DNA self-assembled fluorescent nanoprobes have been developed for bio-imaging owing to their high resistance to enzyme degradation and great cellular uptake capacity. In this work, we designed a new ...Y-shaped DNA fluorescent nanoprobe (YFNP) with aggregation-induced emission (AIE) characteristic for microRNA imaging in living cells. With the modification of the AIE dye, the constructed YFNP had a relatively low background fluorescence. However, the YFNP could emit a strong fluorescence due to the generation of microRNA-triggered AIE effect in the presence of target microRNA. Based on the proposed target-triggered emission enhancement strategy, microRNA-21 was detected sensitively and specifically with a detection limit of 122.8 pM. The designed YFNP showed higher bio-stability and cell uptake than the single-stranded DNA fluorescent probe, which has been successfully applied for microRNA imaging in living cells. More importantly, the microRNA-triggered dendrimer structure could be formed after the recognition of target microRNA, achieving a reliable microRNA imaging with a high spatiotemporal resolution. We expect that the proposed YFNP will become a promising candidate for bio-sensing and bio-imaging.
X-chromosome short tandem repeat (X-STR) markers are a powerful complementary system used for paternity and forensic casework. This study presents the development and validation of a new highly ...efficient multiplex-fluorescent-labeled 19 X-STR typing system, including DXS10079, DXS101, DXS10135, DXS10162, DXS6795, DXS6800, DXS6803, DXS6807, DXS6809, DXS6810, DXS7133, DXS7423, DXS981, DXS9902, DXS9907, GATA165B12, GATA172D05, GATA31E08 and HPRTB along with sex-typing locus, amelogenin. The system was validated according to guidelines issued by the Scientific Working Group on DNA Analysis Methods. Allele frequency and forensic parameters were investigated from 1085 (494 males and 591 females) unrelated Beijing Han individuals, the combined power of discrimination by the 19 X-STR loci in females and males, as well as the combined mean exclusion chance in trios and duos, were 0.999999999999999995, 0.99999999995, 0.9999999995, and 0.9999996, respectively. The results demonstrate that this multiplex system is robust and reliable, and considered to be a powerful tool for forensic application.
In recent years, the cases of tramadol intoxication have become more frequent in many countries. However, most of the previous studies have been based on cases of tramadol intoxication, and the ...detailed information on the differences between postmortem distribution and diffusion of tramadol remains unclear. To investigate this issue systematically, we established a postmortem distribution model and two postmortem diffusion models. Then, gas chromatography-mass spectrometry (GC/MS) was used to measure the concentrations of tramadol in various biological specimens of fluids and tissues. In postmortem distribution, the results showed an uneven distribution of tramadol in various biological specimens, and the concentrations of tramadol in urine were significantly higher than those in other fluids. In postmortem diffusion, the results showed a dosage-dependent increase of tramadol concentration in most specimens; at all time points from 0.25 to 6 h after postmortem administration, the concentrations of tramadol in fluids were not significantly different from those in tissues, and the concentrations of tramadol in urine were lower than those in both tissues and other fluids in most time points. We recommend a quantitative examination of the specimens of both fluids and tissues to provide more evidence for the forensic identification, and the realization that there is a correlation between the concentrations of fluids and tissues is important for determining antemortem and postmortem administration of tramadol. This information can serve as ancillary data in inferring the contribution of a drug to death in cases of suspected tramadol poisoning.
The identification of mixed stains has always been a difficult problem in personal identification in the forensic field. In recent years, tissue-specific methylation sites have proven to be very ...stable biomarkers for distinguishing tissue origin. However, it is still challenging to perform tissue source identification and individual identification simultaneously. In this study, we developed a method that uses tissue-specific methylation markers combined with single-nucleotide polymorphism (SNP) markers to detect semen from mixed biofluids and to identify individuals simultaneously. Semen-specific CpG markers were chosen from the literature and further validated utilizing methylation-sensitive restriction endonuclease (MSRE) combined with PCR technology. The neighboring SNP markers were searched in the flanking sequence of the target CpG within 400 bp, and SNP typing was then carried out through a single-base extension reaction followed by capillary electrophoresis. Eventually, a method of MSRE combined with SNaPshot that could detect 12 compound CpG-SNP markers was developed. Using this system, 10 ng of total DNA and DNA mixture with semen content up to 25% could be typed successfully. Moreover, the cumulative discrimination power of the system in the northern Chinese Han population is 0.9998. This study provides a valuable strategy for forensic practice to perform tissue origin and individual identification from mixed stains simultaneously.
Organophosphorus pesticides (OP
S
) are widely used in the world, and many poisoning cases were caused by them. Phorate intoxication is especially common in China. However, there are currently few ...methods for discriminating phorate poisoning death from phorate exposure after death and interpretation of false-positive results due to the lack of effective biomarkers. In this study, we investigated the metabonomics of rat plasma at different dose levels of acute phorate intoxication using ultra-performance liquid chromatography quadrupole-time of flight mass spectrometry (UPLC-Q-TOF–MS) analysis. A total of 11 endogenous metabolites were significantly changed in the groups exposed to phorate at LD
50
level and three times of LD50 (3LD
50
) level compared with the control group, which could be potential biomarkers of acute phorate intoxication. Plasma metabonomics analysis showed that diethylthiophosphate (DETP) could be a useful biomarker of acute phorate intoxication. The levels of uric acid, acylcarnitine, succinate, gluconic acid, and phosphatidylcholine (PC) (36:2) were increased, while pyruvate level was decreased in all groups exposed to phorate. The levels of ceramides (Cer) (d 18:0/16:0), palmitic acid, and lysophosphatidylcholine (lysoPC) (18:1) were only changed after 3LD
50
dosage. The results of this study indicate that the dose-dependent relationship exists between metabolomic profile change and toxicities associated with apoptosis, fatty acid metabolism disorder, energy metabolism disorder especially tricarboxylic acid (TCA) cycle, as well as liver, kidney, and nervous system functions after acute exposure of phorate. This study shows that metabonomics is a useful tool in identifying biomarkers for the forensic toxicology study of phorate poisoning.
Glutamate is not only a neurotransmitter but also an important neurotoxin in central nervous system (CNS). Chronic elevation of glutamate induces both neuronal and glial cell apoptosis. However, its ...effect on astrocytes is complex and still remains unclear. In this study, we investigated whether morphine, a common opioid ligand, could affect glutamate-induced apoptosis in astrocytes. Primary cultured astrocytes were incubated with glutamate in the presence/absence of morphine. It was found that morphine could reduce glutamate-induced apoptosis of astrocytes. Furthermore, glutamate activated Ca
release, thereby inducing endoplasmic reticulum (ER) stress in astrocytes, while morphine attenuated this deleterious effect. Using siRNA to reduce the expression of κ-opioid receptor, morphine could not effectively inhibit glutamate-stimulated Ca
release in astrocytes, the protective effect of morphine on glutamate-injured astrocytes was also suppressed. These results suggested that morphine could protect astrocytes from glutamate-induced apoptosis via reducing Ca
overload and ER stress pathways. In conclusion, this study indicated that excitotoxicity participated in the glutamate mediated apoptosis in astrocytes, while morphine attenuated this deleterious effect via regulating Ca
release and ER stress.
Abstract
The determination of length of time from the last drinking is critical for cases like drunk driving, sexual assault victims, and also postmortem suspected poisoning cases. The study was ...aimed to established a method of estimating the time of last drinking through the pharmacokinetic study of conjugation metabolites of alcohol in blood after a single oral dose. Twenty-six volunteers (14 males) consumed alcohol with food at a fixed dose of 0.72 g/kg after fasting for 12 h. Five milliliters of blood were collected 120 h after the start of drinking, and all samples were analyzed with headspace-gas chromatography and high-performance liquid chromatography–tandem mass spectrometry. The time point of last drinking was estimated through the relationship between the concentration ratio of ethyl glucuronide to ethyl sulphate and the length of time after drinking. Pharmacokinetic parameters were analyzed by a pharmacokinetic software DAS according to the non-compartment model. A good correlation model was obtained from the relationship between concentration ratio of ethyl glucuronide to ethyl sulphate in blood and the time of alcohol use, and the margin of error was mostly lower than 10%. The time of maximum concentration, maximum concentration, and elimination half-life of ethyl glucuronide in blood were 4.12 ± 1.07 h, 0.31 ± 0.11 mg/L and 2.56 ± 0.89 h; the time of maximum concentration, maximum concentration, and elimination half-life of ethyl sulphate in blood were 3.02 ± 0.70 h, 0.17 ± 0.04 mg/L, and 2.04 ± 0.76 h. The study established a potential method to estimate the length of time after a moderate oral dose, and provided pharmacokinetic parameters of ethyl glucuronide and ethyl sulphate in Chinese population.
The identification of a suspect in a degraded and unbalanced DNA mixture has been a challenge for the standard short tandem repeat polymorphisms (STR) typing. Several methods have been introduced to ...solve this problem, such as DIP-STR, DIP-SNP, and SNP-STR markers. In this study, we proposed DIP-microhaplotype (deletion/insertion linked a chain of SNPs) as a kind of new genetic marker to type the unbalanced and degraded DNA mixture. We established the detection method with ten DIP-microhaplotype markers including 26 SNPs using allele-specific multiplex PCR followed by SNaPshot assay. This novel compound marker allows us to detect the minor DNA with a sensitivity of 1:100 to 1:1000 in a DNA mixture of any gender. Most of the DIP-microhaplotype markers had a relatively high probability of informative alleles with an average informative value (
I
value) of 0.308. In all, we proposed DIP-microhaplotype as a novel type of DNA marker for the detection of minor contributor from unbalanced DNA mixtures. Due to their inherent shorter length, higher polymorphism, and sensitivity, DIP-microhaplotypes are promising markers for the examination of the degraded and unbalanced mixtures in forensic stains or clinical chimeras.
Temozolomide (TMZ) is the first-line drug for the clinical treatment of glioblastoma (GBM), but drug resistance limits its treatment benefits. This study was intended to investigate whether propofol ...could restrict the resistance of GBM cells to TMZ and uncover the underlying mechanisms. Human GBM cell line U251 and TMZ-resistant U251/TMZ cell line were transplanted into mice to construct GBM and TMZ-resistant GBM xenograft tumors. Tumor growth in mice was monitored, and the tumor tissues were collected for biochemical analysis. THP-1 cell differentiated into M0 subtype macrophage using phorbol 12-myristate 13-acetate (PMA). The culture medium of M0 macrophage was collected for treating U251 cells with the presence or absence of propofol or propofol + DMOG (HIF-1α activator). Results showed that propofol significantly enhanced the inhibitory effect of TMZ on tumor growth, macrophage infiltration and inflammation in TMZ-resistant GBM xenograft tumors in vivo. Compared with GBM xenograft tumors, higher expression of HIF-1α, O6-methylguanine-DNA methyltransferase (MGMT), p-p65 and cyclooxygenase 2 (Cox2) was observed in TMZ-resistant GBM xenograft tumors, but propofol co-treatment markedly reduced the expression of these proteins. In in vitro experiments, culture medium from M0 macrophage promoted U251 cell survival, inflammation and expression of HIF-1α, MGMT, p65 and Cox2, whereas inhibited cell apoptosis. However, propofol suppressed the PMA-induced THP-1 M0 macrophage activation, and propofol-treated culture medium from M0 macrophage blocked all the effects of M0 medium on U251 cells. Additionally, DMOG reversed the effect of propofol-treated M0 medium on U251 cells. In conclusion, Propofol restricted TMZ resistance via inhibiting macrophage activation and down-regulating HIF-1α expression in GBM.
•Propofol enhances the sensitivity of TMZ-resistant GBM to TMZ in vivo.•Propofol inhibits macrophage activation-mediated inflammation and apoptosis.•Propofol blocks U251 cell malignant behaviors via down-regulating HIF-1α expression.
Uniparental disomy (UPD) has attracted more attention recently in paternity testing, though it is an infrequent genetic event. Although short tandem repeat (STR) profiling has been widely used in ...paternity testing, it is not sufficient to use STR only to judge the genetic relationship, because the existence of UPD will inevitably affect the results of genotyping. Compared with complete UPD, segmental UPD is more difficult to detect because it does not affect all genotypes on the same chromosome. It is necessary to determine the type of UPD with multiple methods because a single method is not sufficient. Therefore, it is advisable to detect UPD in paternity testing with multiple methods. In this study, after autosomal STR profiling was used, we found that there were several gene loci on the same chromosome that did not conform to Mendelian genetic law, thus we highly suspected the existence of UPD and performed X-STR profiling immediately. Then whole-genome single nucleotide polymorphism (SNP) array analysis was performed to identify the type, and the results provided straightforward evidence for distinguishing complete from segmental UPD. Lastly, we used deletion insertion polymorphism (DIP)-SNP SNaPshot assay and Miseq FGx sequencing (for SNP and STR) to determine whether the mutation source is maternal uniparental disomy (mUPD) or paternal uniparental disomy (pUPD). To avoid false exclusion of kinship, it is vital to determine the type of UPD in paternity testing and effective strategies based on multiple methods to detect the type of UPD are provided in this study.
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