Genome engineering of stem cells for autonomously regulated, closed-loop delivery of biologic drugs

Chronic inflammatory diseases such as arthritis are characterized by dysregulated responses to pro-inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α). Pharmacologic anti-cytokine therapies are often effective at diminishing this inflammatory response but have significant side effects and are used at high, constant doses that do not reflect the dynamic nature of disease activity. Using the CRISPR/Cas9 genome-engineering system, we created stem cells that antagonize IL-1- or TNF-α-mediated inflammation in an autoregulated, feedback-controlled manner. Our results show that genome engineering can be used successfully to rewire endogenous cell circuits to allow for prescribed input/output relationships between inflammatory mediators and their antagonists, providing a foundation for cell-based drug delivery or cell-based vaccines via a rapidly responsive, autoregulated system. The customization of intrinsic cellular signaling pathways in stem cells, as demonstrated here, opens innovative possibilities for safer and more effective therapeutic approaches for a wide variety of diseases

Characterization of the Temporomandibular Joint Disc and Fibrocartilage Engineering using Human Embryonic Stem Cells

Fibrocartilages in the body, including the temporomandibular joint (TMJ) disc and knee meniscus, lack intrinsic healing capacity following trauma or disease. Current treatments only address the symptoms of fibrocartilage damage and do nothing to prevent further degradation of the joint. A tissue engineered replacement, with biochemical and biomechanical properties approaching those of native tissue, could provide a solution. This thesis investigates two components critical to the generation of a tissue engineered TMJ disc: 1) characterization of the native disc to identify a suitable animal model and create design parameters, and 2) development of approaches to use human embryonic stem cells (hESCs) in fibrocartilage tissue engineering. The first step to achieving this goal was to identify an animal model for the human TMJ disc based on quantitative biochemical and biomechanical properties. To this end, rabbit, goat, pig, cow, and human discs were analyzed, and the pig disc was shown to possess properties most similar to the human. The next step was to further characterize the pig TMJ, as many aspects of the joint were still poorly understood. Though the TMJ disc is anchored to the surrounding bony tissue on all sides by discal attachments, little was known about their properties. Biochemical and histological analysis was performed on these attachments and indicated that they are similar to the disc but possess distinct regional matrix content related to joint biomechanics. Finally, though the contribution of collagen to the mechanical properties of the TMJ disc was well characterized, the contribution of the glycosaminoglycans (GAGs) was unknown. By removing sulfated GAGs with chondroitinase ABC, it was found that these molecules contribute to the viscoelastic compressive properties of the disc, but only in regions with the highest native GAG content. The second aspect of this thesis involved producing fibrocartilage tissue from hESCs. The pluripotency and unlimited self-renewal of these cells makes them ideally suited for producing fibrocartilages that contain a spectrum of matrix components. This work began by investigating what factors are necessary for fibrochondrogenic differentiation of hESCs in embryoid bodies (EBs). Growth factors and co-cultures with primary fibrochondrocytes were both shown to be potent modulators of fibrochondrogenesis, although differentiation of hESCs consistently produced a heterogeneous cell population. To purify populations of fibrochondrocytes differentiated form hESCs, two inexpensive and novel techniques were investigated. First, density gradient separation was the first technique attempted. This technique was able to isolate distinct subpopulations of cells, some of which were mechanically similar to native chondrocytes. Second, a chondrogenic tuning technique was applied to differentiated hESCs. Following fibrochondrogenesis in EBs, cells were expanded in monolayer in chondrocyte specific media before being used for tissue engineering. Chondrogenic tuning produced several distinct cell populations during expansion, and, as a result, a spectrum of different cartilaginous tissues was achieved for tissue engineering. Three of the cell populations produced tissues similar to the native TMJ disc, outer meniscus, and inner meniscus. Overall, this thesis identified an animal model for TMJ characterization and in vivo studies, furthered understanding of structure-function relationships of the TMJ disc and its attachments, and developed a technique for producing a spectrum of engineered fibrocartilages from hESCs

NSCAT high-resolution surface wind measurements in Typhoon Violet

NASA scatterometer (NSCAT) measurements of the western Pacific Supertyphoon Violet are presented for revolutions 478 and 485 that occurred in September 1996. A tropical cyclone planetary boundary layer numerical, model, which uses conventional meteorological and geostationary cloud data, is used to estimate the winds at 10-m elevation in the cyclone. These model winds are then compared with the winds inferred from the NSCAT backscatter data by means of a novel approach that allows a wind speed to be recovered from each individual backscatter cell. This spatial adaptive (wind vector) retrieval algorithm employs several unique steps. The backscatter values are first regrouped in terms of closest neighbors in sets of four. The maximum likelihood estimates of speed and direction are then used to obtain speeds and directions for each group. Since the cyclonic flow around the tropical cyclone is known, NSCAT wind direction alias selection is easily accomplished. The selected wind directions are then used to convert each individual backscatter value to a wind speed. The results are compared to the winds obtained from the tropical cyclone boundary layer model. The NSCAT project baseline geophysical model function, NSCAT 1, was found to yield wind speeds that were systematically too low, even after editing for suspected rain areas of the cyclone. A new geophysical model function was developed using conventional NSCAT data and airborne Ku band scatterometer measurements in an Atlantic hurricane. This new model uses the neural network method and yields substantially better agreement with the winds obtained from the boundary layer model according to the statistical tests that were used

Biological resurfacing in a canine model of hip osteoarthritis

[Figure: see text]

RGS21, A Regulator of Taste and Mucociliary Clearance?

Motile cilia of airway epithelial cells help to expel harmful inhaled material. Activation of bitterant-responsive G protein-coupled receptors (GPCRs) is believed to potentiate cilia beat frequency and mucociliary clearance. In this study, we investigated whether regulator of G protein signaling-21 (RGS21) has the potential to modulate signaling pathways connected to airway mucociliary clearance, given that RGS proteins modulate GPCR signaling by acting as GTPase-accelerating proteins (GAPs) for the Gα subunits of heterotrimeric G proteins

Regional variability in peatland burning at mid- to high-latitudes during the Holocene

Acknowledgements This work developed from the PAGES (Past Global Changes) C-PEAT (Carbon in Peat on EArth through Time) working group. PAGES has been supported by the US National Science Foundation, Swiss National Science Foundation, Swiss Academy of Sciences and Chinese Academy of Sciences. We acknowledge the following financial support: UK Natural Environment Research Council Training Grants NE/L002574/1 (T.G.S.) and NE/S007458/1 (R.E.F.); Dutch Foundation for the Conservation of Irish Bogs, Quaternary Research Association and Leverhulme Trust RPG-2021-354 (G.T.S); the Academy of Finland (M.V); PAI/SIA 80002 and FONDECYT Iniciación 11220705 - ANID, Chile (C.A.M.); R20F0002 (PATSER) ANID Chile (R.D.M.); Swedish Strategic Research Area (SRA) MERGE (ModElling the Regional and Global Earth system) (M.J.G.); Polish National Science Centre Grant number NCN 2018/29/B/ST10/00120 (K.A.); Russian Science Foundation Grant No. 19-14-00102 (Y.A.M.); University of Latvia Grant No. AAp2016/B041/Zd2016/AZ03 and the Estonian Science Council grant PRG323 (TrackLag) (N.S. and A.M.); U.S. Geological Survey Land Change Science/Climate Research & Development Program (M.J., L.A., and D.W.); German Research Foundation (DFG), grant MA 8083/2-1 (P.M.) and grant BL 563/19-1 (K.H.K.); German Academic Exchange Service (DAAD), grant no. 57044554, Faculty of Geosciences, University of Münster, and Bavarian University Centre for Latin America (BAYLAT) (K.H.K). Records from the Global Charcoal Database supplemented this work and therefore we would like to thank the contributors and managers of this open-source resource. We also thank Annica Greisman, Jennifer Shiller, Fredrik Olsson and Simon van Bellen for contributing charcoal data to our analyses. Any use of trade, firm, or product name is for descriptive purposes only and does not imply endorsement by the U.S. Government.Peer reviewedPostprin

Application of a risk-management framework for integration of stromal tumor-infiltrating lymphocytes in clinical trials

Stromal tumor-infiltrating lymphocytes (sTILs) are a potential predictive biomarker for immunotherapy response in metastatic triple-negative breast cancer (TNBC). To incorporate sTILs into clinical trials and diagnostics, reliable assessment is essential. In this review, we propose a new concept, namely the implementation of a risk-management framework that enables the use of sTILs as a stratification factor in clinical trials. We present the design of a biomarker risk-mitigation workflow that can be applied to any biomarker incorporation in clinical trials. We demonstrate the implementation of this concept using sTILs as an integral biomarker in a single-center phase II immunotherapy trial for metastatic TNBC (TONIC trial, NCT02499367), using this workflow to mitigate risks of suboptimal inclusion of sTILs in this specific trial. In this review, we demonstrate that a web-based scoring platform can mitigate potential risk factors when including sTILs in clinical trials, and we argue that this framework can be applied for any future biomarker-driven clinical trial setting

Differential cross section measurements for the production of a W boson in association with jets in proton–proton collisions at √s = 7 TeV

Measurements are reported of differential cross sections for the production of a W boson, which decays into a muon and a neutrino, in association with jets, as a function of several variables, including the transverse momenta (pT) and pseudorapidities of the four leading jets, the scalar sum of jet transverse momenta (HT), and the difference in azimuthal angle between the directions of each jet and the muon. The data sample of pp collisions at a centre-of-mass energy of 7 TeV was collected with the CMS detector at the LHC and corresponds to an integrated luminosity of 5.0 fb[superscript −1]. The measured cross sections are compared to predictions from Monte Carlo generators, MadGraph + pythia and sherpa, and to next-to-leading-order calculations from BlackHat + sherpa. The differential cross sections are found to be in agreement with the predictions, apart from the pT distributions of the leading jets at high pT values, the distributions of the HT at high-HT and low jet multiplicity, and the distribution of the difference in azimuthal angle between the leading jet and the muon at low values.United States. Dept. of EnergyNational Science Foundation (U.S.)Alfred P. Sloan Foundatio