Postmortem Diffusion of Aconitum Alkaloids and Their Metabolites in Rabbits.

OBJECTIVES: To explore the postmortem diffusion rule of Aconitum alkaloids and their metabolites in poisoned rabbits, and to provide a reference for identifying the antemortem poisoning or postmortem poisoning of Aconitum alkaloids. METHODS: Twenty-four rabbits were sacrificed by tracheal clamps. After 1 hour, the rabbits were administered with aconitine LD50 in decocting aconite root powder by intragastric administration. Then, they were placed supine and stored at 25 ℃. The biological samples from 3 randomly selected rabbits were collected including heart blood, peripheral blood, urine, heart, liver, spleen, lung and kidney tissues at 0 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h and 96 h after intragastric administration, respectively. Aconitum alkaloids and their metabolites in the biological samples were analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). RESULTS: At 4 h after intragastric administration, Aconitum alkaloids and their metabolites could be detected in heart blood, peripheral blood and major organs, and the contents of them changed dynamically with the preservation time. The contents of Aconitum alkaloids and their metabolites were higher in the spleen, liver and lung, especially in the spleen which was closer to the stomach. The average mass fraction of benzoylmesaconine metabolized in rabbit spleen was the highest at 48 h after intragastric administration. In contrast, the contents of Aconitum alkaloids and their metabolites in kidney were all lower. Aconitum alkaloids and their metabolites were not detected in urine. CONCLUSIONS: Aconitum alkaloids and their metabolites have postmortem diffusion in poisoned rabbits, diffusing from high-content organs (stomach) to other major organs and tissues as well as the heart blood. The main mechanism is the dispersion along the concentration gradient, while urine is not affected by postmortem diffusion, which can be used as the basis for the identification of antemortem and postmortem Aconitum alkaloids poisoning.

A novel method for determining postmortem interval based on the metabolomics of multiple organs combined with ensemble learning techniques.

Determining postmortem interval (PMI) is one of the most challenging and essential endeavors in forensic science. Developments in PMI estimation can take advantage of machine learning techniques. Currently, applying an algorithm to obtain information on multiple organs and conducting joint analysis to accurately estimate PMI are still in the early stages. This study aimed to establish a multi-organ stacking model that estimates PMI by analyzing differential compounds of four organs in rats. In a total of 140 rats, skeletal muscle, liver, lung, and kidney tissue samples were collected at each time point after death. Ultra-performance liquid chromatography coupled with high-resolution mass spectrometry was used to determine the compound profiles of the samples. The original data were preprocessed using multivariate statistical analysis to determine discriminant compounds. In addition, three interrelated and increasingly complex patterns (single organ optimal model, single organ stacking model, multi-organ stacking model) were established to estimate PMI. The accuracy and generalized area under the receiver operating characteristic curve of the multi-organ stacking model were the highest at 93% and 0.96, respectively. Only 1 of the 14 external validation samples was misclassified by the multi-organ stacking model. The results demonstrate that the application of the multi-organ combination to the stacking algorithm is a potential forensic tool for the accurate estimation of PMI.

Pazopanib alleviates neuroinflammation and protects dopaminergic neurons in LPS-stimulated mouse model by inhibiting MEK4-JNK-AP-1 pathway.

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopaminergic (DA) neurons and the accumulation of Lewy bodies (LB) in the substantia nigra (SN). Evidence shows that microglia-mediated neuroinflammation plays a key role in PD pathogenesis. Using TNF-α as an indicator for microglial activation, we established a cellular model to screen compounds that could inhibit neuroinflammation. From 2471 compounds in a small molecular compound library composed of FDA-approved drugs, we found 77 candidates with a significant anti-inflammatory effect. In this study, we further characterized pazopanib, a pan-VEGF receptor tyrosine kinase inhibitor (that was approved by the FDA for the treatment of advanced renal cell carcinoma and advanced soft tissue sarcoma). We showed that pretreatment with pazopanib (1, 5, 10 µM) dose-dependently suppressed LPS-induced BV2 cell activation evidenced by inhibiting the transcription of proinflammatory factors iNOS, COX2, Il-1ß, and Il-6 through the MEK4-JNK-AP-1 pathway. The conditioned medium from LPS-treated microglia caused mouse DA neuronal MES23.5 cell damage, which was greatly attenuated by pretreatment of the microglia with pazopanib. We established an LPS-stimulated mouse model by stereotactic injection of LPS into mouse substantia nigra. Administration of pazopanib (10 mg·kg-1·d-1, i.p., for 10 days) exerted significant anti-inflammatory and neuronal protective effects, and improved motor abilities impaired by LPS in the mice. Together, we discover a promising candidate compound for anti-neuroinflammation and provide a potential repositioning of pazopanib in the treatment of PD.

Engrafted primary type-2 astrocytes improve the recovery of the nigrostriatal pathway in a rat model of Parkinson's disease.

Parkinson's disease (PD) is a disorder characterized by a progressive loss of the dopaminergic neurons in the substantia nigra and a depletion of the neurotransmitter dopamine in the striatum. Our published results indicate that fasciculation and elongation protein zeta-1 (FEZ1) plays a role in the astrocyte-mediated protection of dopamine neurons and regulation of the neuronal microenvironment during the progression of PD. In this study, we examined the effects of engrafted type-2 astrocytes (T2As) with high expression of FEZ1 on the improvement of the symptoms and functional reconstruction of PD rats. T2As were stereotactically transplanted into the striatum of rats with PD induced by 6-hydroxydopamine (6-OHDA). An examination of apomorphine (APO)-induced rotations was performed to evaluate dopamine neuron damage and motor functions. Remarkably, the grafted cells survived in the lesion environment for six weeks or longer after implantation. In addition, the transplantation of T2As decrease the average velocity and the duration time of the APO-induced rotations, and increase the actuation time, as measured in the rotation behavioural tests. In the substantia nigra, the transplantation of T2As reduced the PD-induced GFAP, TH and FEZ1 downregulation. The grafted cells exclusively migrated to other regions near the injection site in the striatum and differentiated into GFAP+ astrocytes or TH+ neurons. Furthermore, by detecting monoamine neurotransmitters through high-performance liquid chromatography, we found that the nigrostriatal pathway had been repaired to some extent. Taken together, these results suggest that engrafted T2As with high expression of FEZ1 improved the symptoms and functional reconstruction of PD rats, providing a theoretical basis for FEZ1 as a potential target and engraftment of T2As as a therapeutic strategy in the treatment of PD.

Estimating Postmortem Interval Using Intestinal Microbiota Diversity Based on 16S rRNA High-throughput Sequencing Technology. / 基于16S rRNAé«˜é€šé‡æµ‹åºæŠ€æœ¯çš„è‚ é“èŒç¾¤å¤šæ ·æ€§æŽ¨æ–­æ­»äº¡æ—¶é—´.

OBJECTIVES: To explore the correlation between intestinal microbiota and postmortem interval(PMI) in rats by using 16S rRNA high-throughput sequencing technology. METHODS: Rats were killed by anesthesia and placed at 16 ℃, and DNA was extracted in caecum at 14 time points of 0, 1, 2, 3, 5, 7, 9, 12, 15, 18, 21, 24, 27 and 30 d after death. The 16S rRNA high-throughput sequencing technology was used to detect intestinal microbiota in rat cecal contents, and the results were used to analyze the rat intestinal microbiota diversity and differences. RESULTS: The total number of intestinal microbial communities did not change significantly within 30 days after death, but the diversity showed an upward trend. A total of 119 bacterial communities were significantly changed at 13 time points after death. The models for PMI estimation were established by using partial least squares (PLS) regression at all time points, before 9 days and after 12 days, reaching an R2 of 0.795, 0.767 and 0.445, respectively; and the root mean square errors (RMSEs) were 6.57, 1.96 and 5.37 d, respectively. CONCLUSIONS: Using 16S rRNA high-throughput sequencing technology, the composition and structure of intestinal microbiota changed significantly within 30 d after death. In addition, the established PLS regression model suggested that the PMI was highly correlated with intestinal microbiota composition, showing a certain time series change.

[Retraction Note: Association of Gene Polymorphisms in Wnt Signal Pathway with Tuberculosis in Chinese Tibetan Population.2016,47(6):920-925.]

This corrects the article DOI: 10.13464/j.scuxbyxb.2016.06.021.

[Methylation Analysis and Validation of Whole Genome DNA in Active Tuberculosis].

OBJECTIVE: To screen and identify the gene of DNA methylation in patients with active tuberculosis. METHODS: ① This study enrolled 9 cases of active tuberculosis patients (including 3 newly diagnosed tuberculosis patients and 6 cases of retreatment of active tuberculosis patients), 3 cases of latent tuberculosis patients and 3 cases of healthy controls. Genome DNA was extracted from Peripheral Blood Mononuclear Cell and following bisulfite conversion treatment. After hybridization with the Illumina HD 450K Infinium Mehtylation BeadChip, the results were compared between patients group and control group, GO and Pathway analysis were performed to evaluate the function of differentially expressed genes; ② We further enrolled 60 cases of active tuberculosis patients and 60 cases of health controls (their age and gender were matched). By using pyrosequencing method to detect the methylation levels of candidate genes (TLR1, TLR2, TLR4) screened by gene chip. RESULTS: ① Compared with healthy controls, we found that most of them were showed demethylation status. GO and Pathway analysis showed that the functions of the differentially methylated regions related genes were mainly enriched in the biological processes of the regulation of leukocyte apoptosis, cytokine regulation and inflammatory response which were closely related to tuberculosis. ②There were 10 CpG sites involved in the verified tuberculosis related genes (TLR1, TLR2, TLR4), the CpG sites of TLR1 gene showed the hypermethylation status (P<0.001), the CpG sites of TLR4 gene showed demethylation status (P=0.012). CONCLUSION: The present study demonstrated that in the course of MTB infection, the methylation status of genomic DNA was altered, and most of the Differentially Methylated Regions (DMRs) were showed status of demethylation. TLR1 gene and TLR4 gene may play an important role in the occurrence and development of tuberculosis.

[Etiology, Prognosis and Risk Factors of 181 Adult Community-acquired Acute Bacterial Meningitis].

OBJECTIVE: To understand the etiology, clinical prognosis and risk factors of adult community-acquired acute bacterial meningitis (ABM) and provide the evidence for clinical diagnosis and treatment. METHODS: We performed a retrospective study of 181 clinically diagnosed hospitalized patients with community-acquired adult ABM from Jan.2010 to Jan.2018. The patients were categorized as non-elderly (16≤age<65 years old, n=156 ) and elderly (age≥65 years old, n=25) group. The etiology, clinical features, prognosis and risk factors of the two groups were compared. RESULTS: Sixty-four of 181 patients (35.4%) had pathogens detected. The most common pathogens were Streptococcus pneumoniae (17.9%), Listeria monocytogenes (13.4%) and Klebsiella pneumoniae (10.5%). The mortality of the elderly group was higher than that of the non-elderly group (P<0.05). Univariate analysis showed that there was a significant difference between the elderly group and the non-elderly group in the incidence of hypertension, hypokalemia, pulmonary infection, ear-nose-throat ( ENT) infection, cerebrospinal fluid (CSF) protein concentration, head CT abnormalities and mortality. Logistic regression analysis showed that pulmonary infection and temperature ≥38.5 ℃ were independent risk factors for poor prognosis in the non-elderly group. CSF pressure ≥200 mmH2O was a independent risk factors for poor prognosis in the elderly group. CONCLUSION: The pathogens that cause acute bacterial meningitis in adult community are mainly Streptococcus pneumoniae, Listeria monocytogenes and Klebsiella pneumoniae.Pulmonary infection and temperature ≥38.5 ℃ are independent risk factors of poor prognosis in the non-elderly patients, as CSF pressure ≥200 mmH2O a independent risk factor in the elderly patients.

[Correlations Between AML1-ETO Fusion Gene and Clinical Features of Acute Myeloid Leukemia in Sichuan.]

OBJECTIVES: To determine the correlations between AML1-ETO fusion gene and clinical characteristics of patients with AML,and its association with the prognosis of AML-M2. METHODS: Medical records of 94 AML-M2 cases with positive AML1-ETO fusion gene and 51 AML-M2 cases with negative AML1-ETO gene were retrospective reviewed.Their clinical characteristics,treatment responses and prognostic outcomes were compared. RESULTS: No significant differences in the clinical symptoms,predominantly anemia,fever and hemorrhage,were found between the AML1-ETO fusion gene positive and negative AML-M2 (P>0.05).However,lower levels of red blood cell (RBC) and platelet (PLT),and higher levels of ratio of grain to red,percentage of bone marrow granulocyte (NC),CD34,human leukocyte antigen DR (HLA-DR),CD56 and CD19 were found in those with positive AML1-ETO fusion gene (P<0.05).The efficacy of treatments and survival curves showed no significant differences between the two groups (P>0.05).CD56 and original percentage of bone marrow granulocyte were predictors of poor long-term survival.Complete remission was the only predictor of better long-term survival. CONCLUSIONS: AML1-ETO fusion gene is neither associated with clinical symptoms,nor with survival and long term prognosis in Sichuan.As many factors affect the efficacy of treatments and prognosis of AML-M2,stratified analysis is needed to determine the role of AML1-ETO.

[Gene Mutation Spectrum Analysis of 170 Patients with Duchenne/Bayesian Muscular Dystrophy in Southwest of China].

OBJECTIVE: To determine gene variations and genotype-phenotype correlations in Duchenne/Bayesian muscular mystrophy (DMD/BMD) patients, and the association between dystrophin gene polymorphisms and clinical phenotype. METHODS: Multiplex ligation-dependent probe amplification (MLPA) was adopted to detect dystrophin gene variations in 170 patients. Sanger sequencing was performed in 3 cases with decreased peaks in MLPA results. RESULTS: The MLPA detected 72.94% mutations in dystrophin gene, including 62.35% (106/170) deletions, 8.82% (15/170) duplications, and 1.76% (3/170) point mutations. 64 different types of mutations were found. 75.47% of deletions occurred in the range from exon 44 to 55. Most 5' breakpoints of exonic variations were located in 2 hotspots (major hotspot: intron 43-55; minor hotspot: intron 1-20), which is different from findings of other studies. Genotype-phenotype analysis showed that the severity of DMD/BMD was associated with frame shift mutation (r = 0.640, P < 0.001) but not with deletions or duplications. CONCLUSION: Deletions and duplications of exon compose the main type of dystrophin gene mutations. DMD/BMD is associated with frame shift mutation.

[Association of Gene Polymorphisms in Wnt Signal Pathway with Tuberculosis in Chinese Tibetan Population.]

OBJECTIVES: To determine the correlation between gene polymorphisms in Wnt signal pathway and susceptibility of Chinese Tibetan people to tuberculosis. METHODS: A total of 488 active tuberculosis patients and 454 healthy subjects(control) were enrolled in this case-control study.Five single nucleotide polymorphisms (SNPs) in Wnt signal pathway (rs4135385 in CTNNB1 gene,rs11001553 in DKK1 gene,rs56900803 in WIF1 gene,rs7832767 in SFRP1 gene and rs11079571 in AXIN2 gene) were genotyped using MassARRAY method.The genotype and allele distributions of these loci were determined using SPSS19.0 and SNP stats software.Significant SNPs were measured in the co-dominant,dominant and recessive genetic models.The polymorphism distributions of Chinese Tibetans were compared with those of Chinese Han populations. RESULTS: The genotype distributions of all SNPs coincided with the Hardy-Weinberg equilibrium in the 2 groups.The frequencies of genotype and allele of rs7832767 in SFRP1 gene were significantly different (P=0.004,0.002,respectively) between the Tibetan patients with tuberculosis and the Tibetan healthy controls.Compared with C allele carriers,those carrying T allele of rs7832767 showed increased risk of tuberculosis [odds ratio (OR)=1.260,95% confidence interval (CI):1.086-1.471,P=0.002].The co-dominant,dominant and recessive models of this locus were also associated with higher risk of tuberculosis.No significant differences in genotype and allele distributions were observed for the other four SNP loci (P all>0.05).The distribution of rs4135385 in CTNNB1 gene in the Chinese Tibetan population differed from the Han population (P=0.035 for genotype,0.021 for allele).There were no obvious differences in genotype and allele distributions for the other four SNPs between the Tibetan and Han populations (P all >0.05). CONCLUSIONS: SFRP1 gene polymorphism in Wnt signal pathway is associated with tuberculosis susceptibility in Chinese Tibetan population.The distribution of CTNNB1 gene polymorphism differs between Chinese Tibetan and Han populations.

[FLT3 Gene Expression and Its Clinical Significance in Acute Myeloid Leukemia.]

OBJECTIVES: To determine the correlation between fms-like tyrosine kinase 3 gene (FLT3) expression and FLT3-internal tandem duplication (ITD) mutations in acute myeloid leukemia patients,and the association between expression of FLT3 gene and clinical and laboratory features of patients. METHODS: The expression of FLT3 mRNA in bone marrow (BM) leukemic cells of 128 acute myeloid leukemia (AML) patients was measured by real-time PCR.The patients were divided into two groups using the 35% FLT3 expression as a cut-off point.The associations between the expression level of FLT3 and clinical and laboratory features of patients were analyzed. RESULTS: The patients had a FLT3 gene expression level of 0.01-180.68 (mean 14.65) at the initial diagnosis,with AML-M1 the most expressed and AML-M6 the least expressed,but without statistical significance.The patients with a high level of FLT3 gene expression had higher peripheral blood white blood cell count (WBC) (P<0.01) and were more likely to become anemic and febrile (P<0.05).WBC [regression coefficient (B)=1.508,odds ratio (OR)=4.518,95% confidence interval(CI):1.465-13.390,P=0.009] and anemia (B=2.142,OR=8.513,95%CI:3.201-22.644,P<0.001)were predictors of higher expression of FLT3.The patients with high levels of FLT3 gene expression had lower complete remission rate (32/83),compared with those (36/44) with low levels of FLT3 gene expression (P<0.05).The Cox regression analysis showed that the patients with higher levels of FLT3 gene expression had a higher risk of death (B=1.338, relative risk=3.810, 95%CI:1.820-7.947,P<0.001).The Kaplan-Meier analysis showed that the patients with higher levels of FLT3 gene expression had lower survival time (56.63%) than those with lower levels of FLT3 expression (70.45%,P<0.05). CONCLUSIONS: FLT3 gene has adverse impacts on complete remission of AML.High expression of FLT3 gene is associated with poor prognosis of patients with AML.

[Molecular Features of SMA-related Genes in Spinal Muscular Atrophy Patients of Han Nationality in Southwest China.]

OBJECTIVES: To investigate the molecular features of spinal muscular atrophy (SMA) related genes in SMA patients of Han nationality of southwest of China. METHODS: We collected 62 unrelated patients of SMA and 50 unrelated healthy individuals in this study.The copy numbers of survival motor neuron gene (SMN) and uronal-apoptosis inhibitory protein gene (NAIP) were measured by using multiplex ligation-dependent probe amplification (MLPA). RESULTS: Of 62 patients,the copy number of SMA1-4 were 30.65% (19/62),41.94%(26/62),16.13% (10/62),11.29% (7/62),respectively.The deletion of SMN1 exon 7 accounts for 98.38% (61/62).The deletion of SMN1 exon 8 accounts for 82.26% (51/62).Among SMA 1 patients,the homozygous deletion of NAIP exon 5 accounts for 68.42% (13/19) and heterzygous deletion accounts for 26.32% (5/19).Among SMA2-4patients,the homozygous deletion of NAIP exon 5 accounts for 13.95% (6/43) and heterzygous deletion accounts for 62.79% (27/43).Furthermore,68.42% (13/19) patients of SMA1have 1 copy and 2 copies of SMN2 gene,84.62% (22/26) patients of SMA 2 have more than 2 copies of SMN2 gene,90.00% (9/10) SMA3 and 85.71% (6/7) SMA4 have over 2 copies of SMN2 gene and even have 5 and 6 copy of SMN2 gene. CONCLUSIONS: The deletion of SMN1 gene is the main cause of SMA,and the change of SMN2 and NAIP copy number can affect the severity of SMA.

[Genetic Polymorphisms in Wnt Signaling Pathway and Acute Leukemia].

OBJECTIVE: To determine the impacts of Wnt signaling pathway products-polymorphisms of rs4135385, rs11079571 and rs7832767 located in ß-catenin gene (CTNNB1), Axin gene (AXIN2), and secreted frizzled-related protein gene (SFRP1) on the risk and treatment outcomes of acute leukemia. METHODS: Bone marrows (volume 1-1. 5 mL) were collected from 372 untreated patients with acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), and peripheral blood samples (2. 0 mL) were obtained from 401 healthy controls for the purpose of total DNA extraction. Polymorphisms of rs4135385, rs11079571 and rs7832767 located in CTNNB1, AXIN2 and SFRP1 were genotyped with high-resolution melting method (HRM). Chi-square analyses were performed to compare the genotype and allele distributions of the three single nucleotides (SNPs) between the leukemia patients and healthy controls. Single factor variance tests were performed to compare the differences in clinical features among different genotype groups. Complete remission (CR) rates after induction chemotherapy were also compared between different genotype groups using Chi-square tests. RESULTS: No significant differences were found beiween the leukemia patients and healthy controls in the frequencies of alleles and genotypes of CTNNB1 rs4135385, SFRP1 rs7832767 polymorphisms. Those with A allele in AXIN2 rs11079571 polymorphism was less likely to have acute myelomonocytic/monocytic leukemia than those with G allele (P = 0. 016, OR=0. 677, 95%CI:0. 439-0. 930). Acute bead monocyte/mononuclear cell leukemia (AML-M4/5)patients with AA genotype presented higher platelet count (P = 0. 040), and higher complete remission rate after chemotherapy (P = 0. 040), compared with the patients with AG and GG genotypes. CONCLUSION: AML-M4/5 patients have less frequency of A allele in AXIN2 rs11079571 polymorphism than healthy controls. Patients carrying A allele have higher platelet counts and higher sensitivity to chemotherapy.

MicroRNA-16 inhibits glioma cell growth and invasion through suppression of BCL2 and the nuclear factor-κB1/MMP9 signaling pathway.

Recent studies have identified a class of small non-coding RNA molecules, named microRNA (miRNA), that is dysregulated in malignant brain glioblastoma. Substantial data have indicated that miRNA-16 (miR-16) plays a significant role in tumors of various origins. This miRNA has been linked to various aspects of carcinogenesis, including cell apoptosis and migration. However, the molecular functions of miR-16 in gliomagenesis are largely unknown. We have shown that the expression of miR-16 in human brain glioma tissues was lower than in non-cancerous brain tissues, and that the expression of miR-16 decreased with increasing degrees of malignancy. Our data suggest that the expression of miR-16 and nuclear factor (NF)-κB1 was negatively correlated with glioma levels. MicroRNA-16 decreased glioma malignancy by downregulating NF-κB1 and MMP9, and led to suppressed invasiveness of human glioma cell lines SHG44, U87, and U373. Our results also indicated that upregulation of miR-16 promoted apoptosis by suppressing BCL2 expression. Finally, the upregulation of miR-16 in a nude mice model of human glioma resulted in significant suppression of glioma growth and invasiveness. Taken together, our experiments have validated the important role of miR-16 as a tumor suppressor gene in glioma growth and invasiveness, and revealed a novel mechanism of miR-16-mediated regulation in glioma growth and invasiveness through inhibition of BCL2 and the NF-κB1/MMP-9 signaling pathway. Therefore, our experiments suggest the possible future use of miR-16 as a therapeutic target in gliomas.

miRNA-381 regulates renal cancer stem cell properties and sunitinib resistance via targeting SOX4.

Cancer stem cells (CSCs) are crucial in the pathogenesis of human cancers. Existing studies reported that microRNA (miRNA) modulates the stemness of CSCs. We discovered that renal cell CSCs have suppressed miR-381. Suppression of miR-381 promotes renal cell tumorigenesis and CSC-like properties. Furthermore, the forced expression of miR-381 prevents the renal cell tumorigenesis and CSC-like properties. Mechanistically, renal cell CSCs have been found to interact with SOX4 through miR-381 directly. miR-381 inhibits renal cell CSC-like properties and tumorigenesis via downregulating SOX4. Examination of the patient-derived xenografts (PDX) and patient cohorts reveals that miR-381 may be able to forecast the advantages of Sunitinib in RCC patients. Moreover, the introduction of SOX4 could reverse the sensitivity of miR-381 overexpression RCC cells to Sunitinib-induced cell apoptosis. These results indicated that miR-381 is critical in renal cell CSC-like properties and tumorigenesis, making it the ideal therapeutic target for RCC.

[Correlation between JAK2-V617F mutations and variation of peripheral blood cells among BCR/ABL-negative MPD patients].

OBJECTIVE: To assess the correlation between JAK2-V617F mutation and complete blood counts among patients with BCR/ABL-negative myeloproliferative diseases (MPD). METHODS: One hundred and ninety one patients were recruited. Retrospectively, their laboratory data were analyzed for the counts of red blood cells (RBC), white blood cells (WBC) and platelets (PLT). And the incidence of JAK2-V617F mutation was determined. RESULTS: There was significant difference in the incidence of JAK2-V617F mutation between patients with different cell counts (P< 0.01). The incidence of JAK2-V617F mutation has increased with the counts of RBC and PLT, which was the highest (92.86%) among those featuring simultaneous increase in all three series. CONCLUSION: The incidence of JAK2-V617F mutation seems to be strongly associated with variation of peripheral blood cell counts among patients with BCR/ABL-negative MPD. Variation of peripheral blood cells, particularly RBC, may be correlated with the rate of JAK2-V617F mutation.

[Detection of epidermal growth factor receptor gene mutation in non-small cell lung cancer by allele-specific oligonucleotide-PCR and bi-loop probe specific primer quantitative PCR].

OBJECTIVE: To compare the detection sensitivity of epidermal growth factor receptor (EGFR) mutations between allele specific oligonucleotide PCR (ASO-PCR) and bi-loop probe and specific primer quantitative PCR (BPSP-qPCR). METHODS: A total of 96 non-small cell lung cancer specimens were selected from West China Hospital from September 2009 to December 2010. ASO-PCR was developed to detect the presence of classical EGFR mutations. A total 39 available specimens were also tested by BPSP-qPCR. RESULTS: EGFR mutation detection rate was 30.2% (26/96) by ASO-PCR. The mutation rate was higher in female than in male patients [45.5% (20/44) vs. 17.3% (9/52), P = 0.003], non-smokers than smokers [44.1% (26/59) vs. 8.1% (3/37), P < 0.001] and adenocarcinomas than other subtypes of lung cancer [37.0% (27/73) vs. 8.7% (2/23), P = 0.01]. Among mutation negative cases by ASO-PCR, BPSP-qPCR increased the rate of detection of 19-del and L858R mutation by 10.3% (4/39) in adenocarcinomas and non-smoking subset. Overall, the mutation detection rate of BPSP-qPCR was higher than that of ASO-PCR [66.7% (26/39) vs. 41.0% (16/39), P = 0.02]. CONCLUSION: BPSP-qPCR has a better detection sensitivity than that of ASO-PCR.

Combining with lab-on-chip technology and multi-organ fusion strategy to estimate post-mortem interval of rat.

Background: The estimation of post-mortem interval (PMI) is one of the most important problems in forensic pathology all the time. Although many classical methods can be used to estimate time since death, accurate and rapid estimation of PMI is still a difficult task in forensic practice, so the estimation of PMI requires a faster, more accurate, and more convenient method. Materials and methods: In this study, an experimental method, lab-on-chip, is used to analyze the characterizations of polypeptide fragments of the lung, liver, kidney, and skeletal muscle of rats at defined time points after death (0, 1, 2, 3, 5, 7, 9, 12, 15, 18, 21, 24, 27, and 30 days). Then, machine learning algorithms (base model: LR, SVM, RF, GBDT, and MLPC; ensemble model: stacking, soft voting, and soft-weighted voting) are applied to predict PMI with single organ. Multi-organ fusion strategy is designed to predict PMI based on multiple organs. Then, the ensemble pruning algorithm determines the best combination of multi-organ. Results: The kidney is the best single organ for predicting the time of death, and its internal and external accuracy is 0.808 and 0.714, respectively. Multi-organ fusion strategy dramatically improves the performance of PMI estimation, and its internal and external accuracy is 0.962 and 0.893, respectively. Finally, the best organ combination determined by the ensemble pruning algorithm is all organs, such as lung, liver, kidney, and skeletal muscle. Conclusion: Lab-on-chip is feasible to detect polypeptide fragments and multi-organ fusion is more accurate than single organ for PMI estimation.

Molecular epidemical characteristics of Lamivudine resistance mutations of HBV in southern China.

BACKGROUND: Lamivudine (LMV), as the preferred oral drug for use in treatment of HBV, always results in development of resistance mutations after long-term treatment. In this study we investigated chronic hepatitis B (CHB) patients in southern China to determine whether different HBV genotypes affect the incidence of LMV resistance mutations. MATERIAL/METHODS: The study recruited 185 CHB patients living in southern China. Enzyme-linked immunosorbent assay was used to test for HBV serological markers, and HBV DNA was quantified by real-time PCR. Sequencing was performed to detect HBV genotypes and mutations. RESULTS: There were 49.19% (91/185) CHB patients with HBV resistant to LMV. Only 2 genotypes were found: B and C; 62.16% (115/185) of patients were infected with genotype B HBV and 37.84% (70/185) of patients were infected with genotype C HBV. The incidence rate of LMV resistance was not significantly different between genotype B and C (49.57% vs. 48.57%, P>0.05). For the mean age and sex ratio, no significant difference was found. The pattern of rtM204I alone was predominantly observed (36.26%, 33/91), followed by rtM204V+rtL180M (23.08%, 21/91). The overall incidence rate of rtM204I mutation in genotype B (45.61%, 26/57) was more frequent than that in genotype C (20.59%, 7/34) (45.61% vs. 20.59%, P<0.05), but the incidence rate of other mutation patterns was not significantly different between genotypes B and C. CONCLUSIONS: Our results emphasize that a LMV resistance test before treatment is of great importance in rational and optimal CHB therapy.