Quantitative Characterization of RCA-based DNA Hydrogels - Towards Rational Design.

DNA hydrogels hold significant promise for biomedical applications and can be synthesized through enzymatic Rolling Circle Amplification (RCA). Due to the exploratory nature of this emerging field, standardized RCA protocols specifying the impact of reaction parameters are currently lacking. This study varied template sequences and reagent concentrations, evaluating RCA synthesis efficiency and hydrogel mechanical properties through quantitative PCR (qPCR) and indentation measurements, respectively. Primer concentration and stabilizing additives showed minimal impact on RCA efficiency, while changes in polymerase and nucleotide concentrations had a stronger effect. Concentration of the circular template exerted the greatest influence on RCA productivity. An exponential correlation between hydrogel viscosity and DNA amplicon concentration was observed, with nucleobase sequence significantly affecting both amplification efficiency and material properties, particularly through secondary structures. This study suggests that combining high-throughput experimental methods with structural folding prediction offers a viable approach for systematically establishing structure-property relationships, aiding the rational design of DNA hydrogel material systems.

Micromechanical Indentation Platform for Rapid Analysis of Viscoelastic Biomolecular Hydrogels.

The advent of biomedical applications of soft bioinspired materials has entailed an increasing demand for streamlined and expedient characterization methods meant for both research and quality control objectives. Here, a novel measurement system for the characterization of biological hydrogels with volumes as low as 75 µL was developed. The system is based on an indentation platform equipped with micrometer drive actuators that allow the determination of both the fracture points and Young's moduli of relatively stiff polymers, including agarose, as well as the measurements of viscosity for exceptionally soft and viscous hydrogels, such as DNA hydrogels. The sensitivity of the method allows differentiation between DNA hydrogels produced by rolling circle amplification based on different template sequences and synthesis protocols. In addition, the polymerization kinetics of the hydrogels can be determined by time-resolved measurements, and the apparent viscosities of even more complex DNA-based nanocomposites can be measured. The platform presented here thus offers the possibility to characterize a broad variety of soft biomaterials in a targeted, fast, and cost-effective manner, holding promises for applications in fundamental materials science and ensuring reproducibility in the handling of complex materials.

Microfluidics for adaptation of microorganisms to stress: design and application.

Microfluidic systems have fundamentally transformed the realm of adaptive laboratory evolution (ALE) for microorganisms by offering unparalleled control over environmental conditions, thereby optimizing mutant generation and desired trait selection. This review summarizes the substantial influence of microfluidic technologies and their design paradigms on microbial adaptation, with a primary focus on leveraging spatial stressor concentration gradients to enhance microbial growth in challenging environments. Specifically, microfluidic platforms tailored for scaled-down ALE processes not only enable highly autonomous and precise setups but also incorporate novel functionalities. These capabilities encompass fostering the growth of biofilms alongside planktonic cells, refining selection gradient profiles, and simulating adaptation dynamics akin to natural habitats. The integration of these aspects enables shaping phenotypes under pressure, presenting an unprecedented avenue for developing robust, stress-resistant strains, a feat not easily attainable using conventional ALE setups. The versatility of these microfluidic systems is not limited to fundamental research but also offers promising applications in various areas of stress resistance. As microfluidic technologies continue to evolve and merge with cutting-edge methodologies, they possess the potential not only to redefine the landscape of microbial adaptation studies but also to expedite advancements in various biotechnological areas. KEY POINTS: • Microfluidics enable precise microbial adaptation in controlled gradients. • Microfluidic ALE offers insights into stress resistance and distinguishes between resistance and persistence. • Integration of adaptation-influencing factors in microfluidic setups facilitates efficient generation of stress-resistant strains.

Surface-Patterned DNA Origami Rulers Reveal Nanoscale Distance Dependency of the Epidermal Growth Factor Receptor Activation.

The nanoscale arrangement of ligands can have a major effect on the activation of membrane receptor proteins and thus cellular communication mechanisms. Here we report on the technological development and use of tailored DNA origami-based molecular rulers to fabricate "Multiscale Origami Structures As Interface for Cells" (MOSAIC), to enable the systematic investigation of the effect of the nanoscale spacing of epidermal growth factor (EGF) ligands on the activation of the EGF receptor (EGFR). MOSAIC-based analyses revealed that EGF distances of about 30-40 nm led to the highest response in EGFR activation of adherent MCF7 and Hela cells. Our study emphasizes the significance of DNA-based platforms for the detailed investigation of the molecular mechanisms of cellular signaling cascades.

Nano- and Microscale Confinements in DNA-Scaffolded Enzyme Cascade Reactions.

Artificial reconstruction of naturally evolved principles, such as compartmentalization and cascading of multienzyme complexes, offers enormous potential for the development of biocatalytic materials and processes. Due to their unique addressability at the nanoscale, DNA origami nanostructures (DON) have proven to be an exceptionally powerful tool for studying the fundamental processes in biocatalytic cascades. To systematically investigate the diffusion-reaction network of (co)substrate transfer in enzyme cascades, a model system of stereoselective ketoreductase (KRED) with cofactor regenerating enzyme is assembled in different spatial arrangements on DNA nanostructures and is located in the sphere of microbeads (MB) as a spatially confining nano- and microenvironment, respectively. The results, obtained through the use of highly sensitive analytical methods, Western blot-based quantification of the enzymes, and mass spectrometric (MS) product detection, along with theoretical modeling, provide strong evidence for the presence of two interacting compartments, the diffusion layers around the microbead and the DNA scaffold, which influence the catalytic efficiency of the cascade. It is shown that the microscale compartment exerts a strong influence on the productivity of the cascade, whereas the nanoscale arrangement of enzymes has no influence but can be modulated by the insertion of a diffusion barrier.

Comparison of all completed suicides in Frankfurt am Main (Hessen) before and during the early COVID-19 pandemic.

To research the effect of the COVID-19 pandemic on mental health, the prevalence and characteristics of all completed suicides in the city of Frankfurt am Main were compared for a 10-month period before the pandemic (March 2019-December 2019) with one during the early pandemic (March 2020-December 2020). Medicolegal data collected in the context of the FraPPE suicide prevention project were evaluated using descriptive statistical methods. In total, there were 81 suicides during the early pandemic period, as opposed to 86 in the pre-pandemic period. Though statistically not significant, the proportion of male suicides (73%) was higher during the early pandemic period than before (63%). The age-at-death was comparable in the pre-pandemic and pandemic periods (average, 54.8 vs. 53.1 years). Between these two periods, there was no difference in respect to the three most commonly used suicide methods by men: fall from a height (26% vs. 22%), intoxication, and strangulation (each 24% vs. 19%). For women, there was, however, a shift in methods from strangulation (38%), intoxication (28%), and fall from a height (19%) to fall from a height (50%), strangulation (18%), intoxication, and collision with a rail vehicle (14% each). There was a trend towards more suicides among non-German nationals during the early pandemic (suicide rate/100,000 inhabitants: German, 14.3 vs. 11.5; non-German, 4.4 vs. 8.8). Before the pandemic, 54% of the suicides were known to have a mental illness in contrast to 44% during the early pandemic. Overall, no increase in completed suicides could be observed in Frankfurt am Main during the early pandemic.

A photobioreactor for production of algae biomass from gaseous emissions of an animal house.

Sustainable approaches to circular economy in animal agriculture are still poorly developed. Here, we report an approach to reduce gaseous emissions of CO2 and NH3 from animal housing while simultaneously using them to produce value-added biomass. To this end, a cone-shaped, helical photobioreactor was developed that can be integrated into animal housing by being freely suspended, thereby combining a small footprint with a physically robust design. The photobioreactor was coupled with the exhaust air of a chicken house to allow continuous cultivation of a mixed culture of Arthrospira spec. (Spirulina). Continuous quantification of CO2 and NH3 concentration showed that the coupled algae reactor effectively purifies the exhaust air from the chicken house while producing algal biomass. Typical production rates of greater than 0.3 g/l*day dry mass were obtained, and continuous operation was possible for several weeks. Morphological, biochemical, and genomic characterization of Spirulina cultures yielded insights into the dynamics and metabolic processes of the microbial community. We anticipate that further optimization of this approach will provide new opportunities for the generation of value-added products from gaseous CO2 and NH3 waste emissions, linking resource-efficient production of microalgae with simultaneous sequestration of animal emissions. KEY POINTS: • Coupling a bioreactor with exhaust gases of chicken coop for production of biomass. • Spirulina mixed culture removes CO2 and NH3 from chicken house emissions. • High growth rates and biodiversity adaptation for nitrogen metabolism. Towards a sustainable circular economy in livestock farming. The functional coupling of a helical tube photobioreactor with exhaust air from a chicken house enabled the efficient cultivation of Spirulina microalgae while simultaneously sequestering the animals' CO2 and NH3 emissions.

Accurate quantification of DNA content in DNA hydrogels prepared by rolling circle amplification.

Accurate quantification of polymerized DNA in rolling circle amplification (RCA)-based hydrogels is challenging due to the high viscosity of these materials, however, it can be achieved with a photometric nucleotide depletion assay or qPCR. We show that the DNA content strongly depends on the template sequence and correlates with the mechanical properties of the hydrogels.

A three-colour stress biosensor reveals multimodal response in single cells and spatiotemporal dynamics of biofilms.

The plethora of stress factors that can damage microbial cells has evolved sophisticated stress response mechanisms. While existing bioreporters can monitor individual responses, sensors for detecting multimodal stress responses in living microorganisms are still lacking. Orthogonally detectable red, green, and blue fluorescent proteins combined in a single plasmid, dubbed RGB-S reporter, enable simultaneous, independent, and real-time analysis of the transcriptional response of Escherichia coli using three promoters which report physiological stress (PosmY for RpoS), genotoxicity (PsulA for SOS), and cytotoxicity (PgrpE for RpoH). The bioreporter is compatible with standard analysis and Fluorescent Activated Cell Sorting (FACS) combined with subsequent transcriptome analysis. Various stressors, including the biotechnologically relevant 2-propanol, activate one, two, or all three stress responses, which can significantly impact non-stress-related metabolic pathways. Implemented in microfluidic cultivation with confocal fluorescence microscopy imaging, the RGB-S reporter enabled spatiotemporal analysis of live biofilms revealing stratified subpopulations of bacteria with heterogeneous stress responses.

Biocatalytic Foams from Microdroplet-Formulated Self-Assembling Enzymes.

Industrial biocatalysis plays an important role in the development of a sustainable economy, as enzymes can be used to synthesize an enormous range of complex molecules under environmentally friendly conditions. To further develop the field, intensive research is being conducted on process technologies for continuous flow biocatalysis in order to immobilize large quantities of enzyme biocatalysts in microstructured flow reactors under conditions that are as gentle as possible in order to realize efficient material conversions. Here, monodisperse foams consisting almost entirely of enzymes covalently linked via SpyCatcher/SpyTag conjugation are reported. The biocatalytic foams are readily available from recombinant enzymes via microfluidic air-in-water droplet formation, can be directly integrated into microreactors, and can be used for biocatalytic conversions after drying. Reactors prepared by this method show surprisingly high stability and biocatalytic activity. The physicochemical characterization of the new materials is described and exemplary applications in biocatalysis are shown using two-enzyme cascades for the stereoselective synthesis of chiral alcohols and the rare sugar tagatose.

Hapten-Decorated DNA Nanostructures Decipher the Antigen-Mediated Spatial Organization of Antibodies Involved in Mast Cell Activation.

The immunological response of mast cells is controlled by the multivalent binding of antigens to immunoglobulin E (IgE) antibodies bound to the high-affinity receptor FcεRI on the cell membrane surface. However, the spatial organization of antigen-antibody-receptor complexes at the nanometer scale and the structural constraints involved in the initial events at the cell surface are not yet fully understood. For example, it is unclear what influence the affinity and nanoscale distance between the binding partners involved have on the activation of mast cells to degranulate inflammatory mediators from storage granules. We report the use of DNA origami nanostructures (DON) functionalized with different arrangements of the haptenic 2,4-dinitrophenyl (DNP) ligand to generate multivalent artificial antigens with full control over valency and nanoscale ligand architecture. To investigate the spatial requirements for mast cell activation, the DNP-DON complexes were initially used in surface plasmon resonance (SPR) analysis to study the binding kinetics of isolated IgE under physiological conditions. The most stable binding was observed in a narrow window of approximately 16 nm spacing between haptens. In contrast, affinity studies with FcεRI-linked IgE antibodies on the surface of rat basophilic leukemia cells (RBL-2H3) indicated virtually no distance-dependent variations in the binding of the differently structured DNP-DON complexes but suggested a supramolecular oligovalent nature of the interaction. Finally, the use of DNP-DON complexes for mast cell activation revealed that antigen-directed tight assembly of antibody-receptor complexes is the critical factor for triggering degranulation, even more critical than ligand valence. Our study emphasizes the significance of DNA nanostructures for the study of fundamental biological processes.

Systematic evaluation of agarose- and agar-based bioinks for extrusion-based bioprinting of enzymatically active hydrogels.

Extrusion-based 3D bioprinting enables the production of customized hydrogel structures that can be employed in flow reactors when printing with enzyme-containing inks. The present study compares inks based on either low-melt agarose or agar at different concentrations (3-6%) and loaded with the thermostable enzyme esterase 2 from the thermophilic organism Alicyclobacillus acidocaldarius (AaEst2) with regard to their suitability for the fabrication of such enzymatically active hydrogels. A customized printer setup including a heatable nozzle and a cooled substrate was established to allow for clean and reproducible prints. The inks and printed hydrogel samples were characterized using rheological measurements and compression tests. All inks were found to be sufficiently printable to create lattices without overhangs, but printing quality was strongly enhanced at 4.5% polymer or more. The produced hydrogels were characterized regarding mechanical strength and diffusibility. For both properties, a strong correlation with polymer concentration was observed with highly concentrated hydrogels being more stable and less diffusible. Agar hydrogels were found to be more stable and show higher diffusion rates than comparable agarose hydrogels. Enzyme leaching was identified as a major drawback of agar hydrogels, while hardly any leaching from agarose hydrogels was detected. The poor ability of agar hydrogels to permanently immobilize enzymes indicates their limited suitability for their employment in perfused biocatalytic reactors. Batch-based activity assays showed that the enzymatic activity of agar hydrogels was roughly twice as high as the activity of agarose hydrogels which was mostly attributed to the increased amount of enzyme leaching. Agarose bioinks with at least 4.5% polymer were identified as the most suitable of the investigated inks for the printing of biocatalytic reactors with AaEst2. Drawbacks of these inks are limited mechanical and thermal stability, not allowing the operation of a reactor at the optimum temperature of AaEst2 which is above the melting point of the employed low-melt agarose.

Does bone mobilization interfere with energy metabolism in transition cows?

The onset of lactation represents a challenge for both mineral homeostasis and energy metabolism in high-performing dairy cows. It has been shown that subclinical and clinical hypocalcemia increases the risk of ketosis and recent studies suggest that bone-derived endocrine factors could play a role in intermediary metabolism. Therefore, we analyzed serum samples from calculated d -7, calculated d -3, d +1, d +3, and d +7 relative to calving from 15 multiparous cows for total Ca, the bone resorption marker CrossLaps, the bone formation marker intact osteocalcin, undercarboxylated osteocalcin (ucOC), insulin, glucose, nonesterified fatty acids, ß-hydroxybutyrate, and insulin-like growth factor 1. Serum concentrations of Ca on d -3 and d +1 were associated with parameters of energy metabolism on d +3 and d +7. As we found large variations for serum concentrations of ucOC already on d -7, we allocated the cows retrospectively to 3 groups: low ucOC, medium ucOC, and high ucOC. These groups differed not only in their ucOC dynamics, but also in insulin sensitivity estimated using the revised quantitative insulin sensitivity index (RQUICKI). High ucOC cows presented with the highest RQUICKI throughout the entire observation period. Our data further support the hypothesis that low serum Ca precedes disturbances of energy metabolism. Furthermore, from our preliminary results it can be assumed that the potential link between mineral homeostasis, bone turnover, and intermediary metabolism should be further investigated.

Orthogonal protein decoration of DNA nanostructures based on SpyCatcher-SpyTag interaction.

We present an efficient and readily applicable strategy for the covalent ligation of proteins to DNA origami by using the SpyCatcher-SpyTag (SC-ST) connector system. This approach showed orthogonality with other covalent connectors and has been used exemplarily for the immobilization and study of stereoselective ketoreductases to gain insight into the spatial arrangement of enzymes on DNA nanostructures.

Enhanced Bioactivity of Tailor-Made Glycolipid Enriched Manuka Honey.

Glycolipids can be synthetized in deep eutectic solvents (DESs) as they possess low water content allowing a reversed lipase activity and thus enables ester formation. Based on this principle, honey can also serve as a media for glycolipid synthesis. Indeed, this supersaturated sugar solution is comparable in terms of physicochemical properties to the sugar-based DESs. Honey-based products being commercially available for therapeutic applications, it appears interesting to enhance its bioactivity. In the current work, we investigate if enriching medical grade honey with in situ enzymatically-synthetized glycolipids can improve the antimicrobial property of the mixture. The tested mixtures are composed of Manuka honey that is enriched with octanoate, decanoate, laurate, and myristate sugar esters, respectively dubbed GOH, GDH, GLH, and GMH. To characterize the bioactivity of those mixtures, first a qualitative screening using an agar well diffusion assay has been performed with methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Candida bombicola, Escherichia coli, and Pseudomonas putida which confirmed considerably enhanced susceptibility of these micro-organisms to the different glycolipid enriched honey mixtures. Then, a designed biosensor E. coli strain that displays a stress reporter system consisting of three stress-specific inducible, red, green, and blue fluorescent proteins which respectively translate to physiological stress, genotoxicity, and cytotoxicity was used. Bioactivity was, therefore, characterized, and a six-fold enhancement of the physiological stress that was caused by GOH compared to regular Manuka honey at a 1.6% (v/v) concentration was observed. An antibacterial agar well diffusion assay with E. coli was performed as well and demonstrated an improved inhibitory potential with GOH upon 20% (v/v) concentration.

Macroporous Silicone Chips for Decoding Microbial Dark Matter in Environmental Microbiomes.

Natural evolution has produced an almost infinite variety of microorganisms that can colonize almost any conceivable habitat. Since the vast majority of these microbial consortia are still unknown, there is a great need to elucidate this "microbial dark matter" (MDM) to enable exploitation in biotechnology. We report the fabrication and application of a novel device that integrates a matrix of macroporous elastomeric silicone foam (MESIF) into an easily fabricated and scalable chip design that can be used for decoding MDM in environmental microbiomes. Technical validation, performed with the model organism Escherichia coli expressing a fluorescent protein, showed that this low-cost, bioinert, and widely modifiable chip is rapidly colonized by microorganisms. The biological potential of the chip was then illustrated through targeted sampling and enrichment of microbiomes in a variety of habitats ranging from wet, turbulent moving bed biofilters and wastewater treatment plants to dry air-based environments. Sequencing analyses consistently showed that MESIF chips are not only suitable for sampling with high robustness but also that the material can be used to detect a broad cross section of microorganisms present in the habitat in a short time span of a few days. For example, results from the biofilter habitat showed efficient enrichment of microorganisms belonging to the enigmatic Candidate Phyla Radiation, which comprise ∼70% of the MDM. From dry air, the MESIF chip was able to enrich a variety of members of Actinobacteriota, which is known to produce specific secondary metabolites. Targeted sampling from a wastewater treatment plant where the herbicide glyphosate was added to the chip's reservoir resulted in enrichment of Cyanobacteria and Desulfobacteria, previously associated with glyphosate degradation. These initial case studies suggest that this chip is very well suited for the systematic study of MDM and opens opportunities for the cultivation of previously unculturable microorganisms.

Linker Engineering of Ligand-Decorated DNA Origami Nanostructures Affects Biological Activity.

News from an old acquaintance: The streptavidin (STV)-biotin binding system is frequently used for the decoration of DNA origami nanostructures (DON) to study biological systems. Here, a surprisingly high dynamic of the STV/DON interaction is reported, which is affected by the structure of the DNA linker system. Analysis of different mono- or bi-dentate linker architectures on DON with a novel high-speed atomic force microscope (HS-AFM) enabling acquisition times as short as 50 ms per frame gave detailed insights into the dynamics of the DON/STV interaction, revealing dwell times in the sub-100 millisecond range. The linker systems are also used to present biotinylated epidermal growth factor on DON to study the activation of the epidermal growth factor receptor signaling cascade in HeLa cells. The studies confirm that cellular activation correlated with the binding properties of linker-specific STV/DON interactions observed by HS-AFM. This work sheds more light on the commonly used STV/DON system and will help to further standardize the use of DNA nanostructures for the study of biological processes.

Intracellular Assembly of Interacting Enzymes Yields Highly-Active Nanoparticles for Flow Biocatalysis.

All-enzyme hydrogel (AEH) particles with a hydrodynamic diameter of up to 120 nm were produced intracellularly with an Escherichia coli-based in vivo system. The inCell-AEH nanoparticles were generated from polycistronic vectors enabling simultaneous expression of two interacting enzymes, the Lactobacillus brevis alcohol dehydrogenase (ADH) and the Bacillus subtilis glucose-1-dehydrogenase (GDH), fused with a SpyCatcher or SpyTag, respectively. Formation of inCell-AEH was analyzed by dynamic light scattering and atomic force microscopy. Using the stereoselective two-step reduction of a prochiral diketone substrate, we show that the inCell-AEH approach can be advantageously used in whole-cell flow biocatalysis, by which flow reactors could be operated for >4 days under constant substrate perfusion. More importantly, the inCell-AEH concept enables the recovery of efficient catalyst materials for stable flow bioreactors in a simple and economical one-step procedure from crude bacterial lysates. We believe that our method will contribute to further optimization of sustainable biocatalytic processes.

A Magnetosome-Based Platform for Flow Biocatalysis.

Biocatalysis in flow reactor systems is of increasing importance for the transformation of the chemical industry. However, the necessary immobilization of biocatalysts remains a challenge. We here demonstrate that biogenic magnetic nanoparticles, so-called magnetosomes, represent an attractive alternative for the development of nanoscale particle formulations to enable high and stable conversion rates in biocatalytic flow processes. In addition to their intriguing material characteristics, such as high crystallinity, stable magnetic moments, and narrow particle size distribution, magnetosomes offer the unbeatable advantage over chemically synthesized nanoparticles that foreign protein "cargo" can be immobilized on the enveloping membrane via genetic engineering and thus, stably presented on the particle surface. To exploit these advantages, we develop a modular connector system in which abundant magnetosome membrane anchors are genetically fused with SpyCatcher coupling groups, allowing efficient covalent coupling with complementary SpyTag-functionalized proteins. The versatility of this approach is demonstrated by immobilizing a dimeric phenolic acid decarboxylase to SpyCatcher magnetosomes. The functionalized magnetosomes outperform similarly functionalized commercial particles by exhibiting stable substrate conversion during a 60 h period, with an average space-time yield of 49.2 mmol L-1 h-1. Overall, our results demonstrate that SpyCatcher magnetosomes significantly expand the genetic toolbox for particle surface functionalization and increase their application potential as nano-biocatalysts.