[Analysis of characteristics of drug resistance gene mutation in HBV RT region of hepatitis B infected patients].

Objective: This article investigated the clinical characteristics and distribution of drug resistance mutation sites in HBV RT region of hepatitis B infected patients. Methods: Retrospective analysis was made on 1 948 patients with HBV infection, who had been tested for NAs resistance mutation and had a medical history of NAs in the Laboratory Department of the Fifth Medical Center of the PLA General Hospital from January 2020 to December 2021. Basic clinical information and drug resistance related mutation information were recorded. Meanwhile, the serological index data of hepatitis B were collected. Drug resistance gene mutant group and non-mutated group were grouped according to whether the drug resistance genes had a mutation in HBV RT region, and the clinical characteristics and genotype distribution of the two groups were statistically analyzed. The pattern of drug resistance gene mutation, number of mutation sites, drug resistance type and mutation of NAs resistance-related sites were analyzed in 917 patients with drug resistance gene mutation in HBV RT region. χ2 Inspection was used for counting data. Meanwhile, two independent samples t-test and Wilcoxon rank sum test were used for measurement data. Results: Among the 1 948 patients with chronic HBV infection, 917 patients had drug resistance gene mutation in RT region (47.07%). The proportion of patients with acute hepatitis B and CHB in HBV RT resistance gene mutant group was lower than that in the non-mutated group, while the proportion of patients with HBV-related cirrhosis was higher than that in the non-mutated group, these differences were statistically significant. Compared with the non-mutated group in HBV RT region, the age, the positive rates of HBeAg and HBV DNA, and HBV DNA load of these patients were increased in drug resistance gene mutant group, these differences were statistically significant. Genotypes of patients in both groups were dominated by C, followed by B and D. The proportion of patients with genotype C in HBV RT drug resistance gene mutant group was higher than that of non-mutated group, the difference was statistically significant. There were 53 gene mutation patterns in 917 patients with drug resistance gene mutation in HBV RT region, and the main pattern was rtL180M+rtM204V+rtS202G (9.70%). The mutation sites were dominated by 3 (20.74%). There were 5 types of drug resistance, LAM+Ldt (21.25%) was the most. Among the 18 sites that were clearly associated with LAM, ADV, ETV and Ldt resistance in the HBV RT region, 14 sites were mutated, and the most common mutation sites were rtL180M, rtM204V, rtM204 and rtS202G. what's more, the proportion of patients with NAs drug resistance was LAM>Ldt>ETV>ADV. Conclusion: In order to prevent adverse consequences of this study such as disease recurrence or disease progression caused by HBV drug resistance, HBV infected patients, who have long-term use of NAs antiviral therapy, should monitor the level of HBV DNA and drug resistance genes in HBV RT region in order to optimize the treatment plan in time or guide individualized treatment.

[Tumor cell-based glycolytic metabolism and single-cell sequencing of urinary exfoliated cells for the diagnosis and molecular profiling of urothelial carcinoma].

Objective: To explore the diagnostic values of HK2 testing and single-cell sequencing in the urothelial carcinoma (UC). Methods: The qualified urine specimens of 265 suspected UC patients or postoperative patients from the Cancer Hospital of Chinese Academy of Medical Sciences, Beijing, China were collected. Both exfoliative cytology and HK2 testing were performed on clinically suspected UC or postoperative patients. The performance of diagnostic cytology and HK2, including consistency, sensitivity, specificity, positive predictive value and negative predictive value, was evaluated based on histopathological, clinical and imaging diagnosis. Isolated HK2 metabolically abnormal cells were subject to single-cell sequencing to verify the reliability of HK2 detection performance and to explore the molecular characteristics of UC. Results: The concordance rate of HK2 testing and cytology for detecting UC was 90.3% (102/113, Kappa=0.604). Compared with cytology, the sensitivity of HK2 was significantly higher (85.2% versus 75.6%, P=0.024). The detection sensitivity of combined HK2 testing and cytology was increased to 91.1%. HK2 testing was significantly more sensitive than cytology for diagnosing UC in the upper urinary tract (81.8% versus 65.5%, P=0.022). It was also more sensitive than cytology for diagnosing early-stage UC (82.6% versus 69.5%, P=0.375) and low-grade UC (69.6% versus 47.8%, P=0.125). Single-cell sequencing of the ten patients, whose samples were positive for HK2, demonstrated highly concordant copy number variations (CNVs) in tumor cells from the same UC patient, with heterogeneity in CNV profiles among different patients. Deletion of chromosome 8p was found in 3 of the 4 urine samples of renal pelvis UC. The 2 patients with benign lesions had no CNVs in all sequenced cells. Conclusions: The test for abnormal urinary glycolytic HK2 metabolism can assist urine cytology to improve the sensitivity of UC diagnosis, and it provides a novel and reliable approach for early detection of upper urinary tract UC and lower grade UC. Meanwhile, this study has preliminarily revealed the feasibility of single-cell sequencing in urinary samples, which is expected to improve the diagnostic specificity of HK2 testing.

Increased Expression of Efflux Pump norA Drives the Rapid Evolutionary Trajectory from Tolerance to Resistance against Ciprofloxacin in Staphylococcus aureus.

The intensively intermittent use of antibiotics promotes the rapid evolution of tolerance, which may lead to resistance acquisition in the following evolutionary trajectory. In addition to directly exporting antibiotics as an instant resistance strategy, efflux pumps are overexpressed in tolerant strains. To investigate how efflux pumps participate in resistance development from tolerance to resistance, we performed in vitro evolutional experiments against the antibiotic ciprofloxacin in norA efflux pump mutants of Staphylococcus aureus. These experiments demonstrated that overexpression of norA rapidly facilitated the development of ciprofloxacin resistance from tolerance to resistance through elevated spontaneous mutations. The generated resistance mutations were further fixed in the population by increasing survival ability. The observed Ser80Phe and Glu84Lys mutations in the topoisomerase IV ParC (GrlA in S. aureus) may be responsible for tolerant strains to develop resistance to ciprofloxacin since it has been reported that such mutations disrupt the water-metal ion bridge between quinolones and ParC. MepA and Sav1866 are related to the same antibiotic (ciprofloxacin) susceptibility as NorA, and they also contributed to resistance development against ciprofloxacin. MgrA positively regulated NorA expression and the development of ciprofloxacin resistance. Importantly, blocking the evolutionary pathway by coadministering ciprofloxacin with the efflux pump inhibitor reserpine effectively delayed the resistance acquisition in an in vitro experiment. This study illustrated the role of efflux pumps in the evolutionary trajectory from tolerance to resistance. The delayed resistance development caused by the efflux pump inhibitor illuminates a possible strategy for postponing the resistance acquisition from tolerance to resistance by disrupting efflux pumps.

[The expression and clinical significance of serum soluble programmed cell death ligand-1 in adult patients with community-acquired pneumonia].

To investigate the clinical significance of serum soluble programmed cell death ligand-1 (PD-L1) in adult patients with community-acquired pneumonia (CAP). A total of 44 CAP patients, 54 severe CAP patients and 30 healthy volunteers were recruited in this study. Serum soluble PD-L1 were detected. Univariate and multivariate regression analyses were used to assess the influence of multiple clinical variables on prognosis. Serum soluble PD-L1 level in severe CAP group was 98.20(57.94, 128.90) ng/L, which was significantly higher than that in the CAP group [59.32(33.55, 92.58) ng/L] and healthy controls [20.44(12.15, 36.20) ng/L] (all P<0.001). PD-L1 level was positively correlated with CRUB-65(r=0.481, P<0.001) and the pneumonia severity index (PSI) score (r=0.442, P<0.001). Univariate regression analysis showed that CURB-65 (HR=2.544, 95%CI 1.324-4.889, P=0.005), PSI score (HR=1.036, 95%CI 1.012-1.061, P=0.004), soluble PD-L1(HR=1.013, 95%CI 1.001-1.026, P=0.041) were risk factors of mortality during hospitalization. Multivariate regression analysis suggested that PSI score (HR=1.042, 95%CI 1.012-1.073, P=0.005), soluble PD-L1 (HR=1.011, 95%CI 1.002-1.071, P=0.020) were independent predictors for mortality risk in CAP patients. CAP patients with soluble PD-L1≥98.20 ng/L had a significantly lower survival rate than those with soluble PD-L1<98.20 ng/L (P=0.033). In conclusion, this study indicates that serum soluble PD-L1 level in CAP patients is correlated with the survival prognosis.

Venturiales.

Members of Venturiales (Dothideomycetes) are widely distributed, and comprise saprobes, as well as plant, human and animal pathogens. In spite of their economic importance, the general lack of cultures and DNA data has resulted in taxa being poorly resolved. In the present study five loci, ITS, LSU rDNA, tef1, tub2 and rpb2 are used for analysing 115 venturialean taxa representing 30 genera in three families in the current classification of Venturiales. Based on the multigene phylogenetic analysis, morphological and ecological characteristics, one new family, Cylindrosympodiaceae, and eight new genera are described, namely Bellamyces, Fagicola, Fraxinicola, Fuscohilum, Neofusicladium, Parafusicladium, Pinaceicola and Sterila. In addition, 12 species are described as new to science, and 41 new combinations are proposed. The taxonomic status of 153 species have been re-evaluated with 20 species excluded from Venturiales. Based on this revision of Venturiales, morphological characteristics such as conidial arrangement (solitary or in chains) or conidiogenesis (blastic-solitary, sympodial or annellidic), proved to be significant at generic level. Venturia as currently defined represents a generic complex. Furthermore, plant pathogens appear more terminal in phylogenetic analyses within Venturiaceae and Sympoventuriaceae, suggesting that the ancestral state of Venturiales is most likely saprobic.

[Neoadjuvant treatment for locally advanced gastric cancer].

Gastric cancer is the second most common and the second leading cause of cancer related mortality in China, which seriously endanger the lives and health of Chinese people. Due to the lack of typical symptoms and screening system of early gastric cancer, about 70% patients with gastric cancer are diagnosed as locally advanced disease in China. Radical resection is the main approach for locally advanced gastric cancer (LAGC), however, the clinical effect of gastrectomy alone or gastrectomy plus adjuvant treatment is limited. As a result, there has been increasing interest in the neoadjuvant treatment, which could potentially downstage tumor, eliminate tumor micrometastasis, reduce intraoperative dissemination and postoperative recurrence, thus improve the prognosis of patients with LAGC. The exploration and application of chemotherapy, radiotherapy, targeted therapy and immunotherapy of neoadjuvant treatment in LAGC have made continuous progress in recent years. To date, neoadjuvant therapy has been developed as a part of multimodality treatment for patients with LAGC.

[Deleted in lymphocytic leukemia 1 promoted proliferation and apoptosis of nephroblastoma cells through regulating miR-513a-5p and RANBP2 pathway].

Objective: To study the regulatory effects and mechanisms of deleted in lymphocytic leukemia 1 (DLEU1), microRNA-513a-5p (miR-513a-5p), and RAN binding protein 2 (RANBP2) in nephroblastoma. Methods: The GHINK-1 cells were transfected with pcDNA (pcDNA group), pcDNA-DLEU1 (pcDNA-DLEU1 group), miR-NC (miR-NC group), miR-513a-5p mimics (miR-513a-5p group), pcDNA-RANBP2 (pcDNA-RANBP2 group), pcDNA-DLEU1 and miR-NC (pcDNA-DLEU1+ miR-NC group), pcDNA-DLEU1 and miR-513a-5p mimics (pcDNA-DLEU1+ miR-513a-5p group), miR-513a-5p mimics and pcDNA (miR-513a-5p+ pcDNA group), miR-513a-5p mimics and pcDNA-RANBP2 (miR-513a-5p + pcDNA-RANBP2 group). Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect the expressions of DLEU1, miR-513a-5p, RANBP2 in nephroblastoma tissues, normal adjacent tissues, normal kidney cell HK2, and hemangioblastoma cell GHINK-1. Western blot was used to detect the expressions of proliferating cell nuclear antigen (PCNA), B cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 related X (Bax). Cell counting kit 8 (CCK-8) was used to detect the cell survival rate. Flow cytometry was used to detect the apoptosis rate. Dual luciferase report test was used to detect the luciferase activity of cells. Results: The expression levels of DLEU1, miR-513a-5p and RANBP2 in adjacent tissues were 1.02±0.08, 1.01±0.06, 1.00±0.05, respectively, significantly lower than 5.16±0.24, 0.23±0.02, 1.67±0.09 in nephroblasts tumor tissues (P<0.05). Their expression levels in HK2 cells were 1.00±0.06, 1.00±0.08, 1.02±0.09, respectively, significantly lower than 3.15±0.21, 0.18±0.01, 1.54±0.10 in GHINK-1 cells (P<0.05). Overexpression of DLEU1 significantly reduced the apoptosis rate (7.35±0.41 vs 12.35±1.12, P<0.05). Overexpression of RANBP2 significantly reduced the apoptosis rate (8.89±0.48 vs 12.64±1.12, P<0.05). Compared with the miR-NC group (1.01±0.06, 0.99±0.06), the luciferase activity of DLEU1-WT (0.43±0.04) and RANBP2-WT (0.61±0.07) in miR-513a-5p group were significantly reduced (P<0.05). Compared with anti-miR-NC group (0.99±0.07, 0.98±0.05), the luciferase activity of DLEU1-WT (1.34±0.11) and RANBP2-WT (1.39 ±0.13) in anti-miR-513a-5p group was significantly increased (P<0.05). Simultaneous overexpression of pcDNA-DLEU1 and miR-513a-5p in GHINK-1 cells significantly reduced the apoptosis rate (11.34±1.03 vs 8.51±0.69, P<0.05). Simultaneous overexpression of miR-513a-5p and RANBP2 in GHINK-1 cells significantly reduced the apoptosis rate (9.96±0.72 vs 15.94±1.00, P<0.05). Conclusions: The long-chain non-coding RNA (lncRNA) DLEU1 can promote the proliferation and inhibit the apoptosis of nephroblastoma cells. The mechanism is related to the targeted regulation of miR-513a-5p and RANBP2 function, which will provide theoretical support for the nephroblastoma treatment.

[Investigation and analysis of occupational exposure factors in an enterprise processing waste electronic products].

Objective: To identify and to analyze occupational exposure factors generating from production process in an enterprise processing waste electronic products, to provide scientific evidences for protection measures. Methods: From June to October 2017, seven waste electrical and electronic product processing enterprises in a province were selected as the research objects. These seven enterprises all include refrigerator dismantling line, washing machine dismantling line, air conditioning dismantling line, TV/computer dismantling line, CRT cutting line. Some enterprises also have circuit board line, wet precious metal recycling line, plastic crushing line, plastic granulating line and other deep processing operations such as precious metal recycling of waste circuit board, waste plastic crushing and recycling. The data were collected by the methods of occupational health field investigation and occupational health testing, and the exposure level of occupational hazard factors was evaluated by combining the effect of the protective facilities of occupational health engineering. Results: the main occupational hazard factors of waste electrical and electronic products treatment enterprises were dust, noise, heavy metal, flame retardant, mercury, fluoride, cyanide, sodium hydroxide, hydrochloric acid and nitric acid. 145 effective samples were obtained from heavy metal sampling, including 102 individual samples and 43 fixed-point long-term samples. Among them, 8 samples lead exceeded the standard, all occurred in the TV dismantling line. In addition to flame retardants, plasticizers, insecticides and other components can be detected by high-throughput qualitative analysis using liquid chromatography-time-of-flight mass spectrometry. Finally, 123 effective 8-h equivalent sound level values were obtained by individual noise detection, 86 of which exceeded the standard, with a rate of 69.9%. Conclusion: there are potential occupational hazards in the process of dismantling waste electrical and electronic products.

[Investigation of mental workload and related factors among nurses from tertiary hospitals in Shandong].

Objective: To investigate mental workload among nurses from tertiary hospitals in Shandong Province, and analyze various factors related to mental workload. Methods: From May to July 2019, a cluster sampling method was used to select 8255 nurses from 20 third class a general hospitals in 16 cities of Shandong Province as the research objects, and 8159 valid questionnaires were collected. The general information and psychological load of nurses were investigated by general information questionnaire and task load index scale. The measurement data were expressed in percentage (%) ; the nurses' psychological load scores were in accordance with normal distribution, and the differences between groups were compared by t-test or ANOVA; the related influencing factors of nurses' psychological load were analyzed by multiple stepwise regression analysis. Results: The average scores of mental workload among nurses was 77.83 (SD=12.88) . Time demands and physical demands were the two highest rated dimensions of mental workload. the average scores were 90.77 (SD=12.47) and 79.92 (SD=15.23) . Multiple stepwise regression analysis showed that Satisfaction with income, monthly average night shift and professional titles were the significant predictors of mental workload (R(2)=0.08) . Conclusion: Nurses with higher psychological load, lower income satisfaction, higher number of night shifts per month and lower title have higher psychological load.

Survival prediction with score model based on clinical characteristics in advanced HCC patients receiving oxaliplatin-containing regimens.

A score model based on clinical characteristics in advanced hepatocellular carcinoma (HCC) patients treated with systemic chemotherapy of oxaliplatin-containing regimens was established to evaluate progression-free survival (PFS) and overall survival (OS). Thirty HCC patients eligible for radical resection were involved in the retrospective study, and these were divided into the good response group (complete response (CR)/partial response (PR) and the poor response group (stable disease (SD)/progression disease (PD). The median PFS and OS were compared in the two groups. PFS and OS combined with clinical characteristics were evaluated by univariate and multivariate analyses. The score model was defined with 1 score for each characteristic, and score model cut-off values were determined by the receiver operating characteristic curve (ROC) which describes treatment response. The median PFS was 10 and 2 months (p<0.001), and the median OS was 13 and 4 months (p=0.011) for the CR/PR and SD/PD groups, respectively. The score of 1 was the optimal cutoff value, with sensitivity ranging from 52.6 to 63.2% and specificity from 81.8 to 100% (AUC= 0.773, p=0.014). The median PFS for good and poor response groups was 9 months and 1month (p<0.001) and the median OS was 22 and 3months at p<0.001, respectively. Patients with scores above 1 had poor response, with median 3 months OS and 1 month PFS, and patients with scores of 0 and 1 established good response, with median 22 months OS and 9 months PFS, respectively.

[Clinical study of brush liquid-based cytology combined with automatic immunohistochemistry in the classification and diagnosis of lung cancer].

Objective: To explore the feasibility of bronchoscopic brushing liquid-based slide cytology combined with automatic immunocytochemistry (ICC) for pathological typing of lung cancer. Methods: A liquid-based thin-prep was prepared from 171 bronchoscopic brushing specimens of patients with pulmonary lesions. ICC was detected by automatic immunohistochemistry instrument while cytomorphological diagnosis was made. The results were compared with those of histopathological diagnosis. Results: Among 171 patients, 130 (76.0%) could be classified by cell morphology alone, including 31 squamous cell carcinomas, 44 adenocarcinomas and 55 small cell carcinomas; 162 (94.7%) could be classified by cell morphology combined with ICC, including 38 squamous cell carcinomas, 61 adenocarcinomas and 63 small cell carcinomas (P<0.001). According to the gold standard of histopathological diagnosis, the coincidence rate of cytomorphology combined with ICC was higher than that of cell morphology alone. The coincidence rate of squamous cell carcinoma was increased from 85.2% to 97.1% (P=0.093), adenocarcinoma from 92.5% to 98.0% (P<0.001), and small cell carcinoma from 96.1% to 98.3% (P=0.465). Conclusion: The combination of liquid-based thin-prep cytology and automatic immunohistochemistry can effectively improve the accuracy of pathological typing of brushing specimens under fiberoptic bronchoscopy, and provide more objective diagnostic results for clinical treatment.

Relationship between Electrical Conductivity and Related Biochemical Indicators of Rat Cerebrum Tissues and Postmortem Interval.

ABSTRACT: Objective To explore the relationship between the electrical conductivity （EC） and biochemical indicators of rat cerebrum tissues and postmortem intervals （PMIs） and discuss the mechanism of applying EC to infer PMI. Methods Forty healthy Sprague-Dawley rats were sacrificed and stored in an environment of about 25 ℃. The whole cerebrum tissues of rats were removed respectively at different PMIs of 0, 1, 2, 3, 4, 5, 6, and 7 d, and then made into homogenized impregnation solution. The EC and related biochemical indicators （potassium, sodium, chloride, calcium, inorganic phosphorus, magnesium, uric acid, urea nitrogen and creatinine） in cerebrum tissue impregnation solution were determined, and the relationships among EC in impregnation solution, related biochemical indicators and PMI were analyzed. Results The EC in cerebrum tissues increased gradually with the extension of PMI, and the content of uric acid, urea nitrogen and inorganic phosphorus in its impregnation solution also increased gradually with the extension of PMI. The correlation of EC, uric acid, urea nitrogen, and inorganic phosphorus with PMI was relatively good （R2 was 0.95-0.99）, and there was a linear correlation between the content change of uric acid, urea nitrogen, inorganic phosphorus and EC （R2 was 0.97-0.99）. The changes of the other 6 kinds of biochemical indicators with the extension of PMI within 7 d after the rats' death were non-significant （P>0.05）. Conclusion The correlation between EC in cerebrum tissues, uric acid, urea nitrogen, inorganic phosphorus and PMI were relatively good, and combining various indicators can also improve the accuracy of PMI estimation.

Relationship between Electrical Conductivity and Chemical Content of Rat Skeletal Muscle Impregnating Solution and Postmortem Interval.

ABSTRACT: Objective To study the mechanism of change of the electrical conductivity （EC） of rat skeletal muscle impregnating solution that occurs with the change of postmortem interval （PMI）. Methods Healthy Sprague-Dawley rats were killed and kept at about 25 ℃. Skeletal muscles were extracted at different PMI--immediate （0 d）, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, and 7 d, then mixed with deionized water to make impregnating solution with a mass concentration of 0.1 g/mL. The solution's EC and nine common chemicals in it, such as potassium ion, calcium ion, and chloride ion, were determined. Results EC increased gradually with the extending of PMI （P=0.024） during the 7 days after the rats' death. The content of uric acid （P=0.032）, urea nitrogen （P=0.013） and phosphorus （P=0.022） also increased during the extension. However, the content of magnesium ions decreased with extending of PMI （P=0.047）. The correlation between potassium ion, sodium ion, chlorine ion, calcium ion, creatinine and PMI were weak （P>0.05）. Conclusion The molecular basis of skeletal muscle EC change in rats after their death is the changes of uric acid, urea nitrogen, inorganic phosphorus and other chemical components. Furthermore, combine use of various indicators can improve the accuracy of the EC method to infer PMI.

Analysis of 28S rRNA and COâ Gene Sequence of Nine Necrophagous Calliphorid Flies from Luoyang.

ABSTRACT: Objective To assess the feasibility of using 28S ribosomal RNA （28S rRNA） and mitochondrial cytochrome c oxidase subunit Ⅰ （COⅠ） gene sequences of nine necrophagous Calliphorid flies for the identification of common necrophagous Calliphorid flies, and to provide technical support for postmortem interval （PMI） estimation. Methods Twenty-three Calliphorid flies were collected and identified morphologically, and DNA were extracted from legs. The gene fragments of 28S rRNA and COⅠ were amplified and sequenced, then the sequence alignment was performed with BLAST. The composition of obtained sequences was analyzed and evolutionary divergence rate between species and intraspecies were established. The phylogeny tree was constructed with neighbor-joining method. Results The 23 necrophagous Calliphorid flies were identified to 9 species of 5 genera. The 715 bp from 28S rRNA and 637 bp from COⅠ gene were obtained and the online BLAST result showed more than 99% of similarity. The phylogeny tree showed that the necrophagous flies could cluster well into 9 groups, which was consistent with morphological identification results. The intraspecific difference in 28S rRNA was 0 and the interspecific difference was 0.001-0.033. The intraspecific difference in COⅠ was 0-0.008 and the interspecific difference was 0.006-0.101. Conclusion Combined use of 28S rRNA and COⅠ gene sequence fragments can effectively identify the nine Calliphorid flies in this study. However, for closely related blowfly species, more genetic markers should be explored and used in combination in future.

[The application of conventional smears and liquid-based cytologic slides of lymphatic metastasis specimens of lung adenocarcinoma acquired by fine needle aspiration to detect the expression of anaplastic lymphoma kinase protein].

Objective: To explore the feasibility of conventional smears and liquid-based cytologic slides of lymphatic metastasis specimens of lung adenocarcinoma acquired by fine needle aspiration cytology (FNAC) to detect the expression of anaplastic lymphoma kinase (ALK/D5F3) by immunocytochemistry (ICC) analysis. Methods: The lymphatic metastasis specimens of 147 lung adenocarcinoma, including 100 liquid-based cytologic slides and 47 conventional smears, were collected in this study. ALK fusion protein was detected by Roche Ventana ICC technology, which was compared with the ALK fusion gene assessed by fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR). Results: The positive rate of ALK (D5F3) fusion protein in advanced lung adenocarcinoma acquired by FNAC was 11.6% (17/147), and 10.6% (5/47) and 12.0% (12/100) were reached in conventional smears and liquid-based cytologic slides, respectively.Among 147 cases, 57 cases including 17 positive cases and 40 negative cases were verified by RT-PCR and FISH. The whole coincidence rate reached 96.5% (55/57). The sensitivity and specificity of ALK (D5F3) fusion protein detected in lung adenocarcinoma acquired by FNAC were 94.1% (16/17) and 97.5% (39/40), respectively. The sensitivity and specificity were both 100% (5/5 of sensitivity and 10/10 of specificity) in conventional smears, while 91.7% (11/12) and 96.7% (29/30) in liquid-based cytologic slides. Conclusion: Conventional smears and liquid-based cytologic slides of FNAC samples can be used to perform ICC analysis of ALK (D5F3) expression in advanced lung adenocarcinoma, especially for patients who have no opportunity for surgery or whose resected samples are difficult to form cell block.

Relationship between Early Postmortem Interval and Electrical Conductivity of Rat Liver, Spleen and Kidney after Death in Rats.

OBJECTIVES: To determine the electrical conductivity （EC） of the liver, spleen and kidney of rats at different postmortem intervals （PMIs） within 24 hours for investigating the relationship between EC of different organs and early PMI. METHODS: Totally 45 SD rats were sacrificed by cervical dislocation and kept at a constant temperature of 25 ℃. Tissues were taken from the liver, spleen, and kidney of rats at 0, 3, 6, 9, 12, 15, 18, 21 and 24 h. Impregnating solution with a mass concentration 0.1 g/mL was prepared using deionized water. The EC value of impregnating solution with different organs was separately determined. The regression equations of EC and PMI for different organs were established, respectively. The relationship between EC of different organs and early PMI was analysed in deceased rats. RESULTS: The relationship between PMI and EC of the liver and spleen was well fitted with the linear equation. The liver showed the best fitting degree followed by the spleen, while the EC of the kidney showed no significant changes within 24 h. There was a good linear relationship between early PMI and the EC of the liver and spleen. CONCLUSIONS: A good linear relationship between early PMI and the EC of the liver and spleen can be found in rats after death, which can be used for the early PMI estimation.

[Molecular Identification of Sarcosaphagous Flies in Luoyang Based on 28S rRNA Gene Sequence].

OBJECTIVES: To detect 715 bp sequence of 28S rRNA in sarcosaphagous flies, and to identify their common species for solving the problem of morphological identification, as well as providing technical support for postmortem interval （PMI） estimation. METHODS: Twenty-nine common sarcosaphagous flies were collected in Luoyang and classified by morphological characteristics. The DNA was extracted from the fly's legs by Chelex-100 method and then the fragments of 28S rRNA were amplified and sequenced. The results were compared with twenty-eight corresponding fly species of GenBank and EMBL databases. All the sequences were analyzed by MEGA7.0 software, and sequence alignment was performed by the searching in BLAST. The nucleotide composition was analysed, and the intraspecific and interspecific genetic distance and phylogenetic tree were established. RESULTS: Twenty-nine sarcosaphagous flies were classified into 6 species of 5 genera, 3 families by morphological characteristics. In the obtained 715 bp sequence of 28S rRNA, the comparison result of online BLAST showed that the similarity was 100%. Five species were well clustered by a phylogenetic tree. Between different groups, the interspecific and intraspecific differences ranged from 0.007 to 0.045 and 0 to 0.001, respectively. CONCLUSIONS: The 28S rRNA target gene sequences shows a good identification capability, which can be a new genetic marker for the identification of sarcosaphagous flies.

Acute hypoxic stress: Effect on blood parameters, antioxidant enzymes, and expression of HIF-1alpha and GLUT-1 genes in largemouth bass (Micropterus salmoides).

Dissolved oxygen (DO) plays a crucial role in survival, growth, and normal physiological functions of aquatic organisms. Nevertheless, the mechanisms involved in hypoxic stress and adaptation have not been fully elucidated in Largemouth bass (Micropterus salmoides). To reveal the effect of acute hypoxia on Largemouth bass, we simulated acute hypoxia (DO: 1.2 ± 0.2 mg/L) in the laboratory and analyzed physiological parameters (RBCs, Hb, SOD, CAT, NA+/K+-ATPase, GPx, and MDA) and gene expression (HIF-1alpha and GLUT-1) in Largemouth bass exposed to various durations of acute hypoxia (0, 1, 2, 4, 8, 12, and 24 h). Our results indicated that acute hypoxic exposure significantly increased RBCs but decreased Hb. In addition, antioxidant enzyme activity was enhanced significantly in the liver and muscles at the initial stage of acute hypoxic exposure, but decreased significantly in gills during the entire process of hypoxic exposure. Furthermore, the expression levels of HIF-1alpha and GLUT-1 mRNA were significantly up-regulated in Largemouth bass under acute hypoxic exposure. In conclusion, our study provides a valuable basis for further elucidation of hypoxic adaptation and facilitates husbandry for an economically valuable species.

[Effects of overexpression of human tissue inhibitor of metalloproteinase-1 on the inflammatory response in rats with myocardial infarction and related mechanisms].

Objective: To observe the effects of recombinant adenovirus with human tissue inhibitor of metalloproteinase-1(Ad-hTIMP-1) on the inflammatory response in rats with myocardial infarction (MI) and explore the related mechanisms. Methods: The male Wistar rats were randomly divided into sham-operated group, saline group, Ad-Track group and Ad-hTIMP-1 group according to the random number table (n=8 each group). MI was induced by ligation of the left anterior descending coronary artery and MI rats were injected with saline, Ad-Track and Ad-hTIMP-1, respectively. Sham-operated rats received similar surgical procedure without ligation of the left anterior descending coronary artery. After 4 weeks, the cardiac function was measured by echocardiography, then rats were sacrificed and hearts were removed for morphological and biological analysis. The morphology of myocardial tissue in each group was detected by HE staining and Masson staining. The mRNA expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and C-reactive protein(CRP) were detected by real-time PCR. Immune histochemical staining was performed to observe the protein expression levels of IL-6 and CRP. Results: (1) Left ventricular end systolic dimension derived from echocardiography was increased in saline group ((5.10±0.72) mm) and Ad-Track group ((4.88±0.64) mm) compared to sham-operated group ((4.25±0.46) mm), which was reduced in Ad-hTIMP-1 group ((4.13±0.35) mm, all P<0.05). The left ventricular ejection fraction was (72.46±5.74)%, (64.27±8.52)%, (64.65±3.90)%, and (71.55±6.95)%, the fractional shortening was (36.90±4.97)%, (29.03±3.40)%, (30.95±2.51)%, and (36.31±5.68)% in sham-operated group, saline group, Ad-Track group and Ad-hTIMP-1 group, respectively. The left ventricular ejection fraction and fractional shortening in saline group and Ad-Track group were lower than those in sham-operated group and Ad-hTIMP-1 group (all P<0.05). (2) Necrosis of myocardial cells was not found and a small amount of immune cell infiltration and interstitial fibrosis were observed on HE and Masson stained myocardial sections of Ad-hTIMP-1 group. (3) Real-time PCR showed that mRNA expressions of TNF-α, IL-6, IL-10 and CRP were lower in Ad-hTIMP-1 group than in saline group. mRNA expressions of TNF-α, IL-10 and CRP were lower in Ad-hTIMP-1 group than in Ad-Track group (all P<0.05). (4) Immune histochemical staining showed that protein expressions of IL-6 and CRP were higher in saline group and Ad-Track group than those in Ad-hTIMP-1 group (all P<0.05). Conclusion: Recombinant adenovirus Ad-hTIMP-1 can improve cardiac function in rats with myocardial infarction via inhibiting the inflammatory response and downregulating the expression of TNF-α, IL-6 and CRP.

[Relationship between Electrical Conductivity and Decomposition Rate of Rat Postmortem Skeletal Muscle].

OBJECTIVES: To analyze the relationship among electrical conductivity （EC）, total volatile basic nitrogen （TVB-N）, which is an index of decomposition rate for meat production, and postmortem interval （PMI）. To explore the feasibility of EC as an index of cadaveric skeletal muscle decomposition rate and lay the foundation for PMI estimation. METHODS: Healthy Sprague-Dawley rats were sacrificed by cervical vertebrae dislocation and kept at 28 ℃. Muscle of rear limbs was removed at different PMI, homogenized in deionized water and then skeletal extraction liquid of mass concentration 0.1 g/mL was prepared. EC and TVB-N of extraction liquid were separately determined. The correlation between EC (x1) and TVB-N (x2) was analyzed, and their regression function was established. The relationship between PMI (y) and these two parameters were studied, and their regression functions were separately established. RESULTS: The change trends of EC and TVB-N of skeletal extraction liquid at different PMI were almost the same, and there was a linear positive correlation between them. The regression equation was x2=0.14x1-164.91(R²=0.982). EC and TVB-N of skeletal muscle changed significantly with PMI, and the regression functions were y=19.38x1³-370.68x1²+2 526.03 x1-717.06(R²=0.994), and y=2.56x2³-48.39x2²+330.60x2-255.04(R²=0.997), respectively. CONCLUSIONS: EC and TVB-N of rat postmortem skeletal muscle show similar change trends, which can be used as an index for decomposition rate of cadaveric skeletal muscle and provide a method for further study of late PMI estimation.