Microscale to Manufacturing Scale-up of Cell-Free Cytokine Production—A New Approach for Shortening Protein Production Development Timelines

Engineering robust protein production and purification of correctly folded biotherapeutic proteins in cell-based systems is often challenging due to the requirements for maintaining complex cellular networks for cell viability and the need to develop associated downstream processes that reproducibly yield biopharmaceutical products with high product quality. Here, we present an alternative Escherichia coli-based open cell-free synthesis (OCFS) system that is optimized for predictable high-yield protein synthesis and folding at any scale with straightforward downstream purification processes. We describe how the linear scalability of OCFS allows rapid process optimization of parameters affecting extract activation, gene sequence optimization, and redox folding conditions for disulfide bond formation at microliter scales. Efficient and predictable high-level protein production can then be achieved using batch processes in standard bioreactors. We show how a fully bioactive protein produced by OCFS from optimized frozen extract can be purified directly using a streamlined purification process that yields a biologically active cytokine, human granulocyte-macrophage colony-stimulating factor, produced at titers of 700 mg/L in 10 h. These results represent a milestone for in vitro protein synthesis, with potential for the cGMP production of disulfide-bonded biotherapeutic proteins. Biotechnol. Bioeng. 2011; 108:1570–1578. © 2011 Wiley Periodicals, Inc

Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity.

Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages

Tomographic Retrievals of Stratospheric Ozone with the Ozone Mapping and Profiler Suite Limb Profiler

Measurements of limb scattered sunlight have been successfully used to obtain vertically resolved profiles of trace species in the stratosphere and upper troposphere for decades. A common simplifying assumption made in inverting limb scatter measurements is that the atmosphere is horizontally homogenous, which may not be valid in regions of large horizontal gradients such as on the edge of the polar vortex. Here we introduce a new radiative transfer model, SASKTRAN-HR, which can solve the radiative transfer equation in two- and three-dimensional atmospheres. A newly developed technique to approximate the Jacobian matrix for two-dimensional atmospheres is also presented. SASKTRAN-HR is then used to perform the first tomographic retrieval of ozone from limb scattered sunlight using measurements from the Ozone Mapping and Profiler Suite (OMPS-LP). The tomographic retrieval is shown to remove biases which are present in traditional one-dimensional retrievals that assume horizontal homogeneity. The two-dimensional retrieval agrees favorably with measurements from the Microwave Limb Sounder to within approximately 5%. The retrieval was applied to the full six years of measurements from OMPS-LP to create a publicly available dataset. The dataset has been included in several merged stratospheric ozone composites which are used in the World Meteorogical Association's 2018 ozone assessment

Tomographic retrievals of ozone with the OMPS Limb Profiler: algorithm description and preliminary results

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Satellite Limb Observations of Unprecedented Forest Fire Aerosol in the Stratosphere

International audienceIntense forest fires in western North America during August 2017 caused smoke plumes that reached the stratosphere. While this phenomenon has often been observed, this particular event caused increases in stratospheric aerosol extinction at higher altitudes with greater magnitude than previously observed in the satellite record. Here we use multiple satellite limb sounding observations, which provide high sensitivity to thin aerosol layers and good vertical resolution, to show that enhancements in aerosol extinction from the fires reached as high as 23 km in altitude and persisted for more than 5 months. Within 1 month, the aerosol is observed to cover latitudes from 20°N to 60°N, which is essentially the northernmost limit of the observations. At midlatitudes between 15‐ and 20‐km altitudes, the sustained level of median aerosol extinction measured at 750 nm increased by almost an order of magnitude, from approximately 10−4 km−1 to nearly 10−3 km−1. Agreement between limb scatter and occultation measurements is generally within 20% despite potential bias due to modified aerosol shape and composition

Tomographic Retrievals of Hunga Tonga‐Hunga Ha'apai Volcanic Aerosol

Abstract The 2022 eruption of the Hunga Tonga‐Hunga Ha'apai volcano caused substantial impacts on the atmosphere, including a massive injection of water vapor, and the largest increase in stratospheric aerosol for 30 years. The Ozone Mapping and Profiler Suite (OMPS) Limb Profiler instrument has been critical in monitoring the amount and spread of the volcanic aerosol in the stratosphere. We show that the rapid imagery from the OMPS instrument enables a tomographic retrieval of the aerosol extinction that reduces a critical bias of up to a factor of two, and improves vertical structure and agreement with coincident lidar and occultation observations. Due to the vertically thin and heterogeneous nature of the volcanic aerosol, this impacts integrated values of aerosol across latitude, altitude, and time for several months. We also investigate the systematic impact of uncertainty in assumed particle size that result in an underestimation of the aerosol extinction at the peak of the volcanic aerosol layer

Estimating and Reporting Uncertainties in Remotely Sensed Atmospheric Composition and Temperature

Remote sensing of atmospheric state variables typically relies on the inverse solution of the radiative transfer equation. An adequately characterized retrieval provides information on the uncertainties of the estimated state variables as well as on how any constraint or a priori assumption affects the estimate. Reported characterization data should be intercomparable between different instruments, empirically validatable, grid-independent, usable without detailed knowledge of the instrument or retrieval technique, traceable and still have reasonable data volume. The latter may force one to work with representative rather than individual characterization data. Many errors derive from approximations and simplifications used in real-world retrieval schemes, which are reviewed in this paper, along with related error estimation schemes. The main sources of uncertainty are measurement noise, calibration errors, simplifications and idealizations in the radiative transfer model and retrieval scheme, auxiliary data errors, and uncertainties in atmospheric or instrumental parameters. Some of these errors affect the result in a random way, while others chiefly cause a bias or are of mixed character. Beyond this, it is of utmost importance to know the influence of any constraint and prior information on the solution. While different instruments or retrieval schemes may require different error estimation schemes, we provide a list of recommendations which should help to unify retrieval error reporting