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

Absolute measurement of the ^{1}S_{0} − ^{3}P_{0} clock transition in neutral ^{88}Sr over the 330 km-long stabilized fibre optic link

We report a stability below $7\times 10{}^{-17}$ of two independent optical lattice clocks operating with bosonic ${}^{88}$Sr isotope. The value (429228066418008.3(1.9)${}_{syst}$(0.9)${}_{stat}$~Hz) of the absolute frequency of the ${}^{1}S_{0}$ - ${}^{3}P_{0}$ transition was measured with an optical frequency comb referenced to the local representation of the UTC by the 330 km-long stabilized fibre optical link. The result was verified by series of measurements on two independent optical lattice clocks and agrees with recommendation of Bureau International des Poids et Mesures

On the Stratospheric Chemistry of Midlatitude Wildfire Smoke

Massive Australian wildfires lofted smoke directly into the stratosphere in the austral summer of 2019/20. The smoke led to increases in optical extinction throughout the midlatitudes of the southern hemisphere that rivalled substantial volcanic perturbations. Previous studies have assumed that the smoke became coated with sulfuric acid and water and would deplete the ozone layer through heterogeneous chemistry on those surfaces, as is routinely observed following volcanic enhancements of the stratospheric sulfate layer. Here, observations of extinction and reactive nitrogen species from multiple independent satellites that sampled the smoke region are compared to one another and to model calculations. The data display a strong decrease in reactive nitrogen concentrations with increased aerosol extinction in the stratosphere, which is a known fingerprint for key heterogeneous chemistry on sulfate/H2O particles (specifically the hydrolysis of N2O5 to form HNO3). This chemical shift affects not only reactive nitrogen but also chlorine and reactive hydrogen species and is expected to cause midlatitude ozone layer depletion. Comparison of the model ozone to observations suggests that N2O5 hydrolysis contributed to reduced ozone, but additional chemical and/or dynamical processes are also important. These findings suggest that if wildfire smoke injection into the stratosphere increases sufficiently in frequency and magnitude as the world warms due to climate change, ozone recovery under the Montreal Protocol could be impeded, at least sporadically. Modeled austral midlatitude total ozone loss was about 1% in March 2020, which is significant compared to expected ozone recovery of about 1% per decade

Updated merged SAGE-CCI-OMPS+ dataset for the evaluation of ozone trends in the stratosphere

In this paper, we present the updated SAGE-CCI-OMPS+ climate data record of monthly zonal mean ozone profiles. This dataset covers the stratosphere and combines measurements by nine limb and occultation satellite instruments – SAGE II (Stratospheric Aerosol and Gases Experiment II), OSIRIS (Optical Spectrograph and InfraRed Imaging System), MIPAS (Michelson Interferometer for Passive Atmospheric Sounding), SCIAMACHY (SCanning Imaging Spectrometer for Atmospheric CHartographY), GOMOS (Global Ozone Monitoring by Occultation of Stars), ACE-FTS (Atmospheric Chemistry Experiment Fourier Transform Spectrometer), OMPS-LP (Ozone Monitor Profiling Suite Limb Profiler), POAM (Polar Ozone and Aerosol Measurement) III, and SAGE III/ISS (Stratospheric Aerosol and Gases Experiment III on the International Space Station). Compared to the original version of the SAGE-CCI-OMPS dataset (Sofieva et al., 2017b), the update includes new versions of MIPAS, ACE-FTS, and OSIRIS datasets and introduces data from additional sensors (POAM III and SAGE III/ISS) and retrieval processors (OMPS-LP). In this paper, we show detailed intercomparisons of ozone profiles from different instruments and data versions, with a focus on the detection of possible drifts in the datasets. The SAGE-CCI-OMPS+ dataset has a better coverage of polar regions and of the upper troposphere and the lower stratosphere (UTLS) than the previous dataset. We also studied the influence of including new datasets on ozone trends, which are estimated using multiple linear regression. The changes in the merged dataset do not change the overall morphology of post-1997 ozone trends; statistically significant trends are observed in the upper stratosphere. The largest changes in ozone trends are observed in polar regions, especially in the Southern Hemisphere

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

Updated trends of the stratospheric ozone vertical distribution in the 60° S–60° N latitude range based on the LOTUS regression model

This study presents an updated evaluation of stratospheric ozone profile trends in the 60° S–60° N latitude range over the 2000–2020 period using an updated version of the Long-term Ozone Trends and Uncertainties in the Stratosphere (LOTUS) regression model that was used to evaluate such trends up to 2016 for the last WMO Ozone Assessment (2018). In addition to the derivation of detailed trends as a function of latitude and vertical coordinates, the regressions are performed with the datasets averaged over broad latitude bands, i.e. 60–35° S, 20° S–20° N and 35–60° N. The same methodology as in the last assessment is applied to combine trends in these broad latitude bands in order to compare the results with the previous studies. Longitudinally resolved merged satellite records are also considered in order to provide a better comparison with trends retrieved from ground-based records, e.g. lidar, ozonesondes, Umkehr, microwave and Fourier transform infrared (FTIR) spectrometers at selected stations where long-term time series are available. The study includes a comparison with trends derived from the REF-C2 simulations of the Chemistry Climate Model Initiative (CCMI-1). This work confirms past results showing an ozone increase in the upper stratosphere, which is now significant in the three broad latitude bands. The increase is largest in the Northern and Southern Hemisphere midlatitudes, with ∼2.2 ± 0.7 % per decade at ∼2.1 hPa and ∼2.1 ± 0.6 % per decade at ∼3.2 hPa respectively compared to ∼1.6 ± 0.6 % per decade at ∼2.6 hPa in the tropics. New trend signals have emerged from the records, such as a significant decrease in ozone in the tropics around 35 hPa and a non-significant increase in ozone in the southern midlatitudes at about 20 hPa. Non-significant negative ozone trends are derived in the lowermost stratosphere, with the most pronounced trends in the tropics. While a very good agreement is obtained between trends from merged satellite records and the CCMI-1 REF-C2 simulation in the upper stratosphere, observed negative trends in the lower stratosphere are not reproduced by models at southern and, in particular, at northern midlatitudes, where models report an ozone increase. However, the lower-stratospheric trend uncertainties are quite large, for both measured and modelled trends. Finally, 2000–2020 stratospheric ozone trends derived from the ground-based and longitudinally resolved satellite records are in reasonable agreement over the European Alpine and tropical regions, while at the Lauder station in the Southern Hemisphere midlatitudes they show some differences