Application of Familial Y-STR Haplotype Mismatch Tolerance in Genealogy Inference.

OBJECTIVES: To provide a guideline for genealogy inference and family lineage investigation through a study of the mismatch tolerance distribution of Y-STR loci in Chinese Han male lineage. METHODS: Three Han lineages with clear genetic relationships were selected. YFiler Platinum PCR amplification Kit was used to obtain the typing data of 35 Y-STR loci in male samples. The variation of Y-STR haplotypes in generation inheritance and the mismatch tolerance at 1-7 kinship levels were statistically analyzed. RESULTS: Mutations in Y-STR were family-specific with different mutation loci and numbers of mutation in different lineages. Among all the mutations, 66.03% were observed on rapidly and fast mutating loci. At 1-7 kinship levels, the number of mismatch tolerance ranged from 0 to 5 on all 35 Y-STR loci, with a maximum step size of 6. On medium and slow mutant loci, the number of mismatch tolerance ranged from 0 to 2, with a maximum step size of 3; on rapidly and fast mutant loci, the number of mismatch tolerance ranged from 0 to 3, with a maximum step size of 6. CONCLUSIONS: Combined use of SNP genealogy inference and Y-STR lineage investigation, both 0 and multiple mismatch tolerance need to be considered. Family lineage with 0-3 mismatch tolerance on all 35 Y-STR loci and 0-1 mismatch tolerance on medium and slow loci can be prioritized for screening. When the number of mismatch tolerance is eligible, family lineages with long steps should be carefully excluded. Meanwhile, adding fast mutant loci should also be handled with caution.

Identification of Human Body Fluid Stains and Non-Biological Stains by Three-Dimensional Fluorescence Spectroscopy.

OBJECTIVES: To establish a rapid and nondestructive identification method for human body fluid stains and non-biological stains using three-dimensional fluorescence spectroscopy. METHODS: The collected three-dimensional fluorescence spectrum data of human saliva, 3% blood, coffee and Fanta® stains were processed with dimensionality reduction. After wavelet transform, spectral denoising and feature extraction, the classification formula was established. The Fisher discriminant was used for spectrum matching and recognition to establish the analysis method to distinguish stain types. RESULTS: According to the results of data training and comparison, all the recognition accuracies of Fanta®, coffee, saliva and blood were more than 91.39%. Among them, saliva reached 100% recognition accuracy. CONCLUSIONS: Three-dimensional fluorescence spectroscopy is a potential method for rapid and nondestructive identification of biological and non-biological stains.

Application of transcriptome in time analysis and donor characterization in blood samples.

As an effective supplement to the current forensic DNA typing and one of the research hotpots in forensic science, the in-depth mining and characterization of biological evidence can provide rich and reliable clues for case investigation. In this study, the time-dependent variations of transcriptome were confirmed in in vitro blood samples within 0-168 days and a random forest model was established to realize the classification of blood samples with different TSD (time since deposition). Meanwhile, significant differences were observed in the transcripts of blood samples with different smoking habits and genders within a certain time period. HLA-DRB1, HLA-DQB1 and HLA-DQA2 were identified as markers for smoking habit identification, while the transcripts for RPS4Y1 and EIF1AY from the non-recombining region of the Y chromosome (NRY) were identified as markers for male sex identification. Thus, this study provides a theoretical foundation and experimental strategy for establishing a transcriptome-based method for characterizing blood sample retention time and donor characteristics in the field of forensic investigation.

Massively Parallel Sequencing of Forensic STRs Using the Ion Chef™ and the Ion S5™ XL Systems.

Next-generation sequencing (NGS) has been used to genotype forensic short tandem repeat (STR) markers for individual identification and kinship analysis. STR data from several NGS platforms have been published, but forensic application trials using the Ion S5™ XL system have not been reported. In this work, we report sensitivity, reproducibility, mixture, simulated degradation, and casework sample data on the Ion Chef™ and S5™ XL systems using an early access 25-plex panel. Sensitivity experiments showed that over 97% of the alleles were detectable with down to 62 pg input of genomic DNA. In mixture studies, alleles from minor contributors were correctly assigned at 1:9 and 9:1 ratios. NGS successfully gave 12 full genotype results from 13 challenging casework samples, compared with five full results using the CE platform. In conclusion, the Ion Chef™ and the Ion S5™ XL systems provided an alternative and promising approach for forensic STR genotyping.

Anti-fibrotic effects of pirfenidone by interference with the hedgehog signalling pathway in patients with systemic sclerosis-associated interstitial lung disease.

AIM: To determine whether pirfenidone attenuates lung fibrosis by interfering with the hedgehog (Hh) signalling pathway in patients with systemic sclerosis-associated interstitial lung disease (SSc-ILD). METHODS: Twenty-five SSc-ILD patients (20 first visit, five who underwent pirfenidone treatment for 6 months) and 10 healthy controls were recruited. Lung tissues were obtained by open-chest surgery, and primary lung fibroblasts were isolated, cultured and stimulated with pirfenidone. The levels of the proteins glioma-associated oncogene 1 (GLI1), suppressor of fused (Sufu), α-smooth muscle actin, and fibronectin in lung tissues or fibroblasts were determined by Western blotting. The messenger RNA levels of GLI1, glioma-associated oncogene 2, protein patched homolog 1, and Sufu in lung tissues or fibroblasts were determined by quantitative reverse-transcription polymerase chain reaction. Meanwhile, the levels of phosphorylation glycogen synthase kinasep-3ß (pGSK-3ß), phosphorylation SMAD2 (pSMAD2), and phosphorylation c-Jun N-terminal kinase (pJNK) in fibroblasts were determined by Western blotting. RESULTS: Hh pathway activation was increased in the lung tissue of SSc-ILD patients and was decreased by pirfenidone, Sufu was upregulated in lung fibroblasts isolated from SSc-ILD patients after pirfenidone challenge, and pirfenidone inhibited the phosphorylation of GSK-3ß signalling. CONCLUSION: Pirfenidone has anti-fibrotic effects in SSc-ILD patients by interfering with both the Hh signalling pathway and the GSK-3ß signalling pathway via the regulation of Sufu expression. These results might promote its use in other Hh driven lung diseases such as idiopathic pulmonary fibrosis and especially the interstitial lung disease associated with connective tissue diseases.

Effect of Leflunomide on the Abnormal Expression of Lipid Rafts and F-Actin in B Lymphocytes from Patients with Systemic Lupus Erythematosus.

PURPOSES: To investigate the possible changes in B cell subsets and in B cell expression patterns of lipid rafts (LRs) and F-actin in patients with SLE and whether leflunomide treatment may have effect on these changes. METHODS: The B cell subsets and LRs expression were determined by flow cytometry and confocal microscopy, and F-actin expression was examined by confocal microscopy. RESULTS: CD27(+)IgD(+) B cell subsets were significantly decreased while CD38(+)CD95(+) B cell subsets increased in SLE patients. The LRs levels of B cells were remarkably increased and positively correlated with SLEDAI and anti-dsDNA titer in SLE patients. The expression level of LRs was significantly higher in CD38(+) B cells than CD38(-) B cells and negatively correlated with C3 levels. The increased expression of LRs was associated with reduced expression of F-actin in the B cells from active SLE patients. Furthermore, in vitro treatment of the cells with A771726 reduced the expression level of LRs, attenuated the overaggregation of LRs, and normalized the distribution of F-actin. CONCLUSIONS: There were abnormalities in B cell subsets and LRs and F-actin expression of B cell from SLE patients. Modulation of B cell expression of LRs and F-actin by LEF could be a potential therapeutic target for SLE.

[Efficacy comparison between transplanting microenvironmental induced and non-induced bone marrow mesenchymal stem cells in ischemic rat hearts].

OBJECTIVE: To compare the efficacy of transplanting bone marrow mesenchymal stem cell (BMSC) or microenvironmental induced BMSC (iBMSC) into the ischemic myocardium of rats with myocardial infarction. METHODS: iBMSC was defined as BMSC co-cultured with myocardial cells for 2 weeks. The stem cells or equal volume PBS were injected into ischemic border zone 1 wk after experimental infarction. Cardiac performance was evaluated at 1, 2, and 4 wk after cell transplantation by echocardiography and analyzed histologically at 4 wk after cell transplantations. RESULTS: Compared with PBS group, both BMSC and iBMSC transplantations reduced infarct size. iBMSC enhanced the beneficial effects of BMSC on improving cardiac function (FS: 28.5% +/- 4.3% in PBS, 29.0% +/- 2.0% in BMSC and 45.1% +/- 3.1% in iBMSC group at 4 weeks post transplantation, iBMSC group vs. PBS group P < 0.05, iBMSC group vs. BMSC group P < 0.05). Immunofluorescence microscopy results revealed co-localization of SPIO-labeled transplanted cells with cardiac markers for cardiomyocytes, indicating regeneration of damaged myocardium. CONCLUSION: Our data suggest that iBMSC implantation is more effective on improving cardiac function than BMSC implantation in this model. iBMSC might serve as a new promising therapeutic cell source for regenerating ischemic myocardium in patients with post-infarction heart failure.

[Combined mesenchymal stem cells and allogenic bone marrow transplantation in treatment of MRL/lpr mice].

OBJECTIVE: In order to study the role of the bone marrow-derived mesenchymal stem cells (MSCs) transplanted with or without bone marrow (BM) in the treatment of lupus mice and the effect of MSCs in the onset of systemic lupus erythematosus (SLE). METHOD: Twenty 12-week-old female MRL/lpr mice were randomly divided into four groups, including simple bone marrow transplantation group (SG, BM 1 x 10(7)), united group-1 (UG1, BM 1 x 10(7) + MSCs 1 x 10(4)), united group-2 (UG2, BM 1 x 10(7) + MSCs 1 x 10()6), the positive control group (PG, no transplantation). BALB/c mice were used as the negative control group (NG, no transplantation). MSCs which were amplified from the bone marrow of male BALB/c mice in vitro were transplanted into the female MRL/lpr mice with or without BM. One month later Y chromosome was detected to confirm if the transplantation was successful or not. The change of weight, white blood cells, urine protein, anti-dsDNA antibody, the pathology and immunofluorescence of renal were observed to evaluate the therapeutic efficacy. RESULTS: Y chromosome was detected in all transplanted female mice. Compared with PG, urine protein concentration in SG, UG1 and UG2 significantly decreased 30 days after transplantation (P < 0.05). 40 days after transplantation, the tite of anti-dsDNA antibodies in SG (0.91 +/- 0.27) was still higher than NG, which OD value was 0.47 +/- 0.10 (P < 0.05), but there was no statistical difference among UG1 (0.76 +/- 0.28), UG2 (0.73 +/- 0.10) and NG (P > 0.05). However, 50 days after transplantation, there was no marked difference of the tite of anti-dsDNA antibodies in SG (0.55 +/- 0.15), UG1 (0.57 +/- 0.14) and UG2 (0.58 +/- 0.05) compared with NG (P > 0.05). After transplantation there was no vasculitis, no inflammatory cell infiltration in matrix and no obvious intercapillary cells proliferation in the kidney. The immunofluorescence became negative or weakly positive. CONCLUSION: MSCs transplantation with or without BM can both improve the pathogenetic condition of MRL/lpr mice. MSCs can accelerate the clearance of anti-dsDNA antibody and promote the restoration of injured organs. We presume that MSCs are important immunological regulation cells in SLE.

[Human bone marrow mesenchymal stem cells cocultured with semi-permeable membrane separated neonatal rat ventricular myocytes differentiated into cardiomyocyte phenotype].

OBJECTIVE: To investigate the ability of human bone marrow mesenchymal stem cells (hBMSCs), cocultured with semi-permeable membrane separated neonatal rat ventricular myocytes, to differentiate into cardiomyocytes. METHODS: hBMSCs were isolated and purified by density gradient centrifugation and adherence screening method. Cells were expanded as undifferentiated cells in culture for more than 3 passages and their phenotypes were identified with flow cytometer. hBMSCs were cocultured with neonatal rat ventricular myocytes in a rate of 1:10 separated by semi-permeable membrane. GATA4 mRNA was detected by RT-PCR; Immunocytochemistry, and Immunostaining were used to detect sarcomeric alpha-actinin, desmin, cTnT, and cTnI protein level. RESULTS: CD29 (98.64% +/- 0.80%) and CD44 (96.70% +/- 1.50%) were the major surface markers of hBMSCs. After coculturing with semi-permeable membrane separated neonatal rat ventricular myocytes, the first contraction of single cells was noted at day 7 and GATA4 expression was detected on these cells by RT-PCR after 1 to 3 weeks coculture. Desmin, sarcomeric alpha-actinin, cTnI and cTnT could be detected by immunocytochemistry and immunostaining on some of these cells. CONCLUSION: hBMSCs possess the potential to differentiate into myocardial cell phenotype in the cardiac microenvironment. Direct contact with cardiomyocytes was not necessary required for hBMSCs differentiation.