77 research outputs found
Single-molecule analysis of circulating cell-free DNA: biological and clinical perspectives
No full textPh.D.The discovery of cell-free DNA molecules in the circulation has opened up opportunities for noninvasive molecular diagnosis. The advent of single-molecule counting techniques, including digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS), has enabled the detection and precise quantification of cell-free DNA. In this study, I have developed new approaches for analyzing circulating cell-free DNA, which enabled us to gain understanding of the biology of circulating DNA. Moreover, the potential clinical applications of these approaches were also demonstrated.Circulating cell-free DNA comes from different tissues. There is a lack of generic methods for measuring the contribution of different organs to the pool of circulating DNA. Section III and IV of this thesis describe a method that makes use of the difference in methylation profiles between different organs to measure the contribution of different organs to the circulating DNA pool. Droplet dPCR assays targeting regions with tissue-specific methylation were designed to determine the contribution of different organs to a DNA mixture. While previous studies showed that the hematopoietic system is a key source of plasma DNA. I further demonstrated that a high proportion of circulating DNA is indeed derived from cells of the erythroid lineage. This method can provide a noninvasive way to monitor the rate of erythropoiesis and is useful for determining the etiology of anemia. In addition, using a NGS-based method, I genotyped the DNA of erythroid origin in the plasma of pregnant women and showed that the fetal-specific allele could not be detected in these plasma DNA molecules.In section IV, I developed a dPCR assay that targeting the liver-specific DMR for quantifying the liver contribution to plasma DNA. This assay can be used for the monitoring of liver-transplant patients, and to determine if liver metastasis would be present in patients with non-liver primary cancers.Section V describes a study on patients with gastrointestinal stromal tumor (GIST). For GIST patients, different mutational profiles in the tumor would affect the responsiveness to different targeted therapies. The cancer-specific mutations can also be used for monitoring the disease after treatment. I have developed a droplet dPCR-based method and an amplicon-based NGS method for detection of common mutations in GIST. The performance of the assays was tested in a batch of formalin-fixed paraffin-embedded (FFPE) tumor tissue samples. Compared with Sanger sequencing, dPCR and NGS analyses showed higher accuracy. I further demonstrated that corresponding mutations could also be detected in the plasma of some of GIST patients using these assays.In conclusion, I developed a series of new molecular diagnostic tests and explored some interesting biological characteristics of circulating cell-free DNA in patients with a variety of pathological conditions.血漿中遊離DNA分子的發現使無創性分子診斷成為可能。包括數字聚合酶鏈式反應（digital polymerase chain reaction, dPCR）和第二代代測序（next-generation sequencing, NGS）等的單分子計數技術的出現，使遊離DNA可以被精確的定量。在本研究中，我開發了一些分析血漿遊離DNA的新方法。使用這些方法，我們對血漿中遊離DNA的生物學有了進一步的瞭解。此外，本文還展示了這些方法在臨床上的潛在應用價值。血漿中的遊離DNA來自不同的組織。目前尚缺乏一種通用方法來測量來自不同器官的遊離DNA對血漿中總遊離DNA貢獻量。本論文的第三章和第四章描述了一種利用不同器官之間的甲基化差異來測量不同器官對血漿遊離總DNA貢獻量的方法。我設計了靶向組織特異性甲基化區域的液滴式dPCR試驗可以對一個混合DNA樣本中自不同器官的DNA進行測量。前人的研究表明，造血系統中的血細胞是血漿遊離DNA的主要來源。本文研究結果進一步表明，血漿遊離DNA中有顯著比例確實來自於造血系統中的紅細胞譜系。這種新的方法可以用來無創性的監測紅細胞生成的速率，並可以用於貧血病病因的診斷。此外，我們使用基於NGS的方法，對孕婦血漿中來自紅細胞譜系的DNA進行基因分型。我們發現孕婦血漿中紅細胞來源的DNA分子不攜帶胎兒特異性的等位基因。在本論文的第四章中，我開發了針對於肝臟特異性差異甲基化區域的dPCR檢測方法，以定量測量肝臟來源的DNA對血漿總DNA的貢獻。該檢測方法可以用於肝移植患者的監測，也可以用於檢測癌癥患者中是否已發生腫瘤肝轉移。本論文的第五章描述了一項針對胃腸道間質瘤（gastrointestinal stromal tumors, GIST）患者的研究。對於GIST患者，具有不同突變類型的腫瘤對靶向治療的反應不同。腫瘤特異性突變也可用於治疾病療後的檢測。我開發了基於液滴式dPCR的測定方法和基於靶向捕獲NGS的測定方法來檢測GIST中的常見突變。我們使用一批腫瘤組織樣品來測試這兩種測定方法的性能。與Sanger測序相比，dPCR和NGS方法具有更高的精確度。我們進一步證明，使用這些測定法也可以在一些GIST患者的血漿中檢測到相應的突變。綜上所述，我開發了一系列新的分子診斷測試方法，發現了血漿遊離DNA的新的生物學特徵，並探討了血漿遊離DNA在各種病理狀況患者中的特徵。Gai, Wanxia.Thesis Ph.D. Chinese University of Hong Kong 2017.Includes bibliographical references (leaves 168-192).Abstracts also in Chinese.Title from PDF title page (viewed on 18, October, 2019)
RTN1 and RTN3 protein are differentially associated with senile plaques in Alzheimer’s brains
No full textAbstractReticulon proteins (RTNs), consisting of RTN1 to RTN4, were previously shown to interact with BACE1 by negatively modulating its secretase activity. In RTN3-null mice, RTN1 expression was slightly elevated. To understand the in vivo role of RTN1, we generated RTN1-null mice and compared the effects of RTN1 and RTN3 on BACE1 modulation. We show that RTN1 is mostly expressed by neurons and not by glial cells under normal conditions, similar to the expression of RTN3. However, RTN1 is more localized in dendrites and is an excellent marker for dendrites of Purkinje cells, while RTN3 expression is less evident in dendrites. This differential localization also correlates with their associations with amyloid plaques in Alzheimer’s brains: RTN3, but not RTN1, is abundantly enriched in dystrophic neurites. RTN3 deficiency causes elevation of BACE1 protein levels, while RTN1 deficiency shows no obvious effects on BACE1 activity due to compensation by RTN3, as RTN1 deficiency causes elevation of RTN3 expression. Hence, expression of RTN1 and RTN3 is tightly regulated in mouse brains. Together, our data show that RTN1 and RTN3 have differential effects on the formation of senile plaques in Alzheimer’s brains and that RTN3 has a more prominent role in Alzheimer’s pathogenesis.</jats:p
Spatial variation of limestone soil minerals in a karst area of northwestern Guangxi
No full textAs extremely important components of soil and constitutors of soil skeleton, soil minerals account for over 95% of the solid mass of soil. They directly participate in the entire process of soil weathering, soil formation, and plant growth and development, impacting significantly on the internal structure, exchange capacity, and fertility status of soil. Studying the spatial distribution of soil mineral elements is of great significance for understanding and mastering soil development, physical and chemical properties, and the supply status of plant nutrients. Because of the special formation matrix and ecological environment conditions of carbonate rock—the parent material of limestone soil in karst areas, soil in karst areas presents a high degree of spatial heterogeneity. At the same time, the shortage of mineral nutrients may be an important limiting factor for the growth and restoration of vegetation in mountainous areas of carbonate rock in southwestern China. However, the spatial distribution characteristics of soil minerals on a large scale in the karst area of northwestern Guangxi are currently unclear. Clarifying the spatial variation characteristics of limestone minerals in the karst area of northwestern Guangxi can provide reference for the effective utilization of mineral resources and ecological restoration and reconstruction. In order to explore the distribution pattern of the main soil mineral components in the karst area of northwestern Guangxi and to guide vegetation restoration and ecological reconstruction in the relevant area, the spatial heterogeneity of mineral components (SiO2, Fe2O3, CaO, MgO, Al2O3, and MnO) in surface soil (0-15 cm) and its influencing factors were studied by the methods of classical statistics and geostatistics. The soil samples were collected by the grid method based on the whole karst regional scale in northwestern Guangxi. The results show that the content differences and variance coefficients of six mineral components in limestone soil of karst area in northwestern Guangxi are large. The average content of SiO2 is up to 55.72%, while the variance coefficient is the smallest (37.50%). The sum of SiO2 and Al2O3 accounts for 85.22% of the total six mineral components. The spatial patterns of the six mineral components are quite different from each other, and fit different models of mineral components. The spatial autocorrelation of SiO2 is medium, but the autocorrelations of Al2O3 and MnO are weak, and their ranges are long in good spatial continuum. CaO, Fe2O3 and MgO are characterized by strong spatial autocorrelations with short ranges. The spatial distribution of minerals is closely related to the main nutrients and topographic characteristics. The spatial variation of Al2O3 and Fe2O3 is mainly affected by the altitude. The bare rock rate is the main topographic factor affecting the spatial variation of SiO2, MnO and MgO, and the gradient is the main factor affecting the spatial distribution of CaO. A principal component analysis show that the soil mineral is an important factor affecting the spatial variation of limestone soil, especially SiO2. On a large regional scale, various topographic factors affect the spatial variation limestone soil minerals, and hence impacting the spatial distribution of limestone soil
BACE1 deletion in the adult mouse reverses preformed amyloid deposition and improves cognitive functions
No full textBACE1 initiates the generation of the β-amyloid peptide, which likely causes Alzheimer’s disease (AD) when accumulated abnormally. BACE1 inhibitory drugs are currently being developed to treat AD patients. To mimic BACE1 inhibition in adults, we generated BACE1 conditional knockout (BACE1fl/fl) mice and bred BACE1fl/fl mice with ubiquitin-CreER mice to induce deletion of BACE1 after passing early developmental stages. Strikingly, sequential and increased deletion of BACE1 in an adult AD mouse model (5xFAD) was capable of completely reversing amyloid deposition. This reversal in amyloid deposition also resulted in significant improvement in gliosis and neuritic dystrophy. Moreover, synaptic functions, as determined by long-term potentiation and contextual fear conditioning experiments, were significantly improved, correlating with the reversal of amyloid plaques. Our results demonstrate that sustained and increasing BACE1 inhibition in adults can reverse amyloid deposition in an AD mouse model, and this observation will help to provide guidance for the proper use of BACE1 inhibitors in human patients.</jats:p
Metformin can alleviate the symptom of patient with diabetic nephropathy through reducing the serum level of Hcy and IL-33
No full textInterleukin-33 (IL-33) and homocysteine (Hcy) were found to be up-regulated in patients with diabetic nephropathy (DN), and the present study aimed to investigate whether metformin (MT) can influence the serum levels of IL-33 and Hcy in patients with DN
BACE1 Regulates Hippocampal Astrogenesis via the Jagged1-Notch Pathway
No full textSummaryBACE1 is the sole secretase for generating β-amyloid (Aβ) in vivo and is being actively pursued as a drug target for the treatment of Alzheimer’s disease. Transmembrane BACE1 exerts its biological activity by cleaving its membrane-bound cellular substrates. Here, we reveal that BACE1 directly regulates the level of membrane-anchored full-length Jagged1 (Jag1), a signaling molecule important for the control of neurogenesis and astrogenesis, via interaction with its cognate Notch receptor. We show that shedding of Jag1 is reduced in BACE1 null mice and upregulated Jag1 enhances Notch signaling via cell-cell juxtacrine interactions. Additional biochemical assays confirmed that overexpression of BACE1 enhanced cleavage of Jag1. Consequently, BACE1 null mice exhibit a significant increase in astrogenesis with a corresponding decrease in neurogenesis in their hippocampi during early development. Hence, BACE1 appears to function as a signaling protease that controls the balance of neurogenesis and astrogenesis via the Jag1-Notch pathway
Adaptive weighted total variation regularized phase retrieval in differential phase-contrast imaging
No full textBACE1 Deficiency Causes Abnormal Neuronal Clustering in the Dentate Gyrus
No full textBACE1 is validated as Alzheimer's β-secretase and a therapeutic target for Alzheimer's disease. In examining BACE1-null mice, we discovered that BACE1 deficiency develops abnormal clusters of immature neurons, forming doublecortin-positive neuroblasts, in the developing dentate gyrus, mainly in the subpial zone (SPZ). Such clusters were rarely observed in wild-type SPZ and not reported in other mouse models. To understand their origins and fates, we examined how neuroblasts in BACE1-null SPZ mature and migrate during early postnatal development. We show that such neuroblasts are destined to form Prox1-positive granule cells in the dentate granule cell layer, and mainly mature to form excitatory neurons, but not inhibitory neurons. Mechanistically, higher levels of reelin potentially contribute to abnormal neurogenesis and timely migration in BACE1-null SPZ. Altogether, we demonstrate that BACE1 is a critical regulator in forming the dentate granule cell layer through timely maturation and migration of SPZ neuroblasts
Impacts of vegetation restoration strategies on soil organic carbon and nitrogen dynamics in a karst area, southwest China
No full textInhibitory Effect of an Acidic Peptide on the Activity of an Antimicrobial Peptide from the Scorpion <i>Mesobuthus martensii</i> Karsch
No full textHighly acidic peptides with no disulfide bridges are widely present in the scorpion venoms; however, none of them has been functionally characterized so far. Here, we cloned the full-length cDNA of a short-chain highly acidic peptide (referred to as HAP-1) from a cDNA library made from the venom glands of the Chinese scorpion Mesobuthus martensii Karsch. HAP-1 contains 19 amino acid residues with a predicted IP value of 4.25. Acidic amino residues account for 33.3% of the total residues in the molecule of HAP-1. HAP-1 shows 76⁻98% identities to some scorpion venom peptides that have not yet been functionally characterized. Secondary structure prediction showed that HAP-1 contains a beta-sheet region (residues 9⁻17), and two coiled coil regions (residues 1⁻8 and 18⁻19) located at the N-terminal and C-terminal regions of the peptide, respectively. Antimicrobial assay showed that HAP-1 does not have any effect on the growth of the bacterium Staphylococcus aureus AB94004. However, it potently inhibits the antimicrobial activity of a 13-mer peptide from M. martensii Karsch against Staphylococcus aureus AB94004. This finding is the first characterization of the function of such highly acidic peptides from scorpions
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