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"Chen, William"
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High-throughput 5′ UTR engineering for enhanced protein production in non-viral gene therapies
Despite significant clinical progress in cell and gene therapies, maximizing protein expression in order to enhance potency remains a major technical challenge. Here, we develop a high-throughput strategy to design, screen, and optimize 5′ UTRs that enhance protein expression from a strong human cytomegalovirus (CMV) promoter. We first identify naturally occurring 5′ UTRs with high translation efficiencies and use this information with in silico genetic algorithms to generate synthetic 5′ UTRs. A total of ~12,000 5′ UTRs are then screened using a recombinase-mediated integration strategy that greatly enhances the sensitivity of high-throughput screens by eliminating copy number and position effects that limit lentiviral approaches. Using this approach, we identify three synthetic 5′ UTRs that outperform commonly used non-viral gene therapy plasmids in expressing protein payloads. In summary, we demonstrate that high-throughput screening of 5′ UTR libraries with recombinase-mediated integration can identify genetic elements that enhance protein expression, which should have numerous applications for engineered cell and gene therapies.
The engineering of 5′ UTRs that modulate protein expression remains a great challenge. Here we leverage synthetic biology and computational design to develop a high-throughput strategy to design, screen, and optimize 5′ UTRs that enhance protein expression for non-viral gene therapies.
Journal Article
Preconditioning of Human Mesenchymal Stem Cells to Enhance Their Regulation of the Immune Response
Mesenchymal stem cells (MSCs) have attracted the attention of researchers and clinicians for their ability to differentiate into a number of cell types, participate in tissue regeneration, and repair the damaged tissues by producing various growth factors and cytokines, as well as their unique immunoprivilege in alloreactive hosts. The immunomodulatory functions of exogenous MSCs have been widely investigated in immune-mediated inflammatory diseases and transplantation research. However, a harsh environment at the site of tissue injury/inflammation with insufficient oxygen supply, abundance of reactive oxygen species, and presence of other harmful molecules that damage the adoptively transferred cells collectively lead to low survival and engraftment of the transferred cells. Preconditioning of MSCs ex vivo by hypoxia, inflammatory stimulus, or other factors/conditions prior to their use in therapy is an adaptive strategy that prepares MSCs to survive in the harsh environment and to enhance their regulatory function of the local immune responses. This review focuses on a number of approaches in preconditioning human MSCs with the goal of augmenting their capacity to regulate both innate and adaptive immune responses.
Journal Article
Practical business negotiation
\"Practical Business Negotiation introduces university students to business negotiation as practiced in the globalized business world. There are no other textbooks which take on this topic in depth with non-native English speakers in mind. Current textbooks about negotiation tend to be dense, academic and less than practical in content. Many are demotivating to students who are not easily able to consume a few hundred pages of academic writing. This textbook takes a step by step approach providing bite-sized presentation of negotiation concepts with practical exercises that include linguistic as well as negotiation content. Explanations are reinforced with practical questions and problem solving and recent examples drawn from a business world that includes much more than North American and Europe\"-- Provided by publisher.
Book
Perivascular cells for regenerative medicine
Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRβ, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte‐like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.
Journal Article
A synthetic transcription platform for programmable gene expression in mammalian cells
Precise, scalable, and sustainable control of genetic and cellular activities in mammalian cells is key to developing precision therapeutics and smart biomanufacturing. Here we create a highly tunable, modular, versatile CRISPR-based synthetic transcription system for the programmable control of gene expression and cellular phenotypes in mammalian cells. Genetic circuits consisting of well-characterized libraries of guide RNAs, binding motifs of synthetic operators, transcriptional activators, and additional genetic regulatory elements express mammalian genes in a highly predictable and tunable manner. We demonstrate the programmable control of reporter genes episomally and chromosomally, with up to 25-fold more activity than seen with the EF1α promoter, in multiple cell types. We use these circuits to program the secretion of human monoclonal antibodies and to control T-cell effector function marked by interferon-γ production. Antibody titers and interferon-γ concentrations significantly correlate with synthetic promoter strengths, providing a platform for programming gene expression and cellular function in diverse applications.
Precise and scalable regulation of gene expression in mammalian cells is challenging. Here, the authors created a highly tunable CRISPR-based synthetic transcription system for programmable control of mammalian gene expression and cellular activity.
Journal Article
Designing flows to resolve human and environmental water needs in a dam-regulated river
Navigating trade-offs between meeting societal water needs and supporting functioning ecosystems is integral to river management policy. Emerging frameworks provide the opportunity to consider multiple river uses explicitly, but balancing multiple priorities remains challenging. Here we quantify relationships between hydrologic regimes and the abundance of multiple native and nonnative fish species over 18 years in a large, dryland river basin in southwestern United States. These models were incorporated into a multi-objective optimization framework to design dam operation releases that balance human water needs with the dual conservation targets of benefiting native fishes while disadvantaging nonnative fishes. Predicted designer flow prescriptions indicate significant opportunities to favor native over nonnative fishes while rarely, if ever, encroaching on human water needs. The predicted benefits surpass those generated by natural flow mimicry, and were retained across periods of heightened drought. We provide a quantitative illustration of theoretical predictions that designer flows can offer multiple ecological and societal benefits in human-altered rivers.
Human and environmental water needs can come into conflict in dam-regulated river systems. Here, Chen and Olden investigate the potential for the use of fish–flow modeling to make recommendations for the management of native and nonnative fish species whilst providing water for society.
Journal Article
Higher order Turán inequalities for the partition function
The Turán inequalities and the higher order Turán inequalities arise in the study of the Maclaurin coefficients of real entire functions in the Laguerre–Pólya class. A sequence {an}n≥0\\{a_{n}\\}_{n\\geq 0} of real numbers is said to satisfy the Turán inequalities or to be log-concave if for n≥1n\\geq 1, an2−an−1an+1≥0a_n^2-a_{n-1}a_{n+1}\\geq 0. It is said to satisfy the higher order Turán inequalities if for n≥1n\\geq 1, 4(an2−an−1an+1)(an+12−anan+2)−(anan+1−an−1an+2)2≥04(a_{n}^2-a_{n-1}a_{n+1})(a_{n+1}^2-a_{n}a_{n+2})-(a_{n}a_{n+1}-a_{n-1}a_{n+2})^2\\geq 0. For the partition function p(n)p(n), DeSalvo and Pak showed that for n>25n>25, the sequence {p(n)}n>25\\{ p(n)\\}_{n> 25} is log-concave, that is, p(n)2−p(n−1)p(n+1)>0p(n)^2-p(n-1)p(n+1)>0 for n>25n> 25. It was conjectured by the first author that p(n)p(n) satisfies the higher order Turán inequalities for n≥95n\\geq 95. In this paper, we prove this conjecture by using the Hardy–Ramanujan–Rademacher formula to derive an upper bound and a lower bound for p(n+1)p(n−1)/p(n)2p(n+1)p(n-1)/p(n)^2. Consequently, for n≥95n\\geq 95, the Jensen polynomials p(n−1)+3p(n)x+3p(n+1)x2+p(n+2)x3p(n-1)+3p(n)x+3p(n+1)x^2+p(n+2)x^3 have only distinct real zeros. We conjecture that for any positive integer m≥4m\\geq 4 there exists an integer N(m)N(m) such that for n≥N(m)n\\geq N(m), the Jensen polynomial associated with the sequence (p(n),p(n+1),…,p(n+m))(p(n),p(n+1),\\ldots ,p(n+m)) has only real zeros. This conjecture was posed independently by Ono.
Journal Article
Visualizing structure and transitions in high-dimensional biological data
The high-dimensional data created by high-throughput technologies require visualization tools that reveal data structure and patterns in an intuitive form. We present PHATE, a visualization method that captures both local and global nonlinear structure using an information-geometric distance between data points. We compare PHATE to other tools on a variety of artificial and biological datasets, and find that it consistently preserves a range of patterns in data, including continual progressions, branches and clusters, better than other tools. We define a manifold preservation metric, which we call denoised embedding manifold preservation (DEMaP), and show that PHATE produces lower-dimensional embeddings that are quantitatively better denoised as compared to existing visualization methods. An analysis of a newly generated single-cell RNA sequencing dataset on human germ-layer differentiation demonstrates how PHATE reveals unique biological insight into the main developmental branches, including identification of three previously undescribed subpopulations. We also show that PHATE is applicable to a wide variety of data types, including mass cytometry, single-cell RNA sequencing, Hi-C and gut microbiome data.
PHATE, a new data visualization tool, better preserves patterns in high-dimensional data after dimensionality reduction.
Journal Article