22,667 research outputs found
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The Expanding Landscape of Alternative Splicing Variation in Human Populations.
Alternative splicing is a tightly regulated biological process by which the number of gene products for any given gene can be greatly expanded. Genomic variants in splicing regulatory sequences can disrupt splicing and cause disease. Recent developments in sequencing technologies and computational biology have allowed researchers to investigate alternative splicing at an unprecedented scale and resolution. Population-scale transcriptome studies have revealed many naturally occurring genetic variants that modulate alternative splicing and consequently influence phenotypic variability and disease susceptibility in human populations. Innovations in experimental and computational tools such as massively parallel reporter assays and deep learning have enabled the rapid screening of genomic variants for their causal impacts on splicing. In this review, we describe technological advances that have greatly increased the speed and scale at which discoveries are made about the genetic variation of alternative splicing. We summarize major findings from population transcriptomic studies of alternative splicing and discuss the implications of these findings for human genetics and medicine
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Loci specific epigenetic drug sensitivity.
Therapeutic targeting of epigenetic modulators offers a novel approach to the treatment of multiple diseases. The cellular consequences of chemical compounds that target epigenetic regulators (epi-drugs) are complex. Epi-drugs affect global cellular phenotypes and cause local changes to gene expression due to alteration of a gene chromatin environment. Despite increasing use in the clinic, the mechanisms responsible for cellular changes are unclear. Specifically, to what degree the effects are a result of cell-wide changes or disease related locus specific effects is unknown. Here we developed a platform to systematically and simultaneously investigate the sensitivity of epi-drugs at hundreds of genomic locations by combining DNA barcoding, unique split-pool encoding, and single cell expression measurements. Internal controls are used to isolate locus specific effects separately from any global consequences these drugs have. Using this platform we discovered wide-spread loci specific sensitivities to epi-drugs for three distinct epi-drugs that target histone deacetylase, DNA methylation and bromodomain proteins. By leveraging ENCODE data on chromatin modification, we identified features of chromatin environments that are most likely to be affected by epi-drugs. The measurements of loci specific epi-drugs sensitivities will pave the way to the development of targeted therapy for personalized medicine
Capturing the βomeβ : the expanding molecular toolbox for RNA and DNA library construction
All sequencing experiments and most functional genomics screens rely on the generation of libraries to comprehensively capture pools of targeted sequences. In the past decade especially, driven by the progress in the field of massively parallel sequencing, numerous studies have comprehensively assessed the impact of particular manipulations on library complexity and quality, and characterized the activities and specificities of several key enzymes used in library construction. Fortunately, careful protocol design and reagent choice can substantially mitigate many of these biases, and enable reliable representation of sequences in libraries. This review aims to guide the reader through the vast expanse of literature on the subject to promote informed library generation, independent of the application
High-throughput, quantitative analyses of genetic interactions in E. coli.
Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli
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Multi-scale cellular engineering: From molecules to organ-on-a-chip.
Recent technological advances in cellular and molecular engineering have provided new insights into biology and enabled the design, manufacturing, and manipulation of complex living systems. Here, we summarize the state of advances at the molecular, cellular, and multi-cellular levels using experimental and computational tools. The areas of focus include intrinsically disordered proteins, synthetic proteins, spatiotemporally dynamic extracellular matrices, organ-on-a-chip approaches, and computational modeling, which all have tremendous potential for advancing fundamental and translational science. Perspectives on the current limitations and future directions are also described, with the goal of stimulating interest to overcome these hurdles using multi-disciplinary approaches
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Computational cytometer based on magnetically modulated coherent imaging and deep learning.
Detecting rare cells within blood has numerous applications in disease diagnostics. Existing rare cell detection techniques are typically hindered by their high cost and low throughput. Here, we present a computational cytometer based on magnetically modulated lensless speckle imaging, which introduces oscillatory motion to the magnetic-bead-conjugated rare cells of interest through a periodic magnetic force and uses lensless time-resolved holographic speckle imaging to rapidly detect the target cells in three dimensions (3D). In addition to using cell-specific antibodies to magnetically label target cells, detection specificity is further enhanced through a deep-learning-based classifier that is based on a densely connected pseudo-3D convolutional neural network (P3D CNN), which automatically detects rare cells of interest based on their spatio-temporal features under a controlled magnetic force. To demonstrate the performance of this technique, we built a high-throughput, compact and cost-effective prototype for detecting MCF7 cancer cells spiked in whole blood samples. Through serial dilution experiments, we quantified the limit of detection (LoD) as 10 cells per millilitre of whole blood, which could be further improved through multiplexing parallel imaging channels within the same instrument. This compact, cost-effective and high-throughput computational cytometer can potentially be used for rare cell detection and quantification in bodily fluids for a variety of biomedical applications
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Έμ΄λ λ±κ³Ό μ λͺ©νμ¬ μ¬μ©λλ€λ©΄ λ³Έ νλ«νΌμ κ°λ₯μ±μ λμ± κ·Ήλν ν μ μμ κ²μΌλ‘ κΈ°λνλ€.Precision or Personalized Medicine is a medical paradigm aimed to determine optimal therapy for individual patient. In particular, clinical oncology has been using methods of molecular profiling for each patient through next-generation sequencing (NGS), mRNA-sequencing, and mass spectrometry, and has been trying to implement personalized treatment. However, personalized treatment based on molecular profiling to each patient is not always possible due to the high level of heterogeneity of tumor that is still not fully understood at the current level and acquired resistance of anti-cancer drug due to cumulative targeted therapy. In such cases, in vitro drug testing platform using primary cells obtained from patients, or patient-derived cells, spheroids, and organoids can make it possible to find a more appropriate treatment for each individual patient. However, though high-throughput drug screening technology for this purpose is of the utmost importance in saving lives, there were many limitations to its wide use in many hospitals. The existing high-throughput drug combination screening technology consumes a large number of samples and consumes a considerable amount of expensive reagents. In addition, expensive automated liquid handlers, which were essential for exploring thousands of different pipetting, were not easy to introduce except for large-sized pharmaceutical companies and research institutes, which limited access to technology.
In this study, I construct a heterogeneous drug-loaded microparticle library by fabricating encoded photocurable polymer particle that has individually identifiable codes to track loaded drug. and I load various drug molecules, which I want to test to target cells, into each coded microparticle. Then, I developed to produce heterogeneous drug-laden microparticle arrays through simple self-assembly without the need for a microarray spotter or dispensing machine for generating microarray. I also have developed cell seeding method of seeding small-volume samples into the microwell-based cell chip. By utilizing the drug-laden microparticle hydrogel array and microwell-based cell chip technology, hundreds to thousands of different assays can be done at once with just a small number of samples and low cost.
Through the implemented platform, the anti-cancer drug sequential combination screening was conducted on the triple-negative breast cooler (TNBC) cells, which are generally known to be difficult to treat due to lack of known drug target, and the results of screening were analyzed by establishing a library of drugs in the EGFR inhibitory type and drugs in the genotoxin type. In addition, another study was conducted to find optimal drug combinations using patient-derived cells derived from tumors in patients with non-small cell lung cancer that have obtained acquired resistance. Finally, as the growing need for three-dimensional culture, such as spheroid and organoid for having a similar response to in vivo drug testing, it was also developed that microwell-based cell chip that is capable of 3D culture with low-cost and small-volume of cells.
The miniaturized in vitro anticancer drug screening platform presented in this study has the following significance. An easy-to-use technique that can be applied to a small number of patient cells or samples, which can dramatically reduce the use of conventional expensive equipment, reagents. The proposed technology in this study can be applied to a variety of academic studies previously inaccessible to high-throughput screening due to the high cost of reagents, the high price of equipment, or the limited amount of samples in conventional drug screening. and this platform can also dramatically increase access to clinical research in hospitals for personalized treatments. In particular, it is expected that the possibility of this platform will be further maximized if it is used in a relatively small and medium-sized research environment by the combined use of various rare samples such as patient-derived cells or patient-derived organoids.Chapter 1 Introduction οΌ
1.1 Motivation of this research οΌ
1.2 Competing technologies and Previous works οΌ
1.3 Main Concept: In vitro drug testing using miniaturized encoded drug-laden hydrogel array technology οΌοΌ
Chapter 2 Platform Development of Drug Releasing Hydrogel Microarray οΌοΌ
2.1 Encoded Drug-Laden Hydrogel & Library construction οΌοΌ
2.2 Array generation of heterogenous drug-laden microparticles. οΌοΌ
2.3 Cell Culturing on Cell Chip and bioassay οΌοΌ
Chapter 3 Sequential Drug Combination Screening Assy on TNBC οΌοΌ
3.1 Background : Sequential Drug Combination as promising therapeutic option οΌοΌ
3.2 Experimental design with sequential drug treatment assay οΌοΌ
3.3 Technical Issue & its engineering solution οΌοΌ
3.4 Assay Result οΌοΌ
Chapter 4 Drug Combination Assay on Patient-Derived Cells οΌοΌ
4.1 Background : Simultaneous Combination Treatment using Patient-Derived Cells οΌοΌ
4.2 Improvement of Platform for facilitating translational study οΌοΌ
4.3 Study Design for small-volume drug combinatorial screening with NSCLC patient derived cell οΌοΌ
4.4 Assay Result οΌοΌ
Chapter 5 Development of platform for 3D culture model οΌοΌ
5.1 3D culturable platform οΌοΌ
5.2 Development of 3D culture platform based Matrigel scaffold. οΌοΌ
5.3 Advantage over conventional 3D culture-based drug testing platform. οΌοΌ
Chapter 6 Conclusion οΌοΌ
Bibliography οΌοΌ
Abstract in Korean οΌοΌDocto
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