Microfluidic fabrication of click chemistry-mediated hyaluronic acid microgels: A bottom-up material guide to tailor a microgel's physicochemical and mechanical properties

The demand for tailored, micrometer-scaled biomaterials in cell biology and (cell-free) biotechnology has led to the development of tunable microgel systems based on natural polymers, such as hyaluronic acid (HA). To precisely tailor their physicochemical and mechanical properties and thus to address the need for well-defined microgel systems, in this study, a bottom-up material guide is presented that highlights the synergy between highly selective bio-orthogonal click chemistry strategies and the versatility of a droplet microfluidics (MF)-assisted microgel design. By employing MF, microgels based on modified HA-derivates and homobifunctional poly(ethylene glycol) (PEG)-crosslinkers are prepared via three different types of click reaction: Diels–Alder [4 + 2] cycloaddition, strain-promoted azide-alkyne cycloaddition (SPAAC), and UV-initiated thiol–ene reaction. First, chemical modification strategies of HA are screened in-depth. Beyond the microfluidic processing of HA-derivates yielding monodisperse microgels, in an analytical study, we show that their physicochemical and mechanical properties—e.g., permeability, (thermo)stability, and elasticity—can be systematically adapted with respect to the type of click reaction and PEG-crosslinker concentration. In addition, we highlight the versatility of our HA-microgel design by preparing non-spherical microgels and introduce, for the first time, a selective, hetero-trifunctional HA-based microgel system with multiple binding sites. As a result, a holistic material guide is provided to tailor fundamental properties of HA-microgels for their potential application in cell biology and (cell-free) biotechnology

Microfluidic Fabrication of Click Chemistry-Mediated Hyaluronic Acid Microgels: A Bottom-Up Material Guide to Tailor a Microgel’s Physicochemical and Mechanical Properties

The demand for tailored, micrometer-scaled biomaterials in cell biology and (cell-free) biotechnology has led to the development of tunable microgel systems based on natural polymers, such as hyaluronic acid (HA). To precisely tailor their physicochemical and mechanical properties and thus to address the need for well-defined microgel systems, in this study, a bottom-up material guide is presented that highlights the synergy between highly selective bio-orthogonal click chemistry strategies and the versatility of a droplet microfluidics (MF)-assisted microgel design. By employing MF, microgels based on modified HA-derivates and homobifunctional poly(ethylene glycol) (PEG)-crosslinkers are prepared via three different types of click reaction: Diels–Alder [4 + 2] cycloaddition, strain-promoted azide-alkyne cycloaddition (SPAAC), and UV-initiated thiol–ene reaction. First, chemical modification strategies of HA are screened in-depth. Beyond the microfluidic processing of HA-derivates yielding monodisperse microgels, in an analytical study, we show that their physicochemical and mechanical properties—e.g., permeability, (thermo)stability, and elasticity—can be systematically adapted with respect to the type of click reaction and PEG-crosslinker concentration. In addition, we highlight the versatility of our HA-microgel design by preparing non-spherical microgels and introduce, for the first time, a selective, hetero-trifunctional HA-based microgel system with multiple binding sites. As a result, a holistic material guide is provided to tailor fundamental properties of HA-microgels for their potential application in cell biology and (cell-free) biotechnology

Combining Hydrophilic and Hydrophobic Materials in 3D Printing for Fabricating Microfluidic Devices with Spatial Wettability

The fabrication of microfluidic flow cells via projection micro-stereolithography (PμSL) has excited researchers in recent years. However, due to the inherent process properties of most commercial PμSL, microfluidic devices are fabricated in a monolithic fashion with uniform material properties across a flow cell. Yet, the large surface-to-volume ratio in microfluidics demands to tailor microchannel surface properties—particularly in planar microchannel arrangements—with spatial control and micron-scale resolution to form a desired flow profile, e.g., emulsion droplets. Here, the fabrication of planar microfluidic devices by PμSLbased 3D printing with spatial control over surface properties is presented. For that, homemade photopolymer formulations being either hydrophilic or hydrophobic are designed. Adding acrylic acid to a resin containing poly(ethylene glycol) diacrylate lowers the contact angle down to 0° against water creating a superhydrophilic surface. By utilizing 1H,1H,2H,2H-perfluorodecyl acrylate, a photopolymer formulation allowing for 3D-printing a hydrophobic microchannel surface with a contact angle >120° against water is obtained. Combining these two materials, microfluidic flow cells with spatially defined wettability are 3D-printed for emulsion formation. Finally, the resin vat of the commercial PμSL printer is switched during the printing process for fabricating multimaterial geometries, as exemplarily applied for realizing a hydrophobic-hydrophilic-hydrophobic device for forming O/W/O double emulsions

Secondary metabolite profiling of the model legume Lotus japonicus during its symbiotic interaction with Mesorhizobium loti

Plant secondary metabolites, particularly flavonoids, are key components in the early stages of nitrogen-fixing symbiosis. Despite their importance, the endogenous secondary metabolites involved in symbiosis have not yet been identified in the model legume Lotus japonicus. We therefore determined changes in the secondary metabolic profile of Lotus japonicus roots in response to its symbiont. Analysis of the root secondary metabolite profiles 1 week after inoculation with Mesorhizobium loti revealed quantitative changes in the level of 14 phenolic peaks when compared with non-inoculated control plants. These changes affected compounds from most phenolic classes, possibly resulting from interconversion between classes since the total phenolic level remained constant. In addition, the use of 2 M. loti strains differing only in their capacity to synthesise Nod factor revealed that, although Nod factor signalling induced accumulation of a specific subset of 4 phenolic peaks, most changes were induced in response to both rhizobial strains.NR was supported by a Training Network funded by the European Training Network EU-RTN-LOTUS-HPRN-CT-2000-00086. KJW was supported by core funding from the Biotechnology and Biological Science Research Council (BBSRC), UK.Peer Reviewe

Stretching and heating cells with light - Nonlinear photothermal cell rheology

Stretching and heating are everyday experiences for skin and tissue cells. They are also standard procedures to reduce the risk for injuries in physical exercise and to relieve muscle spasms in physiotherapy. Here, we ask which immediate and long-term mechanical effects of such treatments are quantitatively detectable on the level of individual living cells. Combining versatile optical stretcher techniques with a well-tested mathematical model for viscoelastic polymer networks, we investigate the thermomechanical properties of suspended cells with a photothermal rheometric protocol that can disentangle fast transient and slow 'inelastic' components in the nonlinear mechanical response. We find that a certain minimum strength and duration of combined stretching and heating is required to induce long-lived alterations of the mechanical state of the cells, which then respond qualitatively differently to mechanical tests than after weaker/shorter treatments or merely mechanical preconditioning alone. Our results suggest a viable protocol to search for intracellular biomolecular signatures of the mathematically detected dissimilar mechanical response modes. © 2020 The Author(s). Published by IOP Publishing Ltd on behalf of the Institute of Physics and Deutsche Physikalische Gesellschaft

Assessment of Global Ocean Biogeochemistry Models for Ocean Carbon Sink Estimates in RECCAP2 and Recommendations for Future Studies

The ocean is a major carbon sink and takes up 25%–30% of the anthropogenically emitted CO2. A state-of-the-art method to quantify this sink are global ocean biogeochemistry models (GOBMs), but their simulated CO2 uptake differs between models and is systematically lower than estimates based on statistical methods using surface ocean pCO2 and interior ocean measurements. Here, we provide an in-depth evaluation of ocean carbon sink estimates from 1980 to 2018 from a GOBM ensemble. As sources of inter-model differences and ensemble-mean biases our study identifies (a) the model setup, such as the length of the spin-up, the starting date of the simulation, and carbon fluxes from rivers and into sediments, (b) the simulated ocean circulation, such as Atlantic Meridional Overturning Circulation and Southern Ocean mode and intermediate water formation, and (c) the simulated oceanic buffer capacity. Our analysis suggests that a late starting date and biases in the ocean circulation cause a too low anthropogenic CO2 uptake across the GOBM ensemble. Surface ocean biogeochemistry biases might also cause simulated anthropogenic fluxes to be too low, but the current setup prevents a robust assessment. For simulations of the ocean carbon sink, we recommend in the short-term to (a) start simulations at a common date before the industrialization and the associated atmospheric CO2 increase, (b) conduct a sufficiently long spin-up such that the GOBMs reach steady-state, and (c) provide key metrics for circulation, biogeochemistry, and the land-ocean interface. In the long-term, we recommend improving the representation of these metrics in the GOBMs

Drivers and uncertainties of future global marine primary production in marine ecosystem models

Past model studies have projected a global decrease in marine net primary production (NPP) over the 21st century, but these studies focused on the multi-model mean rather than on the large inter-model differences. Here, we analyze model-simulated changes in NPP for the 21st century under IPCC's high-emission scenario RCP8.5. We use a suite of nine coupled carbon–climate Earth system models with embedded marine ecosystem models and focus on the spread between the different models and the underlying reasons. Globally, NPP decreases in five out of the nine models over the course of the 21st century, while three show no significant trend and one even simulates an increase. The largest model spread occurs in the low latitudes (between 30° S and 30° N), with individual models simulating relative changes between −25 and +40 %. Of the seven models diagnosing a net decrease in NPP in the low latitudes, only three simulate this to be a consequence of the classical interpretation, i.e., a stronger nutrient limitation due to increased stratification leading to reduced phytoplankton growth. In the other four, warming-induced increases in phytoplankton growth outbalance the stronger nutrient limitation. However, temperature-driven increases in grazing and other loss processes cause a net decrease in phytoplankton biomass and reduce NPP despite higher growth rates. One model projects a strong increase in NPP in the low latitudes, caused by an intensification of the microbial loop, while NPP in the remaining model changes by less than 0.5 %. While models consistently project increases NPP in the Southern Ocean, the regional inter-model range is also very substantial. In most models, this increase in NPP is driven by temperature, but it is also modulated by changes in light, macronutrients and iron as well as grazing. Overall, current projections of future changes in global marine NPP are subject to large uncertainties and necessitate a dedicated and sustained effort to improve the models and the concepts and data that guide their developmen

Constraining the trend in the ocean CO2 sink during 2000–2022

The ocean will ultimately store most of the CO2 emitted to the atmosphere by human activities. Despite its importance, estimates of the 2000−2022 trend in the ocean CO2 sink differ by a factor of two between observation-based products and process-based models. Here we address this discrepancy using a hybrid approach that preserves the consistency of known processes but constrains the outcome using observations. We show that the hybrid approach reproduces the stagnation of the ocean CO2 sink in the 1990s and its reinvigoration in the 2000s suggested by observation-based products and matches their amplitude. It suggests that process-based models underestimate the amplitude of the decadal variability in the ocean CO2 sink, but that observation-based products on average overestimate the decadal trend in the 2010s. The hybrid approach constrains the 2000−2022 trend in the ocean CO2 sink to 0.42 ± 0.06 Pg C yr−1 decade−1, and by inference the total land CO2 sink to 0.28 ± 0.13 Pg C yr−1 decade−1