Doctor of Philosophy

dissertationOne central question in development is how totipotency and pluripotency are established. In mature human sperm, genes of importance for embryo development (i.e. transcription factors) lack DNA methylation and bear nucleosomes with distinctive histone modifications, suggesting the specialized packaging of these developmental genes in the germline. Here, we explored the tractable zebrafish model and found conceptual conservation as well as several new features. Biochemical and mass spectrometric approaches reveal the zebrafish sperm genome packaged in nucleosomes and histone variants (and not protamine), and we find linker histones high and H4K16ac absent-key factors which may contribute to genome condensation. We examined several activating (H3K4me2/3, H3K14ac, H2AFV) and repressing (H3K27me3, H3K36me3, H3K9me3, hypoacetylation) modifications/compositions genome-wide, and find developmental genes packaged in large blocks of chromatin with coincident activating and repressing marks and DNA hypomethylation, revealing complex "multivalent" chromatin. Notably, genes that acquire DNA methylation in the soma (muscle) are enriched in transcription factors for alternative cell fates. Remarkably, we find H3K36me3 located in "silent" developmental gene promoters, and not present at the 3' ends of coding regions of genes heavily transcribed during sperm maturation, suggesting different rules for H3K36me3 in iv the germline and soma. We also reveal the chromatin patterns of transposons, rDNA, and tRNAs. Finally, high levels of H3K4me3 and H3K14ac in sperm are correlated with genes activated in embryos prior to the mid-blastula transition (MBT), whereas multivalent genes are correlated with activation at or after MBT. Taken together, gene sets with particular functions in the embryo are packaged by distinctive types of complex and often atypical chromatin in sperm. Bivalent marks, as the chromatin signature of pluripotency, are not persistent and diluted during early synchronous cell division, making them arguable to be heritable epigenetic marks. Studies in early embryos indicate DNA methylation status is the fundamental to confer totipotency and pluripotency. The anticorrelation between DNA methylation profiles and H2A.Z occupancy is conserved from plants to vertebrates. Here, we examined H2afva occupancy in early embryos in zebrafish by ChIP-seq. We found both H2afva level and enrichment remain consistent from sperm to embryos. H2afva is enriched in proximal promoter region in the first nucleosome. Consistent with previous studies, H2afva occupancy is anticorrelated to DNA methylation both in the promoters and outside of promoters. These data suggest H2afva is potentially a heritable epigenetic mark and sets up DNA methylation profiles of totipotency and pluripotency

Biodiversity shapes tree species aggregations in tropical forests

Spatial patterns of conspecific trees are considered as the consequences of biological interactions and environmental influences. They also reflect species interactions in plant communities. However, biological attributes are often neglected while deliberating the factors shaping species distributions. As rising attentions are paid to spatial patterns of tropical forest trees, we noticed that seven Center of Tropical Forest Sites and four Forest Dynamic Plots in Asia and America have presented analogously high proportions of species with aggregated conspecific individuals coincidently. This phenomenon is distinctive and repudiates fundamental ecology hypotheses which suggested dispersed distributions of conspecific tropical trees due to intensive density and natural enemy pressures in tropical forests. We believe that similar aggregation patterns shared by these tropical forests implies the existence of structuring forces in biogeographical scale instead of habitat heterogeneity in local community scales as scientists have considered. To approach the factors contributing to this cross-continent spatial pattern of trees, we obtained and reviewed ecosystem attributes, including topography, temperature, precipitation, biodiversity, density, and biomass, of these forests. Here we show that the proportions of aggregated species are actually constants independent of any ecosystem attributes regardless the nature of these tropical forests. However, local biodiversity are the major factor determining the number of aggregated species and the aggregation of large individuals of these forests. Aggregation of large trees declines along rising biodiversity, while the numbers of aggregated species increase permanently along lifting biodiversity. We propose a possible equilibrium and saturated status of the tropical forests in accommodating aggregated species. Furthermore, the tight correlations of biodiversity and species aggregation strongly imply the importance of overlooked biological interactions in shaping the spatial patterns in the tropical forests

MCRS2 represses the transactivation activities of Nrf1

<p>Abstract</p> <p>Background</p> <p>Nrf1 [p45 nuclear factor-erythroid 2 (p45 NF-E2)-related factor 1], a member of the CNC-bZIP (CNC basic region leucine zipper) family, is known to be a transcriptional activator by dimerization with distinct partners, such as Maf, FosB, c-Jun, JunD, etc. The transcriptional roles of CNC-bZIP family are demonstrated to be involved in globin gene expression as well as the antioxidant response. For example, CNC-bZIP factors can regulate the expression of detoxification proteins through AREs, such as expression of human gamma-glutamylcysteine synthetases (GCS), glutathione S-transferases (GST), UDP-glucuronosyl transferase (UDP-GT), NADP (H) quinone oxidoreductase (NQOs), etc. To further explore other factor(s) in cells related to the function of Nrf1, we performed a yeast two-hybrid screening assay to identify any Nrf1-interacting proteins. In this study, we isolated a cDNA encoding residues 126–475 of MCRS2 from the HeLa cell cDNA library. Some functions of MCRS1 and its splice variant-MSP58 and MCRS2 have been previously identified, such as transforming, nucleolar sequestration, ribosomal gene regulation, telomerase inhibition activities, etc. Here, we demonstrated MCRS2 can function as a repressor on the Nrf1-mediated transactivation using both in vitro and in vivo systems.</p> <p>Results</p> <p>To find other proteins interacting with the CNC bZIP domain of Nrf1, the CNC-bZIP region of Nrf1 was used as a bait in a yeast two-hybrid screening assay. MCRS2, a splicing variant of p78/MCRS1, was isolated as the Nrf1-interacting partner from the screenings. The interaction between Nrf1 and MCRS2 was confirmed <it>in vitro </it>by GST pull-down assays and <it>in vivo </it>by co-immunoprecipitation. Further, the Nrf1-MCRS2 interaction domains were mapped to the residues 354–447 of Nrf1 as well as the residues 314–475 of MCRS2 respectively, by yeast two-hybrid and GST pull-down assays. By immunofluorescence, MCRS2-FLAG was shown to colocalize with HA-Nrf1 in the nucleus and didn't result in the redistribution of Nrf1. This suggested the existence of Nrf1-MCRS2 complex in vivo. To further confirm the biological function, a reporter driven by CNC-bZIP protein binding sites was also shown to be repressed by MCRS2 in a transient transfection assay. An artificial reporter gene activated by LexA-Nrf1 was also specifically repressed by MCRS2.</p> <p>Conclusion</p> <p>From the results, we showed MCRS2, a new Nrf1-interacting protein, has a repression effect on Nrf1-mediated transcriptional activation. This was the first ever identified repressor protein related to Nrf1 transactivation.</p

Gene identification and transcriptome analysis of cadmium stress in tomato

Cadmium (Cd) is a highly toxic heavy metal that can severely hinder plant growth and development. Tomato is one of the most important economical crops in the world, and its quality and safety are closely related to human health. Therefore, it is important to elucidate the molecular mechanisms involved in tomato plant responses to Cd stress. In this study, tomato plants were treated with or without 100 μM Cd2+ in hydroponic culture for 3 days. Transcriptional changes in tomato roots and shoots were examined by transcriptome sequencing techniques. A total of 1,123 differentially expressed genes (DEGs) were identified in roots and 159 DEGs were identified in shoots after Cd treatment, including 15 DEGs were upregulated and 24 DEGs were downregulated in both roots and shoots. KEGG enrichment analysis showed that DEGs in the roots and shoots under Cd stress were significantly enriched in the glutathione metabolism pathway, sulfur metabolism pathway, phenylpropanoid biosynthesis, plant-pathogen interaction cutin pathway, suberine and wax biosynthesis pathway, and photosynthesis-antenna proteins pathway. 15 DEGs were further validated by quantitative real-time RT-PCR, including ABC transporter genes, WRKY transcription factors, and NAC transcription factors, among others. This study will provide a theoretical basis for further research on the molecular mechanisms involved in tomato responses to Cd stress, and genetic improvement of Cd tolerance

Genes related to the very early stage of ConA-induced fulminant hepatitis: a gene-chip-based study in a mouse model

<p>Abstract</p> <p>Background</p> <p>Due to the high morbidity and mortality of fulminant hepatitis, early diagnosis followed by early effective treatment is the key for prognosis improvement. So far, little is known about the gene expression changes in the early stage of this serious illness. Identification of the genes related to the very early stage of fulminant hepatitis development may provide precise clues for early diagnosis.</p> <p>Results</p> <p>Balb/C mice were used for ConA injection to induce fulminant hepatitis that was confirmed by pathological and biochemical examination. After a gene chip-based screening, the data of gene expression in the liver, was further dissected by ANOVA analysis, gene expression profiles, gene network construction and real-time RT-PCR.</p> <p>At the very early stage of ConA-triggered fulminant hepatitis, totally 1,473 genes with different expression variations were identified. Among these, 26 genes were finally selected for further investigation. The data from gene network analysis demonstrate that two genes, MPDZ and Acsl1, localized in the core of the network.</p> <p>Conclusions</p> <p>At the early stages of fulminant hepatitis, expression of twenty-six genes involved in protein transport, transcription regulation and cell metabolism altered significantly. These genes form a network and have shown strong correlation with fulminant hepatitis development. Our study provides several potential targets for the early diagnosis of fulminant hepatitis.</p

Complications after radical gastrectomy following FOLFOX7 neoadjuvant chemotherapy for gastric cancer

<p>Abstract</p> <p>Background</p> <p>This study assessed the postoperative morbidity and mortality occurring in the first 30 days after radical gastrectomy by comparing gastric cancer patients who did or did not receive the FOLFOX7 regimen of neoadjuvant chemotherapy.</p> <p>Methods</p> <p>We completed a retrospective analysis of 377 patients after their radical gastrectomies were performed in our department between 2005 and 2009. Two groups of patients were studied: the SURG group received surgical treatment immediately after diagnosis; the NACT underwent surgery after 2-6 cycles of neoadjuvant chemotherapy.</p> <p>Results</p> <p>There were 267 patients in the SURG group and 110 patients in the NACT group. The NACT group had more proximal tumours (P = 0.000), more total/proximal gastrectomies (P = 0.000) and longer operative time (P = 0.005) than the SURG group. Morbidity was 10.0% in the NACT patients and 17.2% in the SURG patients (P = 0.075). There were two cases of postoperative death, both in the SURG group (P = 1.000). No changes in complications or mortality rate were observed between the SURG and NACT groups.</p> <p>Conclusion</p> <p>The FOLFOX7 neoadjuvant chemotherapy is not associated with increased postoperative morbidity, indicating that the FOLFOX7 neoadjuvant chemotherapy is a safe choice for the treatment of local advanced gastric cancer.</p

SaPt-CNN-LSTM-AR-EA: a hybrid ensemble learning framework for time series-based multivariate DNA sequence prediction

Biological sequence data mining is hot spot in bioinformatics. A biological sequence can be regarded as a set of characters. Time series is similar to biological sequences in terms of both representation and mechanism. Therefore, in the article, biological sequences are represented with time series to obtain biological time sequence (BTS). Hybrid ensemble learning framework (SaPt-CNN-LSTM-AR-EA) for BTS is proposed. Single-sequence and multi-sequence models are respectively constructed with self-adaption pre-training one-dimensional convolutional recurrent neural network and autoregressive fractional integrated moving average fused evolutionary algorithm. In DNA sequence experiments with six viruses, SaPt-CNN-LSTM-AR-EA realized the good overall prediction performance and the prediction accuracy and correlation respectively reached 1.7073 and 0.9186. SaPt-CNN-LSTM-AR-EA was compared with other five benchmark models so as to verify its effectiveness and stability. SaPt-CNN-LSTM-AR-EA increased the average accuracy by about 30%. The framework proposed in this article is significant in biology, biomedicine, and computer science, and can be widely applied in sequence splicing, computational biology, bioinformation, and other fields

Efficient Commitment to Functional CD34+ Progenitor Cells from Human Bone Marrow Mesenchymal Stem-Cell-Derived Induced Pluripotent Stem Cells

The efficient commitment of a specialized cell type from induced pluripotent stem cells (iPSCs) without contamination from unknown substances is crucial to their use in clinical applications. Here, we propose that CD34+ progenitor cells, which retain hematopoietic and endothelial cell potential, could be efficiently obtained from iPSCs derived from human bone marrow mesenchymal stem cells (hBMMSC-iPSCs) with defined factors. By treatment with a cocktail containing mesodermal, hematopoietic, and endothelial inducers (BMP4, SCF, and VEGF, respectively) for 5 days, hBMMSC-iPSCs expressed the mesodermal transcription factors Brachyury and GATA-2 at higher levels than untreated groups (P<0.05). After culturing with another hematopoietic and endothelial inducer cocktail, including SCF, Flt3L, VEGF and IL-3, for an additional 7–9 days, CD34+ progenitor cells, which were undetectable in the initial iPSC cultures, reached nearly 20% of the total culture. This was greater than the relative number of progenitor cells produced from human-skin-fibroblast-derived iPSCs (hFib-iPSCs) or from the spontaneous differentiation groups (P<0.05), as assessed by flow cytometry analysis. These induced cells expressed hematopoietic transcription factors TAL-1 and SCL. They developed into various hematopoietic colonies when exposed to semisolid media with hematopoietic cytokines such as EPO and G-CSF. Hematopoietic cell lineages were identified by phenotype analysis with Wright-Giemsa staining. The endothelial potential of the cells was also verified by the confirmation of the formation of vascular tube-like structures and the expression of endothelial-specific markers CD31 and VE-CADHERIN. Efficient induction of CD34+ progenitor cells, which retain hematopoietic and endothelial cell potential with defined factors, provides an opportunity to obtain patient-specific cells for iPSC therapy and a useful model for the study of the mechanisms of hematopoiesis and drug screening

Comparative study of mesenchymal stem cells from C57BL/10 and mdx mice

<p>Abstract</p> <p>Background</p> <p>Human mesenchymal stem cells (MSCs) have been studied and applied extensively because of their ability to self-renew and differentiate into various cell types. Since most human diseases models are murine, mouse MSCs should have been studied in detail. The mdx mouse – a Duchenne muscular dystrophy model – was produced by introducing a point mutation in the dystrophin gene. To understand the role of dystrophin in MSCs, we compared MSCs from mdx and C57BL/10 mice, focusing particularly on the aspects of light and electron microscopic morphology, immunophenotyping, and differentiation potential.</p> <p>Results</p> <p>Our study showed that at passage 10, mdx-MSCs exhibited increased heterochromatin, larger vacuoles, and more lysosomes under electron microscopy compared to C57BL/10-MSCs. C57BL/10-MSCs formed a few myotubes, while mdx-MSCs did not at the same passages. By passage 21, mdx-MSCs but not C57BL/10-MSCs had gradually lost their proliferative ability. In addition, a significant difference in the expression of CD34, not Sca-1 and CD11b, was observed between the MSCs from the 2 mice.</p> <p>Conclusion</p> <p>Our current study reveals that the MSCs from the 2 mice, namely, C57BL/10 and mdx, exhibit differences in proliferative and myogenic abilities. The results suggest that the changes in mouse MSC behavior may be influenced by lack of dystrophin protein in mdx mouse.</p

Evaluation of portable colposcopy and human papillomavirus testing for screening of cervical cancer in rural China

OBJECTIVE: To evaluate the use of a portable, rechargeable colposcope combined with human papillomavirus (HPV) testing, as compared with HPV testing alone, for screening of cervical cancer and pre-cancerous lesions. METHODS: This was a cross-sectional study among 488 women in Baoshan County, Yunnan. The women underwent HPV testing followed by Gynocular portable colposcopy with visual inspection with acetic acid. Obvious lesions were biopsied. If portable colposcopy testing was negative but HPV testing was positive, the women underwent follow-up testing with thin-prep cytology and traditional colposcopy. Cervical biopsies were performed for any abnormalities. Histopathology was followed up with diagnosis and treatment. RESULTS: Among 488 women screened with portable colposcopy, 24 women underwent biopsy based on positive colposcopy screening. Of these 24 women, three were HPV positive and 21 were HPV negative. Five women had cervical intra-epithelial neoplasia (CIN) I and one had advanced cervical cancer. Forty-six women tested positive for HPV. Three of these women had screened positive on preliminary colposcopy, with one positive for CIN III/squamous cell carcinoma and one woman with CIN I. Forty-three women underwent follow-up testing with thin-prep cytology. Two women had atypical squamous cells of undetermined significance and five had low-grade squamous intra-epithelial lesions and were biopsied; three women had CIN I, one had CIN II and one had CIN III. HPV testing and portable colposcopy was more sensitive but slightly less specific than portable colposcopy or HPV testing alone. CONCLUSION: While HPV testing has high sensitivity and specificity for the detection of pre-cancerous and cancerous lesions and portable colposcopy has lower specificity, both methods of detection have low positive predictive value and high negative predictive value. In tandem, HPV testing and portable colposcopy had higher sensitivity for detection among women who underwent biopsies. In clinical practice, portable colposcopy was an effective, easy and affordable tool to transport to villages where cytology is not currently feasible