was established to predict the risk of tumor progression based on MRI indices,and the risks of tumor progression were stratified into high and low according to the median prediction.PFS rates were compared between patients with high or low risk of tumor progression.Results Tumor thickness(HR[95％CI]=1.210[1.025,1.429],P=0.024),relationship between the tumor and aorta(HR[95％CI]=4.275[1.064,17.168],P=0.041)and lymph node signal change rate on delayed phase pre-treatment MRI(HR[95％CI]=0.049[0.007,0.362],P=0.003)were all independent factors for predicting PFS.Based on Cox proportional hazards regression model and its predicted value,PFS rate in high risk patients was lower than that in low risk patients(P＜0.05).Conclusion High-resolution MRI could be used to predict prognosis of ESCC after dCRT.

BACKGROUND:Tissue engineering has brought new hope to the clinical challenge of liver failure,and the preparation of plant-derived decellularized fiber scaffolds holds significant importance in liver tissue engineering. OBJECTIVE:To prepare apple tissue decellularized scaffold material by using fresh apple slices and a solution of sodium dodecyl sulfate,and assess its biocompatibility. METHODS:Fresh apples were subjected to decellularization using phosphate buffer saline and sodium dodecyl sulfate solution,separately.Afterwards,the decellularized apple tissues and apple decellularized scaffold materials were decontaminated with phosphate buffer saline.Subsequently,scanning electron microscopy was used to assess the effectiveness of decellularization of the apple materials.Adipose-derived mesenchymal stem cells were extracted from the inguinal fat BALB/C of mice,and their expression of stem cell-related markers(CD45,CD34,CD73,CD90,and CD105)was identified through flow cytometry.The cells were then divided into a scaffold-free control group and a scaffold group.Equal amounts of adipose-derived mesenchymal stem cells were seeded onto both groups.The biocompatibility of the decellularized scaffold with adipose-derived mesenchymal stem cells was evaluated using CCK-8 assay,hematoxylin-eosin staining,and phalloidine staining.Cell adhesion and growth on the scaffold were observed under light microscopy and scanning electron microscopy.Furthermore,the scaffold was subdivided into the non-induced group and the hepatogenic-induced group.Adipose-derived mesenchymal stem cells were cultured on the decellularized apple scaffold,and they were cultured for 14 days in regular culture medium or hepatogenic induction medium for comparison.Immunofluorescent staining using liver cell markers,including albumin,cytokeratin 18,and CYP1A1,was performed.Enzyme-linked immunosorbent assay was used to detect the secretion of alpha fetoprotein and albumin.Additionally,scanning electron microscopy was employed to observe the morphology of the induced cells on the scaffold,verifying the expression of liver cell-related genes on the decellularized scaffold material.Finally,the cobalt-60 irradiated and sterilized decellularized apple scaffolds were transplanted onto the surface of mouse liver and the degradation of the scaffold was observed by gross observation and hematoxylin-eosin staining after 28 days. RESULTS AND CONCLUSION:(1)The scanning electron microscopy results revealed that the decellularized apple scaffold material retained a porous structure of approximately 100 μm in size,with no residual cells observed.(2)Through flow cytometry analysis,the cultured cells were identified as adipose-derived mesenchymal stem cells.(3)CCK-8 assay results demonstrated that the prepared decellularized apple tissue scaffold material exhibited no cytotoxicity.Hematoxylin-eosin staining and phalloidine staining showed that adipose-derived mesenchymal stem cells were capable of adhering and proliferating on the decellularized apple tissue scaffold.(4)The results obtained from immunofluorescence staining and enzyme-linked immunosorbent assay revealed that adipose-derived mesenchymal stem cells cultured on the decellularized apple scaffolds exhibited elevated expression of liver-specific proteins,including albumin,alpha-fetoprotein,cytokeratin 18,and CYP1A1.These results suggested that they were induced differentiation into hepatocyte-like cells possessing functional characteristics of liver cells.(5)The decellularized apple scaffold implanted at 7 days has integrated with the liver,with partial degradation of the scaffold observed.By 28 days,the decellularized apple scaffold has completely degraded and has been replaced by newly-formed tissue.(6)The results indicate that the decellularized scaffold material derived from apple tissue demonstrates favorable biocompatibility,promoting the proliferation,adhesion,and hepatic differentiation of adipose-derived mesenchymal stem cells.

Objective To investigate the in vitro metabolites and metabolic pathways of phenethylamine new psychoactive substances 2C-B-FLY and 25B-NBOH in human liver microsomes.Methods The human liver microsome incubation model was established,and the samples were centrifuged,blow-dried and re-dissolved after 3 h of co-incubation in a water bath at 37℃.The samples were detected by ultra-high performance liquid chromatography Q Exactive mass spectrometry in ESI+mode,and the parent drug and its metabolites were examined by applying Full scan-ddMS2 data-dependent acquisition mode.Results After in vitro incubation with human liver microsomes,2C-B-FLY was metabolised to produce a total of four phase I metabolites and one acetylated phase II metabolite,which underwent metabolic reactions such as hydroxylation,debromination,and N-acetylation.25B-NBOH was metabolised to produce a total of eight phase I metabolites and two glucuronidated phase II metabolites,which comprised the major biotransformation reactions of O-demethylation,hydroxylation,amine dehydrogenation,N-dealkylation and glucuronidation.Conclusion This study describes the metabolites and metabolic pathways of 2C-B-FLY and 25B-NBOH in a human liver microsomal incubation system,providing a scientific basis for the in vivo detection of new psychoactive substance of the phenethylamine group.

Nitazenes are a class of synthetic opioids that have recently emerged in the illegal drug market.They are characterized by high potency and a narrow therapeutic window.Compared with tradi-tional opioids,these substances are more likely to cause severe respiratory depression.In recent years,the abuse of nitazenes has occurred frequently,causing hundreds of overdose deaths worldwide,and posing a serious threat to individual health and public safety.As more nitazenes continue to emerge,research on the identification of these substances has received increasing attention.This paper reviews the structural characteristics,in vivo and in vitro pharmacological characterizations and analysis methods of common nitazenes,aiming to provide a basis and reference for the study of these substances in fo-rensic toxicology.

OBJECTIVES: To establish a simple and rapid qualitative and quantitative detection method of dexmedetomidine in blood. METHODS: Blood was separated on the Allure PFP Propyl liquid chromatography column with isocratic elution after it was precipitated by acetonitrile and filtered. Qualitative and quantitative analysis of dexmedetomidine was performed using positive ion scan mode and multi-reaction monitoring mode. RESULTS: The limit of detection of dexmedetomidine in blood was 0.2 ng/mL and the limit of quantification was 0.5 ng/mL. The linearity of the method was good in the range of 0.5-1 000 ng/mL, and the correlation coefficient was greater than 0.99. The accuracy of the method was 90.34%-112.67% and the extraction recovery was 50.05%-91.08%, with no significant matrix effect. CONCLUSIONS This method is simple, selective and suitable for the qualitative and quantitative analysis of dexmedetomidine in blood, which can provide a reference for drug-facilitated cases involving dexmedetomidine.
Tandem Mass Spectrometry/methods* ; Chromatography, High Pressure Liquid/methods* ; Dexmedetomidine/analysis* ; Reproducibility of Results ; Chromatography, Liquid/methods*

OBJECTIVES: To investigate the cerebral metabolism in the patients with type 2 diabetes mellitus-associated cognitive dysfunction (T2DACD) and explore the mechanism of electroacupuncture (EA) at the acupoints for Tiaozang Xingshen (adjusting zangfu function and rescuing the spirit) in treatment of T2DACD, using magnetic resonance spectroscopy. METHODS: Fifteen patients with T2DACD (observation group) and 22 healthy subjects (control group) were enrolled. In the observation group, the patients were treated with EA for Tiaozang Xingshen at Baihui (GV 20) and Shenting (GV 24), and bilateral Feishu (BL 13), Pishu (BL 20), Shenshu (BL 23), Zusanli (ST 36), Sanyinjiao (SP 6), Hegu (LI 4) and Taichong (LR 3). EA was operated with disperse-dense wave, 2 Hz/100 Hz in frequency and 0.1 mA to 1.0 mA in current intensity; 30 min each time, once daily. One course of EA consisted of 5 treatments, at the interval of 2 days and the intervention lasted 8 courses. Before treatment in the control group, before and after treatment in the observation group, the score of Montreal cognitive assessment scale (MoCA), the score of clinical dementia rating (CDR), Flanker paradigm, Stroop paradigm, Nback paradigm, the score of self-rating anxiety scale (SAS), the score of self-rating depression scale (SDS), and the score of Hamilton depression rating scale (HAMD) were evaluated separately; the glycolipid metabolic indexes (fasting plasma glucose [FPG], glycosylated hemoglobin type A1c [HbA1c], total cholesterol [TC], triacylglycerol [TG], high-density lipoprotein cholesterol [HDL-C] and low-density lipoprotein cholesterol [LDL-C]) were determined；with the magnetic resonance spectroscopy technique adopted, the metabolites in the basal ganglia area were detected. The correlation analysis was performed for the metabolite values with MoCA score, CDR score , Flanker paradigm, Stroop paradigm, and Nback paradigm. RESULTS: Before treatment, compared with the control group, in the observation group, MoCA score was lower (P<0.001), CDR score and the levels of FPG and HbA1c were higher (P<0.001); the reaction times of Flanker non-conflict, Flanker conflict, Stroop neutrality, Stroop congruence, Stroop conflict, and 1-back were prolonged (P<0.05, P<0.001), and the accuracy of Flanker conflict, Stroop conflict, and 1-back decreased (P<0.05, P<0.01); the ratio of N-acetyl aspartate (NAA) to creatine (Cr) in the left basal ganglia area was dropped (P<0.001), and that of myo-inositol (MI) to Cr in the right side increased (P<0.05). In the observation group after treatment, compared with the levels before treatment, MoCA score was higher (P<0.001), the scores of CDR, SAS and HAMD were reduced (P<0.01, P<0.05), the reaction times of Flanker conflict and Stroop conflict shortened (P<0.001, P<0.05), and the accuracy of Flanker conflict and 1-back increased (P<0.001, P<0.05); the ratio of NAA to Cr in the left basal ganglia area and that of the gamma-aminobutyric acid (GABA) to Cr in the right increased (P<0.05), that of MI to Cr in the right decreased (P<0.05). Before treatment, in the observation group, the ratio of MI to Cr in the right basal ganglia area was positively correlated with the reaction time of Stroop congruence (r=0.671, P=0.012) and this ratio was positively correlated with the reaction time of Stroop conflict (r=0.576, P=0.039). CONCLUSIONS Electroacupuncture for "adjusting zangfu function and rescuing the mind" improves the cognitive function of T2DACD patients, which may be related to the regulation of NAA, MI and GABA levels in the basal ganglia.
Humans ; Electroacupuncture ; Acupuncture Therapy ; Acupuncture Points ; Diabetes Mellitus, Type 2/therapy* ; Glycated Hemoglobin ; Cognitive Dysfunction/therapy* ; Cholesterol ; gamma-Aminobutyric Acid