Verification of a loss of heterozygosity at the D8S1179 locus in a paternity case by the MiSeq FGx system.

Capillary electrophoresis is widely used to study short tandem repeats (STRs) in forensic genetics. However, next-generation sequencing platforms have become a new strategy for forensic DNA typing. In this study, we report a false four-step STR mutation between an alleged father (AF) and child in a paternity case. A total of 23 autosomal STR loci were evaluated using the Huaxia™ Platinum and Goldeneye™ 20A kits, revealing a single mismatch in D8S1179 between the AF (10/10) and the male child (14/14). Additional Y-STR typing of the AF and child was performed, and the results were consistent with those based on 27 Y-STR loci. To further confirm the experimental results, we sequenced the individuals using the MiSeq FGx system and detected 10/15 unbalanced alleles in the D8S1179 locus of the AF and 14/15 unbalanced alleles in the D8S1179 locus of the child. Sanger sequencing revealed that both the AF and child had the C→G point mutation in the primer binding region of D8S1179 resulting in allelic dropout. Therefore, the verification of STR typing by different sequencing systems is helpful for the interpretation of results in cases of multistep STR mutations.

Forensic characterisation and polymorphism analysis of 16 X-chromosomal STRs in the Jining Han population in Eastern China.

BACKGROUND: X-chromosomal short tandem repeats (X-STRs) are a useful supplementary approach to analysing autosomal markers in forensics and kinship studies; such markers are not well-characterised in many populations. AIM: To investigate population genetic polymorphism and forensic characterisation of 16 X-STRs in the Jining Han population, and analyse genetic relationships with other Chinese populations. SUBJECTS AND METHODS: Allele frequencies for 16 X-STR loci were obtained from a sample set of 527 unrelated individuals from the Jining Han population. Population genetic analyses of Jining Han and another 10 reference populations were conducted using phylogenetic tree, principal component analysis and multidimensional scaling. RESULTS: We detected 149 alleles, with frequencies ranging from 0.0013 to 0.8242. The combined powers of discrimination in males and females were 0.999999997194774 and 0.999999999999995, respectively. The combined mean exclusion change (MEC)Krüger, MECKishida, MECDesmarais, and MECDesmarais Duos values were 0.999974632649096, 0.999999976997582, 0.999999977013201, and 0.999993755768423, respectively. We detected relatively high genetic homogeneity in populations with similar ethnic or geographic origins, and a close relationship between the Jining Han and Beijing Han populations. CONCLUSIONS: The present findings indicate that the 16 X-STR loci examined are highly polymorphic in the Han population of Jining, providing useful information for forensic science and population genetics studies.

Mechanical stretch and hypoxia inducible factor-1 alpha affect the vascular endothelial growth factor and the connective tissue growth factor in cultured ACL fibroblasts.

PURPOSES: The adult human anterior cruciate ligament (ACL) has poor functional healing response. Hypoxia plays an important role in regulating the microenvironment of the joint cavity after ACL injury, however, its role in mechanical injury is yet to be examined fully in ACL fibroblasts. In this study, we used CoCl2 to induce Hypoxia-inducible factor-1α (HIF-1α) in our experimental model to study its affect on matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) expression in ACL fibroblasts after mechanical stretch. MATERIALS AND METHODS: Cell treatments were performed in the stretch chamber in all experimental groups. Quantitative real-time PCR was used to check mRNA expression levels of MMP-2, CTGF, VEGF, and HIF-1α. Western blot was used to detect the HIF-1α production. Enzyme-Linked immunosorbent assay was performed to check the VEGF and CTGF protein contents in supernatant. MMP-2 activity was assayed by gelatin zymography. RESULTS: The real-time PCR results show that mechanical stretch or CoCl2 treatment increases the expression of MMP-2, VEGF, CTGF, and HIF-1α; however, the combined effects of mechanical stretch and CoCl2-induced HIF-1α increased MMP-2 production but decreased the VEGF and CTGF expression, compared to the CoCl2 treatment group alone. Western blot analysis and ELISA also confirmed these results. CONCLUSIONS: Our results demonstrated that mechanical stretch and CoCl2-induced HIF-1α together increased the level of MMP-2 and decreased the levels of VEGF and CTGF in cultured ACL fibroblasts. The differential expression and production of HIF-1α, VEGF, MMP-2, and CTGF might help to explain the poor healing ability of ACL.

Transcriptomic profiling of the salt-stress response in the wild recretohalophyte Reaumuria trigyna.

BACKGROUND: Reaumuria trigyna is an endangered small shrub endemic to desert regions in Inner Mongolia. This dicotyledonous recretohalophyte has unique morphological characteristics that allow it to tolerate the stress imposed by semi-desert saline soil. However, it is impossible to explore the mechanisms underlying this tolerance without detailed genomic information. Fortunately, newly developed high-throughput sequencing technologies are powerful tools for de novo sequencing to gain such information for this species. RESULTS: Two sequencing libraries prepared from control (C21) and NaCl-treated samples (T43) were sequenced using short reads sequencing technology (Illumina) to investigate changes in the R. trigyna transcriptome in response to salt stress. Among 65340 unigenes, 35495 (52.27%) were annotated with gene descriptions, conserved domains, gene ontology terms, and metabolic pathways with a cut-off E-value of 10-5. These included 44 Gene Ontology (GO) terms, 119 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 25 Clusters of Orthologous Groups families. By comparing the transcriptomes from control and NaCl-treated plants, 5032 genes showed significantly differences in transcript abundance under salt stress (false discovery rate ≤ 0.001 and |log2Ratio| ≥ 1). These genes were significantly enriched in 29 KEGG pathways and 26 GO terms. The transcription profiles indicated that genes related to ion transport and the reactive oxygen species scavenging system were relevant to the morphological and physiological characteristics of this species. The expression patterns of 30 randomly selected genes resulted from quantitative real-time PCR were basically consistent with their transcript abundance changes identified by RNA-seq. CONCLUSIONS: The present study identified potential genes involved in salt tolerance of R. trigyna. The globally sequenced genes covered a considerable proportion of the R. trigyna transcriptome. These data represent a genetic resource for the discovery of genes related to salt tolerance in this species, and may be a useful source of reference sequences for closely related taxa. These results can also further our understanding of salt tolerance in other halophytes surviving under sodic stress.

Evaluation of allelic alterations in short tandem repeats in papillary thyroid cancer.

BACKGROUND: Malignant tissue samples may be the only source of biological material for forensic investigations, including individual identification or paternity testing; however, such samples may lead to uncertainties due to frequent genomic aberrations associated with tumors, including alterations of the short tandem repeat (STR) loci used for forensic casework. METHODS: Short tandem repeat loci routinely used in forensic analysis (n = 23) were analyzed in 68 surgically removed papillary thyroid cancer specimens. Tumor cells and normal stromal cells were separated by laser capture microdissection. RESULTS: Four kinds of changes were detected between normal and tumor tissues: partial loss of heterozygosity (pLOH), complete loss of heterozygosity, an additional allele, and a new allele not found in normal tissue. These changes were distributed across 20 of the tested STRs, with no mutations in VWA, D16S539, or Penta D. The most frequently affected locus was D2S1338, and the most frequent type of alteration was pLOH. Samples from patients aged 40-59 years exhibited the highest frequencies of STR variation. CONCLUSION: Our results suggest that great care should be taken in the evaluation of DNA typing results obtained from malignant tissues, particularly when no normal tissue reference samples are available.

Population genetic data from 23 autosomal STR loci of Huaxia Platinum system in the Jining Han population.

BACKGROUND: Genetic polymorphisms at 23 short tandem repeat (STR) loci were investigated in 1,215 Jining Han individuals from Jining city, Shandong province, eastern China. METHODS: We used population genetic data of 23 autosomal STR loci included in the Huaxia Platinum system to evaluate 1,215 unrelated Chinese Han individuals in the Jining Han population. Allele frequencies and forensic parameters of the STR loci were determined and genetic relationships among the Jining Han and other Chinese populations were evaluated. RESULTS: In total, we observed 321 alleles, with frequencies ranging from 0.00041 to 0.52222. The combined discrimination power and probability of excluding paternity were 0.99999999999999999999999999919 and 0.99999999962, respectively. No deviations from HWE were observed at any loci. Population comparisons showed that the Xinjiang groups (Uyghur and Kazakh) and the Mongolian and Tibetan groups were isolated, while the Jining Han population clustered together with other populations, except the Guizhou Han population. CONCLUSION: This study demonstrated that 23 autosomal STR loci included in the Huaxia Platinum system are highly polymorphic and suitable for personal forensic identification and paternity testing in this population.

Pharmaceutical inhibition of AXL suppresses tumor growth and invasion of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a common type of cancer in a number of regions of the world, including East Asia, South Africa and Iran. It is often associated with poor prognosis rates. Tyrosine-protein kinase receptor UFO (AXL) is overexpressed in a subset of ESCC tumors, therefore the present study aimed to determine the effect of R428, a selective inhibitor of AXL, on ESCC tumor cells. TE1 and KYSE150 cell lines were used as models to investigate the effects of R428 treatment. The proliferative rate of the tumor cells was analyzed using MTT and colony formation assays. In addition, cell migration and invasion rates were analyzed using wound healing and Matrigel assays, respectively. The expression levels of matrix metalloproteinase (MMP)2 and MMP9, and the activation of protein kinase B (AKT), extracellular signal-regulated kinase (ERK) and AXL signaling were analyzed using gelatin zymography and western blotting. The results revealed that R428 inhibited the proliferative and invasive abilities of both cell lines. Furthermore, AXL, AKT and ERK signaling were all decreased in response to R428 treatment, alongside the expression levels of MMP2 and MMP9. In conclusion, the results of the present study suggested that R428 treatment may suppress ESCC tumor cell proliferation and invasion, representing a potential therapeutic target for ESCC.

Genetic diversity and haplotype structure of 27 Y-STR loci in a Yanbian Korean population from Jilin Province, Northeast China.

In this study, 27 Y-STRs were analyzed in 347 male individuals from the Yanbian Korean population. Haplotype diversity (HD) and discrimination capacity (DC) values were calculated. Pairwise Rst values were evaluated in AMOVA analysis and visualized through multidimensional scaling (MDS). Yflier Plus system indicated higher Discrimination Power (DP), HD and DC which is 0.9969, 0.9998 and 0.9769. There is no significant genetic distance between Yanbian Koreans and South Koreans, however, there is a great distance from Chinese Han population. The present results may provide useful information for paternal lineages in forensic cases and increase our understanding of the genetic relationships between Yanbian Korean and other groups.

Effects of arbuscular mycorrhizal symbiosis on growth, nutrient and metal uptake by maize seedlings (Zea mays L.) grown in soils spiked with Lanthanum and Cadmium.

Multiple contaminants can affect plant-microbial remediation processes because of their interactive effects on environmental behaviour, bioavailability and plant growth. Recent studies have suggested that arbuscular mycorrhizal fungi (AMF) can facilitate the revegetation of soils co-contaminated with rare earth elements (REEs) and heavy metals. However, little is known regarding the role of AMF in the interaction of REEs and heavy metals. A pot experiment was conducted to evaluate the effects of Claroideoglomus etunicatum on the biomass, nutrient uptake, metal uptake and translocation of maize grown in soils spiked with Lanthanum (La) and Cadmium (Cd). The results indicated that individual and combined applications of La (100 mg kg-1) and Cd (5 mg kg-1) significantly decreased root colonization rates by 22.0%-35.0%. With AMF inoculation, dual-metal treatment significantly increased maize biomass by 26.2% compared to single-metal treatment. Dual-metal treatment significantly increased N, P and K uptake by 20.1%-76.8% compared to single-metal treatment. Dual-metal treatment significantly decreased shoot La concentration by 52.9% compared to single La treatment, whereas AM symbiosis caused a greater decrease of 87.8%. Dual-metal treatment significantly increased shoot and root Cd concentrations by 65.5% and 58.7% compared to single Cd treatment and the La translocation rate by 142.0% compared to single La treatment, whereas no difference was observed between their corresponding treatments with AMF inoculation. Furthermore, AMF had differential effects on the interaction of La and Cd on metal uptake and translocation under the background concentrations of soil metals. Taken together, these results indicated that AMF significantly affected the interaction between La and Cd, depending on metal types and concentrations in soils. These findings promote a further understanding of the contributions of AMF to the phytoremediation of co-contaminated soil.

Development and characterization of EST-SSR markers in Stipa breviflora (Poaceae).

PREMISE OF THE STUDY: Stipa breviflora (Poaceae) is one of the dominant species of the desert steppe in the eastern Eurasian grasslands. Simple sequence repeat (SSR) markers were developed for use in genetic diversity studies of this species. METHODS AND RESULTS: A total of 1954 potentially polymorphic loci were obtained by comparing transcriptome data of eight different S. breviflora individuals. We selected 81 loci to verify polymorphism and 63 loci amplified, of which 21 loci exhibited polymorphism. The number of alleles per locus varied from two to 24, the observed heterozygosity ranged from 0.083 to 0.958, and the expected heterozygosity ranged from 0.396 to 0.738. CONCLUSIONS: These newly identified SSR loci can be used for population genetic and landscape genetic studies of S. breviflora. In addition, 14 loci also amplified in six related Stipa species (S. grandis, S. krylovii, S. bungeana, S. aliena, S. gobica, and S. purpurea).

Identification of Salt-Stress-Induced Genes from the RNA-Seq Data of Reaumuria trigyna Using Differential-Display Reverse Transcription PCR.

Next generation sequencing (NGS) technologies have been used to generate huge amounts of sequencing data from many organisms. However, the correct choice of candidate genes and prevention of false-positive results computed from digital gene expression (DGE) of RNA-seq data are vital when using these genetic resources. We indirectly identified 18 salt-stress-induced Reaumuria trigyna transcripts from the transcriptome sequencing data using differential-display reverse transcription PCR (DDRT-PCR) combined with local BLAST searches. Highly consistent with the DGE results, the quantitative real-time PCR expression patterns of these transcripts showed strong upregulation by salt stress, suggesting that these genes may play important roles in R. trigyna's survival under high-salt environments. The method presented here successfully identified responsive genes from the massive amount of RNA-seq data. Thus, we suggest that DDRT-PCR could be employed to mine NGS data in a wide range of applications in transcriptomic studies. In addition, the genes identified in the present study are promising candidates for further elucidation of the salt tolerance mechanisms in R. trigyna.

64-Slice spiral CT perfusion combined with vascular imaging of acute ischemic stroke for assessment of infarct core and penumbra.

The aim of this study was to determine the value of computed tomography perfusion (CTP) parameters, including cerebral blood flow (CBF), cerebral blood volume (CBV), mean transit time (MTT) and time-to-peak (TP), in a clinical study of patients with stroke. Additionally, we determined which parameter or combination of parameters are reliable in detecting the presence of an infarct and penumbra. CTP was performed within 24 h of the onset of symptoms in 20 patients with possible stroke. Magnetic resonance imaging (MRI) was performed 3-7 days later and the threshold of the CTP was adjusted according to the results to provide CT images that correlated with the MRI; the MRI results were taken as the gold standard. CBV, CBF and TP contrast agent enhancement were calculated using the CT results. The CTP results were compared with the MRI findings. All CTP parameters were reliable in detecting the penumbra (P<0.001). In these parameters, changes of MTT were the most useful. CTP revealed various changes in CBF, CBV, MTT and TP in ischemic areas. CTP parameters were also reliable in detecting the infarct core (P<0.001). We determined that when detecting the penumbra, all CTP parameters are reliable, and when detecting cerebral ischemia, a combination of parameters should be used.