Investigating the effect of fusion partners on the enzymatic activity and thermodynamic stability of poly(ethylene terephthalate) degrading enzymes.

Plastics are a cornerstone of the modern world, yet the durable material properties that we have come to depend upon have made them recalcitrant environmental pollutants. Biological solutions in the form of engineered enzymes offer low energy and sustainable approaches to recycle and upcycle plastic waste, uncoupling their production and end of life from fossil fuels and greenhouse gases. These enzymes however, encounter immense challenges acting on plastics: facing hydrophobic surfaces, molecular crowding, and high levels of substrate heterogeneity. There have been mixed reports about the benefits of fusing partner domains to polyethylene terephthalate (PET) degrading enzymes, with moderate improvements identified under specific conditions, but no clarity into the factors that underlie the mechanisms. Here, we use the SpyCatcher003:SpyTag003 technology, which demonstrates a profound 47 °C shift in Tm upon irreversible complex formation, to investigate the influence of the thermal stability of the fusion partner on a range of PETases selected for their optimal reaction temperatures. We find that the thermal stability of the fusion partner does not have a positive correlation on the activity of the enzymes or their evident kinetic and thermal stabilities. Instead, it appears that the fusion to less stable SpyCatcher003 tends to increase the measured activation energy of unfolding compared to the more stable complex and wildtype enzymes. Despite this, the fusions to SpyCatcher003 do not show significantly better catalytic activity on PET films, with or without SpyTag003, and were found to be sometimes disruptive. The approach we highlight here, in using a fusion partner with controllable melting temperature, allowed us to dissect the impact of the stability of a fusion partner on enzyme properties. Although fusion stability did not appear to be coupled with identifiable trends in enzymatic activities, careful analysis of the unfolding pathways, and solid and solution activities of a wider range of enzymes may yield a more detailed understanding.

Functionalizing DNA Origami by Triplex-Directed Site-Specific Photo-Cross-Linking.

Here, we present a cross-linking approach to covalently functionalize and stabilize DNA origami structures in a one-pot reaction. Our strategy involves adding nucleotide sequences to adjacent staple strands, so that, upon assembly of the origami structure, the extensions form short hairpin duplexes targetable by psoralen-labeled triplex-forming oligonucleotides bearing other functional groups (pso-TFOs). Subsequent irradiation with UVA light generates psoralen adducts with one or both hairpin staples leading to site-specific attachment of the pso-TFO (and attached group) to the origami with ca. 80% efficiency. Bis-adduct formation between strands in proximal hairpins further tethers the TFO to the structure and generates "superstaples" that improve the structural integrity of the functionalized complex. We show that directing cross-linking to regions outside of the origami core dramatically reduces sensitivity of the structures to thermal denaturation and disassembly by T7 RNA polymerase. We also show that the underlying duplex regions of the origami core are digested by DNase I and thus remain accessible to read-out by DNA-binding proteins. Our strategy is scalable and cost-effective, as it works with existing DNA origami structures, does not require scaffold redesign, and can be achieved with just one psoralen-modified oligonucleotide.

Concentration-Dependent Inhibition of Mesophilic PETases on Poly(ethylene terephthalate) Can Be Eliminated by Enzyme Engineering.

Enzyme-based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis (IsPETase) is capable of PET hydrolysis under mild conditions but suffers from concentration-dependent inhibition. In this study, this inhibition is found to be dependent on incubation time, the solution conditions, and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET-degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable IsPETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability.

Micromechanical mapping of the intact ovary interior reveals contrasting mechanical roles for follicles and stroma.

Follicle development in the ovary must be tightly regulated to ensure cyclical release of oocytes (ovulation). Disruption of this process is a common cause of infertility, for example via polycystic ovary syndrome (PCOS) and premature ovarian insufficiency (POI). Recent ex vivo studies suggest that follicle growth is mechanically regulated, however, crucially, the actual mechanical properties of the follicle microenvironment have remained unknown. Here we use atomic force microscopy (AFM) spherical probe indentation to map and quantify the mechanical microenvironment in the mouse ovary, at high resolution and across the entire width of the intact (bisected) ovarian interior. Averaging over the entire organ, we find the ovary to be a fairly soft tissue comparable to fat or kidney (mean Young's Modulus 3.3±2.5 kPa). This average, however, conceals substantial spatial variations, with the overall range of tissue stiffnesses from c. 0.5-10 kPa, challenging the concept that a single Young's Modulus can effectively summarize this complex organ. Considering the internal architecture of the ovary, we find that stiffness is low at the edge and centre which are dominated by stromal tissue, and highest in an intermediate zone that is dominated by large developmentally-advanced follicles, confirmed by comparison with immunohistology images. These results suggest that large follicles are mechanically dominant structures in the ovary, contrasting with previous expectations that collagen-rich stroma would dominate. Extending our study to the highest resolutions (c. 5 µm) showed substantial mechanical variations within the larger zones, even over very short (sub-100 µm) lengths, and especially within the stiffer regions of the ovary. Taken together, our results provide a new, physiologically accurate, framework for ovarian biomechanics and follicle tissue engineering.

Comb-like Pseudopeptides Enable Very Rapid and Efficient Intracellular Trehalose Delivery for Enhanced Cryopreservation of Erythrocytes.

Cell cryopreservation plays a key role in the development of reproducible and cost-effective cell-based therapies. Trehalose accumulated in freezing- and desiccation-tolerant organisms in nature has been sought as an attractive nontoxic cryoprotectant. Herein, we report a coincubation method for very rapid and efficient delivery of membrane-impermeable trehalose into ovine erythrocytes through reversible membrane permeabilization using pH-responsive, comb-like pseudopeptides. The pseudopeptidic polymers containing relatively long alkyl side chains were synthesized to mimic membrane-anchoring fusogenic proteins. The intracellular trehalose delivery efficiency was optimized by manipulating the side chain length, degree of substitution, and concentration of the pseudopeptides with different hydrophobic alkyl side chains, the pH, temperature, and time of incubation, as well as the polymer-to-cell ratio and the concentration of extracellular trehalose. Treatment of erythrocytes with the comb-like pseudopeptides for only 15 min yielded an intracellular trehalose concentration of 177.9 ± 8.6 mM, which resulted in 90.3 ± 0.7% survival after freeze-thaw. The very rapid and efficient delivery was found to be attributed to the reversible, pronounced membrane curvature change as a result of strong membrane insertion of the comb-like pseudopeptides. The pseudopeptides can enable efficient intracellular delivery of not only trehalose for improved cell cryopreservation but also other membrane-impermeable cargos.

An Electroactive Oligo-EDOT Platform for Neural Tissue Engineering.

The unique electrochemical properties of the conductive polymer poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) make it an attractive material for use in neural tissue engineering applications. However, inadequate mechanical properties, and difficulties in processing and lack of biodegradability have hindered progress in this field. Here, the functionality of PEDOT:PSS for neural tissue engineering is improved by incorporating 3,4-ethylenedioxythiophene (EDOT) oligomers, synthesized using a novel end-capping strategy, into block co-polymers. By exploiting end-functionalized oligoEDOT constructs as macroinitiators for the polymerization of poly(caprolactone), a block co-polymer is produced that is electroactive, processable, and bio-compatible. By combining these properties, electroactive fibrous mats are produced for neuronal culture via solution electrospinning and melt electrospinning writing. Importantly, it is also shown that neurite length and branching of neural stem cells can be enhanced on the materials under electrical stimulation, demonstrating the promise of these scaffolds for neural tissue engineering.

Rationalization of the X-ray photoelectron spectroscopy of aluminium phosphates synthesized from different precursors.

The aim of this paper is to clarify the assignments of X-ray photoelectron spectra of aluminium phosphate materials prepared from the reaction of phosphoric acid with three different aluminium precursors [Al(OH)3, Al(NO3)3 and AlCl3] at different annealing temperatures. The materials prepared have been studied by X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), infrared spectroscopy and high-resolution solid-state 31P NMR spectroscopy. A progressive polymerization from orthophosphate to metaphosphates is observed by XRD, ATR-FTIR and solid state 31P NMR, and on this basis the oxygen states observed in the XP spectra at 532.3 eV and 533.7 eV are assigned to P-O-Al and P-O-P environments, respectively. The presence of cyclic polyphosphates at the surface of the samples is also evident.

Direct solution-phase synthesis of 1T' WSe2 nanosheets.

Crystal phase control in layered transition metal dichalcogenides is central for exploiting their different electronic properties. Access to metastable crystal phases is limited as their direct synthesis is challenging, restricting the spectrum of reachable materials. Here, we demonstrate the solution phase synthesis of the metastable distorted octahedrally coordinated structure (1T' phase) of WSe2 nanosheets. We design a kinetically-controlled regime of colloidal synthesis to enable the formation of the metastable phase. 1T' WSe2 branched few-layered nanosheets are produced in high yield and in a reproducible and controlled manner. The 1T' phase is fully convertible into the semiconducting 2H phase upon thermal annealing at 400 °C. The 1T' WSe2 nanosheets demonstrate a metallic nature exhibited by an enhanced electrocatalytic activity for hydrogen evolution reaction as compared to the 2H WSe2 nanosheets and comparable to other 1T' phases. This synthesis design can potentially be extended to different materials providing direct access of metastable phases.

Thickness-Dependent Characterization of Chemically Exfoliated TiS2 Nanosheets.

Monolayer TiS2 is the lightest member of the transition metal dichalcogenide family with promising applications in energy storage and conversion systems. The use of TiS2 has been limited by the lack of rapid characterization of layer numbers via Raman spectroscopy and its easy oxidation in wet environment. Here, we demonstrate the layer-number-dependent Raman modes for TiS2. 1T TiS2 presents two characteristics of the Raman active modes, A1g (out-of-plane) and Eg (in-plane). We identified a characteristic peak frequency shift of the Eg mode with the layer number and an unexplored Raman mode at 372 cm-1 whose intensity changes relative to the A1g mode with the thickness of the TiS2 sheets. These two characteristic features of Raman spectra allow the determination of layer numbers between 1 and 5 in exfoliated TiS2. Further, we develop a method to produce oxidation-resistant inks of micron-sized mono- and few-layered TiS2 nanosheets at concentrations up to 1 mg/mL. These TiS2 inks can be deposited to form thin films with controllable thickness and nanosheet density over square centimeter areas. This opens up pathways for a wider utilization of exfoliated TiS2 toward a range of applications.

Realization of ground state in artificial kagome spin ice via topological defect-driven magnetic writing.

Arrays of non-interacting nanomagnets are widespread in data storage and processing. As current technologies approach fundamental limits on size and thermal stability, enhancing functionality through embracing the strong interactions present at high array densities becomes attractive. In this respect, artificial spin ices are geometrically frustrated magnetic metamaterials that offer vast untapped potential due to their unique microstate landscapes, with intriguing prospects in applications from reconfigurable logic to magnonic devices or hardware neural networks. However, progress in such systems is impeded by the inability to access more than a fraction of the total microstate space. Here, we demonstrate that topological defect-driven magnetic writing-a scanning probe technique-provides access to all of the possible microstates in artificial spin ices and related arrays of nanomagnets. We create previously elusive configurations such as the spin-crystal ground state of artificial kagome dipolar spin ices and high-energy, low-entropy 'monopole-chain' states that exhibit negative effective temperatures.