Application of Multiple Genetic Markers in Determination of Full and Half Sibling Relationship： A Case Report.

ABSTRACT: Objective To investigate the application of the comprehensive use of multiple genetic markers in full and half sibling relationship testing through the identification of a case of suspected sibling relationship. Methods Genomic DNA were extracted from bloodstain samples from 4 subjects （ZHANG-1, ZHANG-2, male; ZHANG-3, ZHANG-4, female）. Autosomal STR loci, X-STR, Y-STR loci and polymorphisms of mtDNA HV-Ⅰ and Ⅱwere genotyped by EX20 STR kit, X19 kit, Data Y24 STR kit, and Sanger sequencing, respectively. Results According to autosomal STR based IBS scoring results, full sibling relationships were indicated among ZHANG-2, ZHANG-3 and ZHANG-4, but those were not indicated between ZHANG-1 and ZHANG-2 or ZHANG-3 or ZHANG-4. According to autosomal STR based FSI and HSI, with ITO method and discriminant function method, full sibling relationships among ZHANG-2, ZHANG-3 and ZHANG-4 were indicated, and half sibling relationships between ZHANG-1 and ZHANG-2 or ZHANG-3 or ZHANG-4 were also indicated. X-STR and mtDNA sequencing results showed that all the 4 samples came from a same maternal line, and Y-STR results showed that ZHANG-1 and ZHANG-2 did not come from a same paternal line, which supported the half sibling relationship between ZHANG-1 and ZHANG-2 or ZHANG-3 or ZHANG-4, verified by parental genotype reconstruction based on autosomal STR genotyping. Conclusion For the identification of sibling relationships, it is effective to have reliable results with the mutual verification and support of multiple genetic markers （autosomal STR, sex chromosomal STR and mtDNA sequence） and calculations （IBS, ITO, discriminant function method and family reconstruction）.

[Inhibition of G9a attenuates cell proliferation via the mitochondrial apoptosis pathway in lung adenocarcinoma].

Objective: The aim of this study is to investigate the effect of G9a inhibitor BIX-01294 on attenuating cell proliferation in human lung adenocarcinoma A549 cell line and the underlying molecular mechanism. Methods: Treated with BIX-01294, the growth and proliferation of A549 cells were detected by MTT assay and colony formation assay, and its impact on cell apoptosis was analyzed using flow cytometry. By Western blot, we explored the alterations in the expression of apoptosis-related proteins and the G9a catalysate, H3K9me and H3K9me2. In addition, in the pretreatment with caspase inhibitor Z-VAD-FMK, we detected the apoptotic dependence of BIX-01294 attenuating impact on A549 cell proliferation. Results: Compared with the control group, the histone methyltransferase G9a inhibitor BIX-01294 attenuated cell proliferation in A549 cells in a dose- and time-dependent manner. There were 42.5±8.7 colonies after BIX-01294 (10 µmol/L) treatment for 7 days, while 172.7±23.0 colonies in the control group, with a statistical significance (P<0.05). After treatment with BIX-01294 (10 µmol/L) for 24 hours, the cell apoptotic rate was(47.6±8.4)%, with a significant difference in comparison with the control group [(7.2±3.6)%, P<0.05]. The expression of G9a catalysate, H3K9me and H3K9me2 was downregulated, the same with anti-apoptotic protein Bcl-2, while the proteins in mitochondrial apoptosis pathway, Bax, Bak and cleaved caspase-9, were upregulated, so was the expression of cleaved caspase-3 and cleaved PARP, and there was no alteration in the expression of cleaved caspase-8, which is a protein related with death receptor apoptosis pathway. Furthermore, after Z-VAD-FMK pretreatment, the cell apoptotic rate was decreased significantly, and the expression of apoptosis-related proteins were downregulated. Conclusions: Our results indicate that BIX-01294 can attenuate cell proliferation in lung adenocarcinoma, and it can be considered as one of the underlying mechanisms, the apoptosis may be induced by activating mitochondrial pathway.

[Genotyping of ABO Blood Group in Partial Population of Yunnan Province by SNaPshot Technology].

OBJECTIVES: To detect the genotype of ABO blood group by SNaPshot technology. METHODS: DNA were extracted from the peripheral blood samples with known blood groups （obtained by serology） of 107 unrelated individuals in Yunnan. Six SNP loci of the 261th, 297th, 681th, 703th, 802th, and 803th nucleotide positions were detected by SNaPshot Multiplex kit, and relevant genetics parameters were calculated. RESULTS: In 107 blood samples, the allele frequencies of types A, B, OA, and OG were 0.355 1, 0.168 2, 0.230 0 and 0.247 6, respectively, while that of types AG and cis AB were not detected. The genotyping results of ABO blood group were consistent with that of serologic testing. CONCLUSIONS: SNaPshot technology can be adapted for genotyping of ABO blood group.

Genome-wide identification, phylogenetic relationships, and expression analysis of the carotenoid cleavage oxygenase gene family in pepper.

Carotenoid cleavage oxygenases (CCOs) are a family of dioxygenases, which specifically catalyze the cleavage of conjugated double bonds in carotenoids and apocarotenoids in plants. In this study, genome-wide analysis of CCO genes in pepper plants was performed using bioinformatic methods. At least 11 members of the CCO gene family were identified in the pepper genome. Phylogenetic analysis showed that pepper and tomato CCO genes could be divided into two groups (CCDs and NCEDs). The CCD group included five sub-groups (CCD1, CCD4, CCD7, CCD8, and CCD-like). These results indicate that there is a close genetic relationship between the two species. Sequence analysis using the online tool, Multiple Expectation Maximization for Motif Elicitation (MEME), showed that the CCO proteins comprise multiple conserved motifs, with 20 to 41 amino acids. In addition, multiple cis-acting elements in the promoter of CCO genes were identified using the online tool PlantCARE, and were found to be involved in light responsiveness, plant hormone regulation, and biotic and abiotic stresses, suggesting potential roles of these proteins under different conditions. RNA-seq analysis revealed that the CCO genes exhibit distinct patterns of expression in the roots, stems, leaves, and fruit. These findings suggest that the CCO genes have important roles in the vegetative and reproductive development of pepper plants.

In silico identification and analysis of phytoene synthase genes in plants.

In this study, we examined phytoene synthetase (PSY), the first key limiting enzyme in the synthesis of carotenoids and catalyzing the formation of geranylgeranyl pyrophosphate in terpenoid biosynthesis. We used known amino acid sequences of the PSY gene in tomato plants to conduct a genome-wide search and identify putative candidates in 34 sequenced plants. A total of 101 homologous genes were identified. Phylogenetic analysis revealed that PSY evolved independently in algae as well as monocotyledonous and dicotyledonous plants. Our results showed that the amino acid structures exhibited 5 motifs (motifs 1 to 5) in algae and those in higher plants were highly conserved. The PSY gene structures showed that the number of intron in algae varied widely, while the number of introns in higher plants was 4 to 5. Identification of PSY genes in plants and the analysis of the gene structure may provide a theoretical basis for studying evolutionary relationships in future analyses.

Assessment of the genetic diversity of tomato yellow leaf curl virus.

The objective of the present study was to analyze the genetic diversity of tomato yellow leaf curl virus (TYLCV). Representative TYLCV sequences were searched in the National Center for Biotechnology Information database. Comprehensive analysis of TYLCV was performed using bioinformatics by examining gene structure, sequence alignments, phylogeny, GC content, and homology. Forty-eight representative TYLCV sequences were selected from 48 regions in 29 countries. The results showed that all TYLCV sequences were 2752-2794 nucleotides in length, which encoded 6 open reading frames (AV1, AV2, AC1, AC2, AC3, and AC4). GC content ranged from 0.41-0.42. Sequence alignment showed a number of insertions and deletions within these TYLCV sequences. Phylogenetic tree results revealed that the sequences were divided into 10 classes; homology of the sequences ranged from 72.8 to 98.6%. All 48 sequences contained the typical structure of TYLCV, including open reading frames and intergenic regions. These results provide a theoretical basis for the identification and evolution of the virus in the future.

In silico analysis of gene content in tomato genomic regions mapped to the Ty-2 resistance gene.

Tomato yellow leaf curl virus is one of the main diseases affecting tomato production worldwide. Previous studies have shown that Ty-2 is an important resistance gene located between molecular markers C2_At2g28250 (82.3 cM) and T0302 (89.0 cM), and exhibits strong resistance to tomato yellow leaf curl virus in Asia. In this study, Ty-2 candidate genes were subjected to bioinformatic analysis for the sequenced tomato genome. We identified 69 genes between molecular markers C2_At2g28250 and T0302, 22 of which were disease-related resistant genes, including nucleotide binding site-leucine-rich repeat disease resistance genes, protease genes (protein kinase, kinase receptor, and protein isomerase), cytochromes, and transcription factors. Expressed sequence tag analysis revealed that 77.3% (17/22) of candidate disease-resistance genes were expressed, involving 143 expressed sequence tags. Based on full-length cDNA sequence analysis, 7 candidate genes were found, 4 of which were involved in tomato responses to pathogens. Microarray expression analysis also showed that most candidate genes were involved in the tomato responses to multiple pathogens, including fungi, viruses, and bacteria. RNA-seq expression analysis revealed that all candidate genes participated in tomato growth and development.

[Studies on chemical constituents from solvent extracts of Poria cocos (Schw.) Wolf].

Crystals I-N were isolated from the ethereal extracts of Poria cocos and identified respectively as O-acetyl-pachymic acid (I), 3 beta-hydroxy-lanosta-7,9(11),24-trien-21-oic acid (II), beta-amyrin acetate (III) and 3 beta-hydroxy-16 alpha-acetoxy-lanosta-7,9(11),24-trien-21-oic acid (N). Crystal III was obtained from P. cocos for the first time, and crystal N was newly discovered.

Treatment of advanced breast cancer with chinese medicinal herbs of Fei decoction: a case report.

A 46-year-old female underwent surgery for cancer of the right breast mammary (T3N2M0) in Sep 2010. Following post surgery, adjuvant chemotherapy of CAF regimens (cyclophosphamide+adriamycin+fluorouracil) was administered. Two years later, multiple pulmonary and skeletal metastatic lesions had been found by CT (computerized tomography) and ECT (emission computed tomograph) imaging. She received the treatment of second-line chemotherapy regimens of GP (cisplatin + gemcitabine). In the meantime, we administered Chinese traditional herb drugs (Fei Decoction, mixed a variety of effective herbal components) to help her recover from the poor condition. After taking the Chinese herbs for 2 months, the tumour marker (CEA, CA15-3) dramatically decreased, resulting in the normal range. Both lung and bone metastatic sites reduced according to CT and ECT imaging, and the patient felt free from the complaint of pulmonary and cardiac discomfort. Over time, the quality of life has been greatly improved, we have managed to prolong the PFS (progression-free-survival) and TTP (time-to-progression) from the onset to date. CTM (Chinese traditional medicine) considers human body as a dynamic platform in which all organs are correlative and bind each other. Relationship between heart, liver, spleen, lung and kidney is like an interlink between mother and son, and runs in cycle as a circle. In the course of this combined treatment, we showed that Chinese herbal medicine played an important role in the therapy of breast cancer. Chinese herbs might be an additional choice with their better benefits and tolerability in the treatment of recurrent breast cancer.