Comparative of Forensic DNA Identification Using Cell Lysis Method and Magnetic Beads Method.

OBJECTIVES: To compare the effects of cell lysis method and magnetic beads method in forensic DNA identification and to explore these two methods in forensic DNA identification. METHODS: The genome DNA of THP-1 cells in different quantities was extracted by the cell lysis method and magnetic beads method, and the DNA content was quantified by real-time quantitative PCR. The cell lysis method and magnetic beads method were used to type the STR of human blood with different dilution ratios. RESULTS: When the numbers of THP-1 cell were 100, 400 and 800, the DNA content extracted by cell lysis method were (1.219±0.334), (5.081±0.335), (9.332±0.318) ng, respectively; and the DNA content extracted by magnetic beads method were (1.020±0.281), (3.634±0.482), (7.896±0.759) ng, respectively. When the numbers of THP-1 cells were 400 and 800, the DNA content extracted by the cell lysis method was higher than that by the magnetic beads method. The sensitivity of cell lysis method and magnetic beads method was similar in STR typing of human blood at different dilution ratios. Complete STR typing could be obtained at 100, 300 and 500-fold dilutions of blood samples, but could not be detected at 700-fold dilution. STR typing of undiluted human blood could not be detected by cell lysis method. CONCLUSIONS: The cell lysis method is easy to operate and can retain template DNA to the maximum extend. It is expected to be suitable for trace blood evidence tests.

Endothelial NOX4 aggravates eNOS uncoupling by decreasing dihydrofolate reductase after subarachnoid hemorrhage.

Endothelial malfunction is a major contributor to early or delayed vasospasm after subarachnoid hemorrhage (SAH). As a representative form of endothelial dysfunction, endothelial nitric oxide synthase (eNOS) uncoupling leads to a reduction in nitric oxide (NO) generated by endothelial cells. In this study, we investigated how the interaction between endothelial NOX4 (nicotinamide adenine dinucleotide phosphate oxidase 4) and DHFR (dihydrofolate reductase) contributes to eNOS uncoupling after SAH. Setanaxib and the adeno-associated virus (AAV) targeting brain vascular endothelia were injected through the tail vein and the expression and localization of proteins were examined by western blot and immunofluorescence staining. The NO content was measured using the NO assay kit, and laser speckle contrast imaging was used to assess cortical perfusion. ROS (reactive oxygen species) level was detected by DHE (dihydroethidium) staining, DCFH-DA (2',7'-dichlorofluorescin diacetate) staining and H2O2 (hydrogen peroxide) measurement. The Garcia score was employed to examine neurological function. Setanaxib is widely used for its preferential inhibition for NOX1/4 over other NOX isoforms. After endothelial NOX4 was inhibited by Setanaxib in a mouse model of SAH, the endothelial DHFR level was significantly elevated, which attenuated eNOS uncoupling, increased cortical perfusion, and improved the neurological function. The protective role of inhibiting endothelial NOX4, however, disappeared after knocking down endothelial DHFR. Our results suggest that endothelial DHFR decreased significantly because of the elevated level of endothelial NOX4, which aggravated eNOS uncoupling after SAH, leading to decreased cortical perfusion and worse neurological outcome.

Tumor-associated macrophages are associated with response to neoadjuvant chemotherapy and poor outcomes in patients with triple-negative breast cancer.

Background and objective: Tumor-associated macrophages (TAMs) play an essential role in tumor progression and metastasis. However, the role of TAMs in neoadjuvant chemotherapy (NAC) is unclear and need to be identified. The main subject of this study was to investigate whether TAMs are related to the chemotherapeutic response with triple-negative breast cancers (TNBC). Methods: We retrospectively analyzed pretreatment tissue from patients who received NAC and followed by a mastectomy or breast-conservation for stage II-III TNBC in this study. The association between TAMs and the pathological complete response (pCR) rate of TNBC to NAC was analyzed. In addition, the correlation of the TAMs with recurrence-free survival (RFS) in patients with TNBC was also evaluated. Results: Of the 91 patients, 31 (34.1%) patients experienced pathological complete response (pCR) after completion of NAC. Regarding the chemotheraptic response, patients with low infiltration of CD163+ macrophages achieved a significantly higher rate of pCR. Importantly, Kaplan-Meier survival shown that patients with high infiltration of CD163+ macrophages and non-pCR had poor OS and RFS. Conclusions: our data showed that TAMs may predict chemotherapeutic response and can be used as a promising prognostic candidate for poor survival in TNBC patients treated with NAC.

Elevated Expression of CAV1 is Associated with Unfavorable Prognosis of Patients with Breast Cancer Who Undergo Surgery and Neoadjuvant Chemotherapy.

INTRODUCTION: Neoadjuvant chemotherapy (NACT), which is standard treatment for locally advanced breast cancer, improves the resectability of patients with early breast cancer and reduces the extent of breast and axillary surgery. Caveolin-1 (CAV1) is implicated in human cancers, although its utility for cancer prognosis is unknown. Here, we investigated the expression of CAV1 in breast cancer tissues to evaluate its prognostic significance on patients with breast cancer administered NACT. METHODS: CAV1 expression in 80 breast cancer tissue samples was evaluated using immunohistochemistry (IHC). The association between CAV1 levels and clinical factors was analyzed using the chi-square test and that between CAV1 and prognosis was evaluated using multivariate Cox regression and Kaplan-Meier analyses. RESULTS: High levels of CAV1 were significantly associated with survival, and patients with overexpression of CAV1 had a poor prognosis. Adjusted multivariate Cox regression analyses revealed that a high level of CAV1 expression was an independent, significant prognostic factor for patients with breast cancer treated with NACT. DISCUSSION: Overexpression of CAV1 in patients with breast cancer administered NACT was associated with shorter disease-free survival and overall survival. Therefore, high levels of CAV1 may serve as a prognostic biomarker for such patients.

Lack of association of neprilysin gene polymorphisms with Alzheimer's disease in a southern Chinese community.

BACKGROUND: Increasing evidence suggests that neprilysin (NEP) may be the major degrading enzyme of amyloid beta (Abeta) in the brain and the NEP gene has been proposed as a candidate gene for Alzheimer's disease (AD). Association results between the NEP gene and AD are still preliminary. This study investigates the effect of the polymorphisms of -204G/C and 159C/T in the NEP gene on the development of sporadic Alzheimer's disease (SAD) in a southern Chinese community. METHOD: 236 sporadic late-onset AD patients were recruited from Guangzhou Psychiatric Hospital in southern China, and 332 healthy elderly controls were enrolled from three old age homes in suburban Guangzhou. NEP and ApoE genotype were determined by PCR-RFLP. RESULTS: No differences in genotypic and allelic frequencies of -204G/C and 159C/T polymorphisms in NEP were found between AD and control group (for -204G/C genotype: chi2 = 2.34, P > 0.05; for allele: chi2 = 2.31, P > 0.05; for 159C/T genotype: chi2 = 1.34, P > 0.05; for allele: chi2 = 0.88, P > 0.05). Neither was any difference found in genotypic and allelic frequency when stratified by sex or by ApoE epsilon4 allele (P > 0.05). CONCLUSIONS: Our results suggest that -204G/C and 159C/T polymorphisms of the NEP gene may not be associated with SAD. Moreover, both sex and ApoE epsilon4 allele do not affect the distribution of NEP gene polymorphisms.

[Meta-analysis of the association of the LRP C766T polymorphism with the risk of Alzheimer's disease].

A C-to-T polymorphism in exon 3 of the low density lipoprotein receptor-related protein 1 (LPR-1) gene has been implicated as a risk factor for Alzheimer's disease (AD). The authors performed a meta-analysis to investigate the association between the C766T polymorphism in the LPR-1 gene and the risk for AD. Nineteen references were retrieved through Medline, Cochran Library and CBM search from 1997 to 2004. Similar search strategies were applied to each of these databases. Studies which were eligible for the meta-analysis should meet the following inclusion criteria: presentation of original data and a cross-sectional design, AD as the outcome of interest, an odds ratio (or enough information to calculate it) reported to quantify the association between the frequencies of genotypes and/or alleles of LPR-1 gene C766T polymorphism and the risk for AD. All analyses were performed with Review Manager 4.2. A total of 3,560 AD patients and 3,476 control subjects were analyzed according to the random effect model because some between-study heterogeneity was found (P<0.01). The combined data statistics revealed that there was no statistical difference (test for overall effect: Z=1.74, P=0.08, OR=1.17, 95% CI: 0.98-1.39; Z=1.31, P=0.19, OR=1.11, 95% CI: 0.95-1.31) in the frequencies of allele and genotype between the AD patients and the controls. The meta-analysis showed that the LPR-1 polymorphism was not a major risk factor for AD, although a small effect of the polymorphism for AD risk could not be excluded.

[The C224T polymorphism in the cathepsin D gene is not associated with sporadic Alzheimer's disease in Chinese].

Cathepsin D is the major lysosomal/endosomal aspartic protease and exhibits beta- and gamma-secretase-like activity in vitro. Data from German suggest that the C224T polymorphism in the Cathepsin D gene (CTSD) exon 2 is strongly associated with the risk for Alzheimer's disease (AD). Meanwhile other studies have not been able to replicate the result. It's necessary to determine the genotype of the polymorphism in CTSD in Chinese sporadic AD patients and age-matched controls with normal cognition and examine possible association of the polymorphism with the disease. We find no strong evidence of association between the CTSD C224T polymorphism and Chinese sporadic AD. Whereas there may be a weak synergistic interaction between ApoE epsilon4 and CTSD T allele.

[Possible association between 278C/A single nucleotide polymorphism of FKBP6 and idiopathic azoospermia].

OBJECTIVE: To investigate 2 polymorphism sites in exon 3 and intron 2 of FKBP6 in Chinese population, while screening the gene mutations and polymorphisms in exons 3, 4 of FKBP6, and the association of these polymorphisms with azoospermia. METHODS: Possible variations of exons 3, 4 and genotypes and frequencies of 2 polymorphic loci were examined by denaturing high-performance liquid chromatography(DHPLC) and PCR-restriction fragment length polymorphism(PCR-RFLP) technique in 177 azoospermia patients and 231 control individuals. RESULTS: The observed allele frequencies conformed well to Hardy-Weinberg equilibrium. The frequency of 278A allele was significantly higher in controls than that in patients (P<0.05). C/T(s7797242) polymorphism was not found in either group and variations in exons 3, 4 were not detected. CONCLUSION: 278A polymorphism of FKBP6 gene was associated with idiopathic azoospermia, while C/T, 370G/A, 430G/C, 467T/C, 468G/A polymorphisms might be very rare in Chinese population.

8302A/C and (TTA)n polymorphisms in the HMG-CoA reductase gene may be associated with some plasma lipid metabolic phenotypes in patients with coronary heart disease.

HMG-CoA reductase (HMGCR) is a rate-limiting enzyme that participates in cholesterol metabolism. Here we analyzed the 8302A/C and the (TTA)n polymorphisms in the HMGCR gene in 169 Chinese patients with coronary heart disease (CHD) and 161 age-matched controls. Results indicated that the levels of plasma VLDL and TG in patients with the AA genotype of the 8302A/C locus were significantly higher than in patients with other genotypes (P < 0.05). In addition, the frequency of allele A4 of the (TTA)n locus was higher (P < 0.05) and the frequency of allele A5 was lower (P = 0.002) in CHD patients than in the controls. This suggests that both polymorphisms in the HMGCR gene may be associated with lipid and lipoprotein abnormalities in CHD in the Chinese.

[Gln192Arg polymorphism of the paraoxonase-1 gene is not associated with Alzheimer's disease in Chinese].

OBJECTIVE: To explore the relationship between paraoxonase-1 (PON1) gene Gln192Arg polymorphism and sporadic Alzheimer's disease (AD) in Chinese. METHODS: A total of 165 AD patients and 174 age-matched control subjects were enrolled in this study for examination of PON1 Gln192Arg and apolipoprotein E gene polymorphisms using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The distribution of PON1 allelic and genotypic frequencies did not significantly differ between AD patients and the control subjects, even after the stratification by ApoE-epsilon4 status. CONCLUSION: Gln192Arg polymorphism of the PON1 gene is not associated with sporadic AD in Chinese.

[Association of HMG-CoA reductase gene polymorphism with levels of lipids].

OBJECTIVE: To study the distribution of ScrF1 restriction polymorphism in intron 2 of the 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA) reductase gene in Chinese Han population and the association of the polymorphism with coronary heart disease(CHD). METHODS: HMG-CoA reductase genotyping was performed using polymerase chain reaction-restriction fragment polymorphism. RESULTS: HMG-CoA reductase allelic frequencies of A, a were 0.519, 0.481; 0.440, 0.560 in CHD group and control group respectively. There was no significant difference in frequencies of allele and genotype in ScrF1 polymorphism between CHD group and control group(P>0.05). However, the levels of plasma very low density lipoprotein (VLDL) and TG in CHD patients with AA genotype were higher than those in CHD patients with other genotypes(P<0.05). The frequencies of A, a alleles at ScrF1 polymorphic site were significantly different from those reported in European Caucasians (0.44 vs 0.55, 0.56 vs 0.45, P<0.05). CONCLUSION: No direct association was found between the ScrF1 polymorphism and CHD, but there is a significant correlation between the AA genotype of the HMG-CoA reductase gene and the levels of plasma VLDL and TG in CHD group.