[Cytotoxicity and underlying mechanism of evodiamine in HepG2 cells].

OBJECTIVE: To investigate evodiamine (EVO)-induced hepatotoxicity and the underlying mechanism. METHODS: HepG2 cells were treated with EVO (0.04-25 µmol/L) for different time intervals, and the cell survival rate was examined by cell counting kit-8 (CCK-8) method. After HepG2 cells were treated with EVO (0.2, 1 and 5 µmol/L) for 48 h, the alanine transaminase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) activities and total bilirubin (TBIL) content of supernatant were detected. A multifunctional microplate reader was used to detect the intracellular superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in HepG2 cells to evaluate the level of cell lipid peroxidation damage. The interactions between EVO and apoptosis, autophagy or ferroptosis-associated proteins were simulated by molecular docking. The HepG2 cells were stained by mitochondrial membrane potential (MMP) fluorescent probe (JC-10) and annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI), and MMP and apoptosis in HepG2 cells were detected by flow cytometry. The protein expression levels of caspase-9, caspase-3, bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2) were detected by Western blot. RESULTS: The cell survival rate was significantly reduced after the HepG2 cells were exposed to EVO (0.04-25 µmol/L) in a time- and dose-dependent manner. The half maximal inhibitory concentration (IC50) of the HepG2 cells treated with EVO for 24, 48 and 72 h were 85.3, 6.6 and 4.7 µmol/L, respectively. After exposure to EVO (0.2, 1 and 5 µmol/L) for 48 h, the ALT, AST, LDH, ALP activities and TBIL content in the HepG2 cell culture supernatant, and the MDA content in the cells were increased, and SOD enzyme activity was decreased. Molecular docking results showed that EVO interacted with apoptosis-associated proteins (caspase-9 and caspase-3) better. JC-10 and Annexin V-FITC/PI staining assays demonstrated that EVO could decrease MMP and promote apoptosis in the HepG2 cells. Western blot results indicated that the protein expressions of cleaved caspase-9 and cleaved caspase-3 were upregulated in the HepG2 cell treated with EVO for 48 h. In contrast, the protein expressions of pro-caspase-3, BSEP and MRP2 were downregulated. CONCLUSION: These results suggested that 0.2, 1 and 5 µmol/L EVO had the potential hepatotoxicity, and the possible mechanism involved lipid peroxidation damage, cell apoptosis, and cholestasis.

[Exploratory screening of potential pan-cancer biomarkers based on The Cancer Genome Atlas database].

OBJECTIVE: To screen potential pan-cancer biomarkers based on The Cancer Genome Atlas (TCGA) database, and to provide help for the diagnosis and prognosis assessment of a variety of cancers. METHODS: "GDC Data Transfer Tool" and "GDCRNATools" packages were used to obtain TCGA database. After data sorting, a total of 13 cancers were selected for further analysis. False disco-very rate (FDR) < 0.05 and fold change (FC) >1.5 were used as the differential expression criteria to screen genes and miRNAs that were up- or down-regulated in all the 13 cancers. In the receiver operating characteristic curve (ROC curve), the area under the curve (AUC), the best cut-off value and the corresponding sensitivity and specificity were used to reflect diagnostic significance. The Kaplan-Meier method was used to calculate the survival probability and then the log-rank test was performed. Hazard ratio (HR) was calculated to reflect prognostic evaluation significance. DAVID tool were used to perform GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis for differentially expressed genes. STRING and TargetScan tools were used to analyze the regulatory network of differentially expressed genes and miRNAs. RESULTS: A total of 48 genes and 2 miRNAs were differentially expressed in all the 13 cancers. Among them, 25 genes were up-regulated, 23 genes and 2 miRNAs were down-regulated. Most differentially expressed genes and miRNAs had good ability to distinguish between the cases and controls, with AUC, sensitivity and specificity up to 0.8-0.9. Survival analysis results show that differentially expressed genes and miRNAs were significantly associated with patient survival in a variety of cancers. Most up-regulated genes were risk factors for patient survival (HR>1), while most down-regulated genes were protective factors for patient survival (0 < HR < 1). The enrichment analysis of GO and KEGG showed that the differentially expressed genes were mostly enriched in biological events related to cell proliferation. In the regulatory network analysis, a total of 13 differentially expressed genes and 2 differentially expressed miRNAs had regulatory and interaction relationships. CONCLUSION: The 48 genes and 2 miRNAs that were differentially expressed in 13 cancers may serve as potential pan-cancer biomarkers, providing help for the diagnosis and prognosis evaluation of a variety of cancers, and providing clues for the development of broad-spectrum tumor therapeutic targets.

Determination of 23 perfluorinated alkylated substances in water and suspended particles by ultra-performance liquid chromatography/tandem mass spectrometry.

A practical multi-residue method based on ultra-performance liquid chromatography/tandem mass spectrometry was developed for the simultaneous determination of 23 perfluorinated alkylated substances (PFASs) in water and suspended particles. Suspended particle samples were extracted with 1% formic acid-acetonitrile and cleaned by matrix solid phase dispersion extraction using a C18 sorbent and graphitized carbon black. Water samples were filtered through 0.7-µm glass fiber membranes and enriched utilizing weak anion exchange cartridges. The eluent was dried under a gentle stream of N2 at 45°C and suspended in 1 mL acetonitrile-5 mM ammonium acetate (1:1, vol:vol). Gradient elution for chromatographic separation utilized acetonitrile and 5 mM ammonium acetate as mobile phases on a reverse phase C18 column. The compounds were quantified using an internal standard method in multiple reaction-monitoring mode. Limits of detection and quantitation of the 23 PFAS test compounds in water samples were 0.5-10 ng L-1 and 2-20 ng L-1, respectively. Recoveries at three fortified levels of 20, 50, and 200 ng L-1 ranged from 68.5% to 118% with relative standard deviations below 9.6%. We used this method to determine PFAS levels in real water and suspended particle samples and found high sensitivity and good reproducibility.

Significant prognostic values of nuclear genes encoding mitochondrial complex I subunits in tumor patients.

In cancer biology, it remains still open question concerning the oncogenic versus oncosuppressor behavior of metabolic genes, which includes those encoding mitochondrial complex I (CI) subunits. The prognostic value of nuclear genome mRNAs expression of CI subunits is to be evaluated in the tumor patients. We used the Kaplan Meier plotter database, the cBio Cancer Genomics Portal, and the Oncomine in which gene expression data and survival information were from thousands of tumor patients to assess the relevance of nuclear genome mRNAs level of CI subunits to patients' survival, as well as their alterations in gene and expression level in tumors. We presented that the relative expression level of overwhelming majority of the nuclear genes of CI subunits with survival significance (overall survival, relapse free survival, progression free survival, distant metastasis free survival, post progression survival, and first progression), had consistent effects for patients in each type of four tumors separately, including breast cancer, ovarian cancer, lung cancer, and gastric cancer. However, in gene level, frequent cumulative or individual alteration of these genes could not significantly affect patients' survival and the overexpression of the individual gene was not ubiquitous in tumors versus normal tissues. Given that reprogrammed energy metabolism was viewed as an emerging hallmark of tumor, thus tumor patients' survival might potentially to be evaluated by certain threshold for overall expression of CI subunits. Comprehensive understanding of the nuclear genome encoded CI subunits may have guiding significance for the diagnosis and prognosis in tumor patients.

Pre-B-cell colony-enhancing factor is markedly elevated in childhood hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome involving a final common pathway of hypercytokinemia, in which tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and soluble interleukin 2-receptor-alpha (sIL-2Rα) are the key cytokines. Pre-B-cell colony-enhancing factor (PBEF) is an inflammatory cytokine involved in several inflammatory diseases. However, its role in HLH is unknown. In this study, we examined the role of PBEF in HLH. Plasma was collected from 22 children with HLH and 14 healthy children. The concentrations of plasma PBEF, TNF-α, IFN-γ, and sIL-2Rα were determined using an enzyme-linked immunosorbent assay. All clinical data were derived from medical records. In the acute phase, children with HLH had much higher PBEF, TNF-α, IFN-γ, and sIL- 2Rα levels than did healthy children (P < 0.05). After treatment, 13 HLH children improved and PBEF, TNF-α, and IFN-γ levels decreased to normal levels (P < 0.05); sIL-2Rα levels also decreased (P < 0.05), but remained above the normal level (P < 0.05). Two patients were lost to follow-up, while 7 patients showed a bad response to therapy and eventually died, showing high PBEF levels above those of the survivors (P < 0.01). PBEF level was significantly positively correlated with TNF-α, IFN-γ, sIL-2Rα, serum ferritin, and triglycerides (all P < 0.05), and was negatively correlated with fibrin (P < 0.05). PBEF appears to be involved in the inflammatory process of HLH, and elevated PBEF is related to disease activity. We are currently evaluating the role of PBEF as a marker for the diagnosis and management of patients.

Association of the LMNA gene single nucleotide polymorphism rs4641 with bdilated cardiomyopathy.

Recently, studies on the pathogenesis of dilated cardiomyopathy (DCM) have focused on the underlying molecular biology and the association between single nucleotide polymorphisms (SNPs) and disease. This study was designed to explore the association between the rs4641 SNP of the LMNA gene and DCM in order to identify a new gene locus related to DCM. Polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing were employed to detect and genotype rs4641 in 198 patients with DCM and 160 healthy controls. Genotype and allele frequencies were compared to discover their relationship and logistic regression was used to assess the risk of DCM associated with the polymorphic variants. In the DCM group, the frequencies of the TC and TT genotypes and the T allele of rs4641 were remarkably higher than those in the control group (P < 0.01). According to risk analysis, taking the CC genotype as a reference, both the TC and TT genotypes increased the risk of DCM pathogenesis, with OR (95%CI) values of 5.957 (2.903- 12.222) and 6.424 (2.156-19.141), respectively. Taking the C allele as the reference, presence of the T allele was found to increase DCM risk, with OR (95%CI) of 5.295 (3.121-8.983). These results suggested that the C to T mutation at the rs4641 locus of LMNA could enhance the risk of DCM, and that rs4641 represented a genetic susceptibility locus. Therefore, it was concluded that the LMNA rs4641 SNP was associated with DCM risk, which indicated that LMNA is a susceptibility gene for DCM.

[Comparison of BCR::ABL (P210) mRNA levels detected by dPCR and qPCR methods in patients with chronic myeloid leukemia].

Objective: To compare digital polymerase chain reaction (dPCR) and real-time quantitative PCR (qPCR) measurements of BCR::ABL (P210) mRNA expression in patients with chronic myeloid leukemia (CML) . Methods: In this non-interventional, cross-sectional study, BCR::ABL (P210) mRNA was simultaneously measured by dPCR and qPCR in peripheral blood samples collected from patients with CML who underwent tyrosine kinase inhibitor therapy and who achieved at least a complete cytogenetic response from September 2021 to February 2023 at Peking University People's Hospital. The difference, correlation, and agreement between the two methods were evaluated using the Wilcoxon signed-rank test, Spearman's correlation, and Bland-Altman analysis, respectively. Results: In total, 459 data pairs for BCR::ABL mRNA expression measured by dPCR and qPCR from 356 patients with CML were analyzed. There was a significant difference in BCR::ABL mRNA expression between the two methods (P<0.001). When analyzed by the depth of the molecular response (MR), a significant difference only existed for patients with ≥MR4.5 (P<0.001). No significant difference was observed for those who did not achieve a major MR (no MMR; P=0.922) or for those who achieved a major MR (MMR; P=0.723) or MR4 (P=0.099). There was a moderate correlation between the BCR::ABL mRNA expression between the two methods (r=0.761, P<0.001). However, the correlation gradually weakened or disappeared as the depth of the MR increased (no MMR: r=0.929, P<0.001; MMR: r=0.815, P<0.001; MR4: r=0.408, P<0.001; MR4.5: r=0.176, P=0.176). In addition, the agreement in BCR::ABL mRNA expression between the two methods in those with MR4.5 was weaker than other groups (no MMR: ▉= 0.042, P=0.846; MMR:▉=0.054, P=0.229; MR4:▉=-0.020, P=0.399; MR4.5:▉=-0.219, P<0.001) . Conclusions: dPCR is more accurate than qPCR for measuring BCR::ABL (P210) mRNA expression in patients with CML who achieve a stable deep MR.

Reducing Injury During Restraint by Crisis Intervention in Psychiatric Wards in Singapore.

OBJECTIVE: The application of restraints during psychiatric crises is a serious adverse event. We aimed to reduce the number of injuries sustained by patients during the application of restraints. METHODS: Structured interviews were conducted with 10 staff to determine six root causes of patient injury during restraint. Three plan-do-study-act cycles were implemented: (1) reorganising shift rosters to pair trained staff with inexperienced staff, (2) holding monthly session for practising de-escalation and restraint techniques as a team in a supervised setting, and (3) rotating the responsibility for leading the de-escalation in real crises. RESULTS: Pre-intervention period was from January 2014 to December 2014 (28 251 inpatient bed days). Intervention period was from January 2015 to March 2015 (7121 inpatient bed days). Post-intervention period was from April 2015 to December 2016 (51 735 inpatient bed days). Data extracted included the dates and numbers of crises, activation of the crisis team, use of restraints, and injuries. During pre-intervention and intervention periods, only two minor and three moderate injuries were recorded. During post-intervention period, no injury was recorded and the number of restraints decreased gradually although the number of crisis team activations increased in the early phase. Eventually restraints were used only upon arrival of the crisis team. CONCLUSION: Our quality improvement project identified six root causes and implemented three plan-do-study-act cycles to successfully eliminated patient injuries during the use of restraints.

[An interlaboratory comparison study on the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels].

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People's Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories' results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion: The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.

A minimally invasive model of myocardial infarction made by video-assisted thoracoscopic surgery.

The general myocardial infarction model was made by ligating the left anterior descending coronary artery through traditional large opening thoracotomy, although there were many drawbacks such as severe injuries and pain in this method. The present study tried to find a new method to establish a minimally invasive canine model of myocardial infarction that was less injurious and more accurate. Twelve mongrel dogs were used in this study. After three 10-mm-long incisions were made on the left thoracic wall, pericardium was cut, left anterior descending coronary artery was found and titanium nips were clamped through video-assisted thoracoscopy. Two or three titanium nips were used until ECG showed a definite ST-segment elevation more than 0.1 mV, then the thoracic wall was sutured. The survival time of the dogs was 4 weeks. During this period, a series of thoracoscopy were performed. White blood count, biochemical analysis of oxidatively modified proteins, creatine kinase and cardiac Troponin I were examined at the baseline and in the 1st and 6th hour, and 1st, 4th, 7th, 14th and 28th day after surgery. The dogs were then killed and the ratio of fibrosis area to whole left ventricular area was measured and calculated to assess the extent of fibrosis. The sections from different parts of the heart were stained with Masson trichrome stain to assess degree of fibrosis. In the model group we observed that ST-segment of ECG elevated more than 0.1 mV instantly when titanium nips were clamped and the elevation could last for 28 days after surgery. Significant difference between the model and control group was discerned in white blood count, the levels of oxidatively modified proteins, creatine kinase, cardiac Troponin I and the ratio of fibrosis area to whole left ventricular area. Masson trichrome staining showed a large amount of collagen deposition in the fibrosis area. All these results demonstrated that a new canine model of myocardial infarction could be established with a minimally invasive procedure through video-assisted thoracoscopy. This minimally invasive pharmacological animal model was perhaps a more promising animal model for some newly rising science fields such as the study of Metabonomics.

Subacute bloodstream blockade effect by supercontraction of spider silk.

The present experiment was designed to test the subacute bloodstream blockade effect of supercontraction of spider silk on the femoral artery of rats. Observation on ligated femoral artery lasted 10 days. Blood flow, systolic/diastolic blood pressure, and oxidatively modified proteins in serum were measured before and after ligation. Meanwhile, histological manifestations of femoral artery at different times were observed by HE stain. We found that blood flow and systolic/diastolic blood pressure of femoral artery showed a descending tendency. Biochemical assay showed that oxidatively modified proteins significantly increased in the first 3 days and reached peak on the third day. Histological examination demonstrated that there was a progressive procedure from partial to complete occlusion of lumen of blood vessel and there was possibility of recanalization at last in the occlusive vessel. Our study indicated that ligation by spider silk might be applied to models of subacute ischemic disease such as subacute femoral artery ischemia, subacute or chronic ischemic cardiocerebrovascular disease, etc.

Room-temperature phosphorimetry studies of some addictive drugs following dansyl chloride labelling.

A room-temperature phosphorimetric (RTP) method for the analysis of barbital, codeine, morphine and practolol after labelling with dansyl chloride (DNS-Cl) is described. The drug-DNS derivatives were obtained by refluxing with drug-ethyl acetate solutions and solid DNS-Cl in the presence of anhydrous potassium carbonate. The reaction conditions were investigated in detail. The fluorescence emission of drug-DNS derivatives shifted to longer wavelengths compared with that of DNS-Cl. The RTP phenomena observed for these derivatives by using a micellar stabilized room-temperature phosphorescence technique were examined and optimum conditions for their RTP emission were studied using an orthogonal array design. Derivative RTP spectra were obtained and successfully used to determine practolol by the established method without further separation.

Determination of photophysical rate constants for the non-protected fluid room temperature phosphorescence of several naphthalene derivatives.

The determination of kinetic parameters for luminescence processes is very important in understanding the phosphorescence process and the mechanisms of the heavy atom effect (HAE). In our previous work, we reported that room temperature phosphorescence (RTP) emission of many naphthalene derivatives can be induced directly from their aqueous solution without using any kind of protective medium, and the name Non-Protected Fluid Room Temperature Phosphorescence (NP-RTP) is suggested for this new type of RTP emission. In order to further understand this kind of luminescence phenomenon, the influence of heavy atom perturber (HAP) concentration on RTP lifetime of several naphthalene derivatives was studied in detail in this paper. The possibility of determination of photophysical parameters for emission of NP-RTP was explored based on the definition on the phosphorescence lifetime and the relation with the concentration of HAP in this paper. A static Stern-Volmer equation for phosphorescence was derived and the luminescence kinetic parameters were calculated. The results obtained by two different ways proved that photophysical parameters for RTP emission can be determined based on the changes of the RTP lifetime.

[The trends and preventive strategies of esophageal cancer in high-risk areas of Taihang Mountains, China].

OBJECTIVE: To analyze and predict the trends of incidence and mortality rates of esophageal cancer in areas of Taihang Mountains, and explore the strategies for facilitating the reduction of the incidence and mortality of esophageal cancer. METHODS: The data were collected from Linxian and Cixian Counties registries between 1988-1997. The trends of the incidence and mortality rates for esophageal cancer had been fitted and predicted by the model of BP (Back Propagation) Neural Networks. Then Linqi Xiang in Linxian County was taken as an example, to determine the power for reducing incidence and mortality of esophageal cancer using endoscopic technology as an early detection and treatment method. RESULTS: There were slowly declining trends for both incidence and mortality rates during past 10 years in Taihang Mountains. The predicting rates of the esophageal cancer incidence in 2,002 are 115. 70/100,000 for male and 79.88/100,000 for female; mortality rates are 94.00/100,000 and 56.29/100,000 respectively. Both incidence and mortality rates a still in high level. But when using endoscopic technology as an early detection and treatment method, the incidence rate is nearly 1/3 of a control group and the power is 88.30% at the end of eight years. The mortality rate is 1/4 of the control group and power is 89.44% after five years intervention (both powers based on 0.05 significant levels). CONCLUSIONS: The incidence and mortality rates for esophageal cancer are still on high levels in Taihang Mountains. We should continue to enhance our preventive effort there. After using endoscopic technology as an early detection and treatment method, the incidence and mortality rates will be decreased markedly during 5 to 8 years. It is recommended to implement a large-scale secondary prevention strategy in these areas.

Quantitative chimerism: an independent acute leukemia prognosis indicator following allogeneic hematopoietic SCT.

This study evaluates the prognostic significance of quantitative chimerism to monitor minimal residual disease and predict relapse in acute leukemia (AL) patients following allogeneic hematopoietic SCT (HSCT). The quantitative chimerism levels of 129 AL patients were measured using RQ-PCR based on 29 sequence polymorphisms. Receiver-operating characteristic curve indicated that the optimal cutoff point to predict an inevitable relapse was 1.0%, which results in 100.0% sensitivity and 79.6% specificity.The relapse rate of patients with chimerism >1.0% at 2 years was 55.0%, whereas that for patients with chimerism <1.0% was 0%(P=0.000). Quantitative chimerism >1.00% indicated a higher probability of relapse. Cox multivariate analysis indicated that quantitative chimerism >1.00% was associated with lower disease-free survival (hazard ratio (HR)=10.825; 95% confidence interval (CI) =4.704-24.912, P=0.000) and lower OS (HR=8.681; 95% CI=3.728-20.212, P=0.000). Patients (24/47 with quantitative chimerism >1.00%) who received preemptive modified DLI immunotherapy had significantly lower relapse rate (37.5%) than those (n=9) who did not (100%; P=0.001). Thus, quantitative chimerism is an independent prognostic factor that predicts clinical outcomes after HSCT and provides a guide for suitable interventions.