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 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 establish an analytical method of LC-MS/MS based on micro-segmentation analysis of multiple hairs,verify the detection of 20 benzodiazepines in 1mm hair,and finally use it for case detection analysis.Methods 5 hairs were cut into 1mm long segments each,and the corresponding segments were immersed and sonicated in an extract containing dithiothreitol,methanol,etc.Mobile phase A was 0.1%formic acid in water,and mobile phase B was acetonitrile.Data were collected using electrospray ion source positive ion mode in multiple reaction monitoring mode.Results The 20 benzodiazepines in multiple hairs had a good linear relationship(r>0.99),detection limit of 0.2～5 pg/mm,minimum quantification limit of 0.5 to 20 pg/mm,the intro-day and inter-day precisions of 2.03%to 13.81%,the intro-day and inter-day accuracies of 87.98%～111.29%,matrix effect of 71.15%～110.43%,and extraction recovery of 69.34%to 99.94%.Using this method,hair samples were analyzed in volunteers taking a single dose of clonazepam for 20 days,and the detected location of clonazepam in 15 hairs was located in segment 6～11 from the hair root,with the concentration range of 1.06～3.45 pg/mm.By this method,the hair of the victims suspected of having taken sleeping pills in the case was analyzed.In 15 hairs,clonazepam was detected in segments 9～13 of the hair root.The quantitative results were 1.07～19.06 pg/mm.The experimental results were basically consistent with the medication time of the victim.Conclusion The micro-segmentation analysis technique of multiple hairs can be applied to the investigation of drug-assisted crime cases,and can roughly estimate the medication time.

Objective To establish an analytical method of LC-MS/MS based on micro-segmentation analysis of multiple hairs,verify the detection of 20 benzodiazepines in 1mm hair,and finally use it for case detection analysis.Methods 5 hairs were cut into 1mm long segments each,and the corresponding segments were immersed and sonicated in an extract containing dithiothreitol,methanol,etc.Mobile phase A was 0.1%formic acid in water,and mobile phase B was acetonitrile.Data were collected using electrospray ion source positive ion mode in multiple reaction monitoring mode.Results The 20 benzodiazepines in multiple hairs had a good linear relationship(r>0.99),detection limit of 0.2～5 pg/mm,minimum quantification limit of 0.5 to 20 pg/mm,the intro-day and inter-day precisions of 2.03%to 13.81%,the intro-day and inter-day accuracies of 87.98%～111.29%,matrix effect of 71.15%～110.43%,and extraction recovery of 69.34%to 99.94%.Using this method,hair samples were analyzed in volunteers taking a single dose of clonazepam for 20 days,and the detected location of clonazepam in 15 hairs was located in segment 6～11 from the hair root,with the concentration range of 1.06～3.45 pg/mm.By this method,the hair of the victims suspected of having taken sleeping pills in the case was analyzed.In 15 hairs,clonazepam was detected in segments 9～13 of the hair root.The quantitative results were 1.07～19.06 pg/mm.The experimental results were basically consistent with the medication time of the victim.Conclusion The micro-segmentation analysis technique of multiple hairs can be applied to the investigation of drug-assisted crime cases,and can roughly estimate the medication time.

It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro. However, the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown. In this study, we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2 (PC2) located on the primary cilia on the surface of ROBs. First, immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs, and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting. Subsequently, we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP), as well as the expression of Runx-2, Bmp-2, Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI), a PC2 blocker. Moreover, we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference. The results showed that PC2 was localized on the primary cilia of ROBs, and PEMFs treatment increased the expression of PC2 protein. When PC2 was blocked by AMI, PEMFs could no longer increase PC2 protein expression and ALP activity, and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset. After inhibiting the expression of PC2 using RNA interference, PEMFs can no longer increase the expression of genes related to bone formation. The results showed that PC2, located on the surface of primary cilia of osteoblasts, plays an indispensable role in perceiving and transmitting the physical signals from PEMFs, and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2. This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.
Alkaline Phosphatase/metabolism* ; Animals ; Electromagnetic Fields ; Osteoblasts/metabolism* ; Osteogenesis/genetics* ; Rats ; TRPP Cation Channels/physiology*

Objective:To study the effects of a simulated plateau environment on fracture healing in rats.Methods:A rat model of mid-femoral fracture was established by hacksaw truncation and intramedullary fixation with Kirschner wires in 60 male Wistar rats which were divide into 2 groups ( n=30) by the random number table method. The rats in the control group were raised in the animal experiment center of The 940 Hospital of Joint Logistic Support Force of Chinese PLA at an altitude of 1,400 m, while the rats in the plateau group were placed in an animal experimental cabin in a simulated plateau environment at a simulated altitude of 5,000 m. The body weight was weighed once a week and X-ray films were taken every 2 weeks. Blood samples were collected after 4 weeks for detection of biochemical indicators of bone metabolism. After 8 weeks, the femurs of the surgical side were taken for bone biomechanical detection and the bone mineral density of the healthy side was detected. After 4 and 8 weeks, the femurs of the surgical side were taken for in vitro Micro-CT scanning and angiography detection. After 1, 2, 4 and 8 weeks, the femurs of the surgical side were taken for bone histopathologic detection. Results:During the entire experiment, no rats in the control group died while the mortality rate of the rats in the plateau group was as high as 26.7% (8/30). In the plateau group, some organs were pathologically damaged in the rats, fracture union was delayed, and the callus differentiated and matured slowly with the chondrocytes still dominant at the 8th week. The bone mineral density and the maximum load of the femur in the plateau group were significantly lower than those in the control group ( P< 0.05). Angiography showed that the rats in the plateau group had microvascular proliferation which did not penetrate the fracture end at the 8th week. The bone formation indexes like osteocalcin, procollagen type Ⅰ N-terminal propeptide (PⅠNP), and osteoprotegerin of the rats in the plateau group were significantly lower than those in the control group at the 4th week ( P<0.05). The bone resorption indexes like tartrate resistant acid phosphatase 5b (TRACP-5b) and receptor activator for nuclear factor-κB ligand (RANKL) in the plateau group were significantly higher than those in the control group ( P<0.05). Conclusion:A simulated plateau environment at an altitude of 5,000 m may lead to delayed fracture healing in rats.

OBJECTIVETo investigate whether transcription factor-kappaB (NF-κ B) is involved in the modulation of P-glycoprotein (P-gp) by glucosylceramide synthase (GCS) in a multidrug resistance leukemia cell line K562/A02 and to explore the relationship between NF-κ B and extracelluar signal-regulated kinase (ERK).

METHODSK562/A02 cells were treated with GCSsiRNA, pyrrolidine dithiocarbamate (PDTC, a NF-κ B specific inhibitor) and U0126 (a MEK1/2 inhibitor), respectively. The expression of GCS and multidrug resistance protein 1 (MDR1) mRNA were analyzed with qRT-PCR. Various proteins of different groups were measured by Western blotting.

RESULTSAfter transfected with GCSsiRNA for 48 h, GCS mRNA were reduced by 62% (51%-73%) and MDR1 mRNA was reduced by 52% (43%-61%) in the K562/A02 cells. Compared with the negative control, relative expression of NF-κ B p65 in nuclear and P-ERK1/2 were both down-regulated, and P-gp was also inhibited significantly at 72 h after transfected with GCSsiRNA (P< 0.05). In addition, the expression of P-gp was decreased at 24 h with 80 μ mol/L PDTC and 48 h with 20 μ mol/L PDTC. P-ERK1/2 was inhibited significantly when the cells were treated with 20 μ mol/L U0126 for 48 h. The expression of NF-κ B p65 in nuclear and P-gp were also down-regulated.

CONCLUSIONNF-κ B can modulate the effect of GCS on P-gp in K562/A02 cells. P-ERK1/2 can activate NF-κ B in above signal transduction pathway.

ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Cell Line, Tumor ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Glucosyltransferases ; genetics ; metabolism ; Humans ; K562 Cells ; Leukemia ; genetics ; NF-kappa B ; genetics ; metabolism

BACKGROUNDBladder cancer is widely known as the most common malignant tumor in the urinary tract, with 75%-85% of patients suffering from nonmuscle invasive bladder cancer (NMIBC). However, the optimal dose of Bacillus Calmette-Guérin (BCG) remains controversial. The aim of this study was to compare the therapeutic efficacy of full dose (FD) with the reduced dose (RD) of BCG.

METHODSRandomized controlled trials (RCTs) were selected through the Cochrane Library, PubMed and Embase and were supplemented by hand searching of bibliographies. The end points include overall survival rate, recurrence rate, progression rate and side effects.

RESULTSFive RCTs that included a total of 1 473 patients (727 in the reduced dose group vs 746 in the full dose group), with a median follow-up period from 33.5 month to 7.1 year. Disease in 80 of 687 (11.6%) patients assigned to the RD group progress to the muscular layer or distant metastasis, compared with 81 of 698 (11.6%) patients assigned to the FD group (RR = 1.02; 95% CI, 0.77-1.36; P = 0.89). The incidence of recurrence at three year was reported in all five studies to be 41.1% (299 of 727) and 36.1% (269 of 746) in the RD and FD groups, respectively (RR = 1.13; 95% CI, 1.00-1.29; P = 0.05). The 5-year survival rate was 75.9% (502 of 662) in the RD group, and 75.8% (510 of 673) in the FD group. In the RD group 41 of 655 (6.3%) patients and 56 of 663 (8.7%) patients in the FD group did not complete the treatment due to systemic or local side effects (RR = 0.75; 95% CI, 0.51-1.10; P = 0.14) CONCLUSIONS: In general, the results of our study demonstrate a trend towards a reduction of the toxicity in reduced dose group without affecting the efficacy of treatment when compared with full dose. More trials with large sample size are still necessary to explore the prognosis of the patients with high risk of tumor in different dose group.

BCG Vaccine ; therapeutic use ; Female ; Humans ; Male ; Neoplasm Recurrence, Local ; prevention & control ; Randomized Controlled Trials as Topic ; Urinary Bladder Neoplasms ; prevention & control

ObjectiveTo investigate the influence factors of operative effect in complex acetabular fracture. MethodsSixty-seven patients with complex acetabular fractures were selected. All patients were used surgical treatment,the clinical curative effect by Merle d'Aubigne-Postel hip scoring function to evaluate,set the age,sex, operation time,operative approach,the quality of fracture reduction,complex dislocation of the hip fracture and type of complex acetabular fractures with comparative index,the results were statistically analyzed. ResultsPatients were followed up for 6-24 months,the mean time was 14.6 months. Univariate analysis showed that the fine efficacy of surgery taken in no more than 2 weeks [84.0％ (42/50)]was better than more than 2 weeks [64.7％ ( 11/17 )](P ＜ 0.05 ). The high-quality of reduction [86.4％ ( 19/22 )]was better than low-quality of reduction [44.4％ (4/9)] (P ＜ 0.01 ). The compare of age,sex,operative approach,complex dislocation of the hip fracture were no statistically different (P ＞0.05).Complex acetabular fractures of T-shaped fracture healing rate was 85.7％ (12/14),the excellent rate was significantly higher than posterior column with posterior wall fractures[77.8％ (7/9)],transverse with posterior wall fractures [75.0％ (15/20)], anterior column with half of transverse fractures [66.7％ (2/3)],double-column fractures[71.4％ (15/21)],the results were statistically different. Conclusion The necessary conditions for ensuring fracture prognosis is the quality of reduction,timing of surgery,and type of fracture.

AIM: To explore the effects of Angelicae sinensis preparation and sodium ferulate on inflammatory liver injury induced by lipopolysaccharide (LPS) and their molecule mechanism. METHODS: ICR mice were divided into five groups: Angelicae sinensis group, sodium ferulate group, dexamethasone group, inflammation control group and normal group. The model of inflammatory injury in mice was set up by tail vein injection with the bacillus calmette-guerin (BCG) and LPS respectively prior and posterior to administration of those tested drugs. The tested drugs (the preparations of Angelica sinensis, sodium ferulate and dexamethasone) and normal saline were given respectively to the corresponding group. The pathological observation of the liver tissues in mice was made for the intensity of inflammatory liver injuries. The immunohistochemical detection and comparison were performed for the expression of ICAM-1 and E-selectin proteins in the liver tissues in those mice. RESULTS: The intensities of liver inflammatory injuries in mice from drug-treated groups were obviously lighter than that from the inflammation control group (P