Driving skyrmions in flow regime in synthetic ferrimagnets.

The last decade has seen significant improvements in our understanding of skyrmions current induced dynamics, along with their room temperature stabilization, however, the impact of local material inhomogeneities still remains an issue that impedes reaching the regime of steady state motion of these spin textures. Here, we study the spin-torque driven motion of skyrmions in synthetic ferrimagnetic multilayers with the aim of achieving high mobility and reduced skyrmion Hall effect. We consider Pt|Co|Tb multilayers of various thicknesses with antiferromagnetic coupling between the Co and Tb magnetization. The increase of Tb thickness in the multilayers reduces the total magnetic moment and increases the spin-orbit torques allowing to reach velocities up to 400 ms-1 for skyrmions with diameters of about 160 nm. We demonstrate that due to reduced skyrmion Hall effect combined with the edge repulsion of the magnetic track, the skyrmions move along the track without any transverse deflection. Further, by comparing the field-induced domain wall motion and current-induced skyrmion motion, we demonstrate that the skyrmions at the largest current densities present all the characteristics of a dynamical flow regime.

Optimized Spatial Configuration of Heterogeneous Biocatalysts Maximizes Cell-Free Biosynthesis of ω-Hydroxy and ω-Amino Acids.

Cell-free biocatalysis is gaining momentum in producing value-added chemicals, particularly in stepwise reaction cascades. However, the stability of enzyme cascades in industrial settings is often compromised when free enzymes are involved. In this study, we have developed a stable multifunctional heterogeneous biocatalyst coimmobilizing five enzymes on microparticles to transform 1,ω-diols into 1,ω-hydroxy acids. We improved the operational efficiency and stability of the heterogeneous biocatalyst by fine-tuning the enzyme loading and spatial organization. Stability issues are overcome through postimmobilization polymer coating. The general applicability of this heterogeneous biocatalyst is demonstrated by its scale-up in both batch and packed bed reactors, allowing a product yield of >80%. The continuous process is fed with H2O2 as the oxygen source, reaching a space-time yield (STY) of 0.76 g·L-1·h-1, maintained for the first 12 h. Finally, this flow system is telescoped with a second plug-flow reactor packed with a different heterogeneous biocatalyst integrating an additional transaminase. As a result, this 6-enzyme 2-reactor system sequentially transforms 1,ω-diols into 1,ω-amino acids while in situ recycling NAD+, depleting H2O2, and generating O2.

Recognition and normalization of multilingual symptom entities using in-domain-adapted BERT models and classification layers.

Due to the scarcity of available annotations in the biomedical domain, clinical natural language processing poses a substantial challenge, especially when applied to low-resource languages. This paper presents our contributions for the detection and normalization of clinical entities corresponding to symptoms, signs, and findings present in multilingual clinical texts. For this purpose, the three subtasks proposed in the SympTEMIST shared task of the Biocreative VIII conference have been addressed. For Subtask 1-named entity recognition in a Spanish corpus-an approach focused on BERT-based model assemblies pretrained on a proprietary oncology corpus was followed. Subtasks 2 and 3 of SympTEMIST address named entity linking (NEL) in Spanish and multilingual corpora, respectively. Our approach to these subtasks followed a classification strategy that starts from a bi-encoder trained by contrastive learning, for which several SapBERT-like models are explored. To apply this NEL approach to different languages, we have trained these models by leveraging the knowledge base of domain-specific medical concepts in Spanish supplied by the organizers, which we have translated into the other languages of interest by using machine translation tools. The results obtained in the three subtasks establish a new state of the art. Thus, for Subtask 1 we obtain precision results of 0.804, F1-score of 0.748, and recall of 0.699. For Subtask 2, we obtain performance gains of up to 5.5% in top-1 accuracy when the trained bi-encoder is followed by a WNT-softmax classification layer that is initialized with the mean of the embeddings of a subset of SNOMED-CT terms. For Subtask 3, the differences are even more pronounced, and our multilingual bi-encoder outperforms the other models analyzed in all languages except Swedish when combined with a WNT-softmax classification layer. Thus, the improvements in top-1 accuracy over the best bi-encoder model alone are 13% for Portuguese and 13.26% for Swedish. Database URL: https://doi.org/10.1093/database/baae087.

Microtiter Plate Immobilization Screening for Prototyping Heterogeneous Enzyme Cascades.

Immobilization is a key enabling technology in applied biocatalysis that facilitates the separation, recovery, and reuse of heterogeneous biocatalysts. However, finding a consensus immobilization protocol for several enzymes forming a multi-enzyme system is extremely difficult and relies on a combinatorial trial-and-error approach. Herein, we describe a protocol in which 17 different carriers functionalized with different reactive groups are tested in a 96-well microtiter plate to screen up to 21 immobilization protocols for up to 18 enzymes. This screening includes an activity and stability assay to select the optimal immobilization chemistry to achieve the most active and stable heterogeneous biocatalysts. The information retrieved from the screening can be rationalized using a Python-based application CapiPy. Finally, through scoring the screening results, we find the consensus immobilization protocol to assemble an immobilized four-enzyme system to transform vinyl acetate into (S)-3-hydroxybutyric acid. This methodology opens a path to speed up the prototyping of immobilized multi-enzyme pathways for chemical manufacturing.

Engineering bio-brick protein scaffolds for organizing enzyme assemblies.

Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi-enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi-enzyme systems. Two SCAB systems are developed, one employing head-to-tail interactions with reversible covalent disulfide bonds, the other relying on non-covalent metal-driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non-reducing environment or by metal presence. A proof-of-concept with formate dehydrogenase (FDH) and L-alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6-fold compared to free enzymes, with the covalent stapling outperforming the metal-driven assembly. This enhancement likely stems from higher-order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial-organizing tools, for more efficient enzymatic cascade reactions.

Toward Reliable Synthesis of Superconducting Infinite Layer Nickelate Thin Films by Topochemical Reduction.

Infinite layer (IL) nickelates provide a new route beyond copper oxides to address outstanding questions in the field of unconventional superconductivity. However, their synthesis poses considerable challenges, largely hindering experimental research on this new class of oxide superconductors. That synthesis is achieved in a two-step process that yields the most thermodynamically stable perovskite phase first, then the IL phase by topotactic reduction, the quality of the starting phase playing a crucial role. Here, a reliable synthesis of superconducting IL  nickelate films is reported after successive topochemical reductions of a parent perovskite phase with nearly optimal stoichiometry. Careful analysis of the transport properties of the incompletely reduced films reveals an improvement in the strange metal behavior of their normal state resistivity over subsequent topochemical reductions, offering insight into the reduction process.

Size-Dependence and High Temperature Stability of Radial Vortex Magnetic Textures Imprinted by Superconductor Stray Fields.

Swirling spin textures, including topologically nontrivial states, such as skyrmions, chiral domain walls, and magnetic vortices, have garnered significant attention within the scientific community due to their appeal from both fundamental and applied points of view. However, their creation, controlled manipulation, and stability are typically constrained to certain systems with specific crystallographic symmetries, bulk or interface interactions, and/or a precise stacking sequence of materials. Recently, a new approach has shown potential for the imprint of magnetic radial vortices in soft ferromagnetic compounds making use of the stray field of YBa2Cu3O7-δ superconducting microstructures in ferromagnet/superconductor (FM/SC) hybrids at temperatures below the superconducting transition temperature (TC). Here, we explore the lower size limit for the imprint of magnetic radial vortices in square and disc shaped structures as well as the persistence of these spin textures above TC, with magnetic domains retaining partial memory. Structures with circular geometry and with FM patterned to smaller radius than the superconductor island facilitate the imprinting of magnetic radial vortices and improve their stability above TC, in contrast to square structures where the presence of magnetic domains increases the dipolar energy. Micromagnetic modeling coupled with a SC field model reveals that the stabilization mechanism above TC is mediated by microstructural defects. Superconducting control of swirling spin textures, and their stabilization above the superconducting transition temperature by means of defect engineering holds promising prospects for shaping superconducting spintronics based on magnetic textures.

Deciphering the immobilization of lipases on hydrophobic wrinkled silica nanoparticles.

In this work, the adsorption of Candida antarctica B (CALB) and Rhizomucor miehei (RML) lipases into hydrophobic wrinkled silica nanoparticles (WSNs) is investigated. WSNs are hydrophobized by chemical vapor deposition. Both proteins are homogeneously distributed inside the pores of the nanoparticles, as confirmed by Transmission Electron Microscopy and Energy Dispersive X-ray measurements. The maximum enzyme load of CALB is twice that obtained for RML. Fourier Transform Infrared Spectroscopy confirms the preservation of the enzyme secondary structure after immobilization for both enzymes. Adsorption isotherms fit to a Langmuir model, resulting in a binding constant (KL) for RML 4.5-fold higher than that for CALB, indicating stronger binding for the former. Kinetic analysis reveals a positive correlation between enzyme load and RML activity unlike CALB where activity decreases along the enzyme load increases. Immobilization allows for enhancing the thermal stability of both lipases. Finally, CALB outperforms RML in the hydrolysis of ethyl-3-hydroxybutyrate. However, immobilized CALB yielded 20 % less 3-HBA than free lipase, while immobilized RML increases 3-fold the 3-HBA yield when compared with the free enzyme. The improved performance of immobilized RML can be explained due to the interfacial hyperactivation undergone by this lipase when immobilized on the superhydrophobic surface of WSNs.

In-Hydrogel Cell-Free Protein Expression System as Biocompatible and Implantable Biomaterial.

Biomaterials capable of delivering therapeutic proteins are relevant in biomedicine, yet their manufacturing relies on centralized manufacturing chains that pose challenges to their remote implementation at the point of care. This study explores the viability of confined cell-free protein synthesis within porous hydrogels as biomaterials that dynamically produce and deliver proteins to in vitro and in vivo biological microenvironments. These functional biomaterials have the potential to be assembled as implants at the point of care. To this aim, we first entrap cell-free extracts (CFEs) from Escherichia coli containing the transcription-translation machinery, together with plasmid DNA encoding the super folded green fluorescence protein (sGFP) as a model protein, into hydrogels using various preparation methods. Agarose hydrogels result in the most suitable biomaterials to confine the protein synthesis system, demonstrating efficient sGFP production and diffusion from the core to the surface of the hydrogel. Freeze-drying (FD) of agarose hydrogels still allows for the synthesis and diffusion of sGFP, yielding a more attractive biomaterial for its reconstitution and implementation at the point of care. FD-agarose hydrogels are biocompatible in vitro, allowing for the colonization of cell microenvironments along with cell proliferation. Implantation assays of this biomaterial in a preclinical mouse model proved the feasibility of this protein synthesis approach in an in vivo context and indicated that the physical properties of the biomaterials influence their immune responses. This work introduces a promising avenue for biomaterial fabrication, enabling the in vivo synthesis and targeted delivery of proteins and opening new paths for advanced protein therapeutic approaches based on biocompatible biomaterials.

Single-Particle and Single-Molecule Characterization of Immobilized Enzymes: A Multiscale Path toward Optimizing Heterogeneous Biocatalysts.

Heterogeneous biocatalysis is highly relevant in biotechnology as it offers several benefits and practical uses. To leverage the full potential of heterogeneous biocatalysts, the establishment of well-crafted protocols, and a deeper comprehension of enzyme immobilization on solid substrates are essential. These endeavors seek to optimize immobilized biocatalysts, ensuring maximal enzyme performance within confined spaces. For this aim, multidimensional characterization of heterogeneous biocatalysts is required. In this context, spectroscopic and microscopic methodologies conducted at different space and temporal scales can inform about the intraparticle enzyme kinetics, the enzyme spatial distribution, and the mass transport issues. In this Minireview, we identify enzyme immobilization, enzyme catalysis, and enzyme inactivation as the three main processes for which advanced characterization tools unveil fundamental information. Recent advances in operando characterization of immobilized enzymes at the single-particle (SP) and single-molecule (SM) levels inform about their functional properties, unlocking the full potential of heterogeneous biocatalysis toward biotechnological applications.

Coenzyme A Thioester Intermediates as Platform Molecules in Cell-Free Chemical Biomanufacturing.

The in vitro synthesis of Coenzyme A (CoA)-thioester intermediates opens new avenues to transform simple molecules into more complex and multifunctional ones by assembling cell-free biosynthetic cascades. In this review, we have systematically cataloged known CoA-dependent enzyme reactions that have been successfully implemented in vitro. To faciliate their identification, we provide their UniProt ID when available. Based on this catalog, we have organized enzymes into three modules: activation, modification, and removal. i) The activation module includes enzymes capable of fusing CoA with organic molecules. ii) The modification module includes enzymes capable of catalyzing chemical modifications in the structure of acyl-CoA intermediates. And iii) the removal module includes enzymes able to remove the CoA and release an organic molecule different from the one activated in the upstream. Based on these reactions, we constructed a reaction network that summarizes the most relevant CoA-dependent biosynthetic pathways reported until today. From the information available in the articles, we have plotted the total turnover number of CoA as a function of the product titer, observing a positive correlation between both parameters. Therefore, the success of a CoA-dependent in vitro pathway depends on its ability to regenerate CoA, but also to regenerate other cofactors such as NAD(P)H and ATP.

Region-Directed Enzyme Immobilization through Engineering Protein Surface with Histidine Clusters.

Enzyme immobilization is a key enabling technology for a myriad of industrial applications, yet immobilization science is still too empirical to reach highly active and robust heterogeneous biocatalysts through a general approach. Conventional protein immobilization methods lack control over how enzymes are oriented on solid carriers, resulting in negative conformational changes that drive enzyme deactivation. Site-selective enzyme immobilization through peptide tags and protein domains addresses the orientation issue, but this approach limits the possible orientations to the N- and C-termini of the target enzyme. In this work, we engineer the surface of two model dehydrogenases to introduce histidine clusters into flexible regions not involved in catalysis, through which immobilization is driven. By varying the position and the histidine density of the clusters, we create a small library of enzyme variants to be immobilized on different carriers functionalized with different densities of various metal chelates (Co2+, Cu2+, Ni2+, and Fe3+). We first demonstrate that His-clusters can be as efficient as the conventional His-tags in immobilizing enzymes, recovering even more activity and gaining stability against some denaturing agents. Furthermore, we find that the enzyme orientation as well as the type and density of the metal chelates affect the immobilization parameters (immobilization yield and recovered activity) and the stability of the immobilized enzymes. According to proteomic studies, His-clusters enable a different enzyme orientation as compared to His-tag. Finally, these oriented heterogeneous biocatalysts are implemented in batch reactions, demonstrating that the stability achieved by an optimized orientation translates into increased operational stability.

The Lambda Variant in Argentina: Analyzing the Evolution and Spread of SARS-CoV-2 Lineage C.37.

The second wave of COVID-19 occurred in South America in early 2021 and was mainly driven by Gamma and Lambda variants. In this study, we aimed to describe the emergence and local genomic diversity of the SARS-CoV-2 Lambda variant in Argentina, from its initial entry into the country until its detection ceased. Molecular surveillance was conducted on 9356 samples from Argentina between October 2020 and April 2022, and sequencing, phylogenetic, and phylogeographic analyses were performed. Our findings revealed that the Lambda variant was first detected in Argentina in January 2021 and steadily increased in frequency until it peaked in April 2021, with continued detection throughout the year. Phylodynamic analyses showed that at least 18 introductions of the Lambda variant into the country occurred, with nine of them having evidence of onward local transmission. The spatial--temporal reconstruction showed that Argentine clades were associated with Lambda sequences from Latin America and suggested an initial diversification in the Metropolitan Area of Buenos Aires before spreading to other regions in Argentina. Genetic analyses of genome sequences allowed us to describe the mutational patterns of the Argentine Lambda sequences and detect the emergence of rare mutations in an immunocompromised patient. Our study highlights the importance of genomic surveillance in identifying the introduction and geographical distribution of the SARS-CoV-2 Lambda variant, as well as in monitoring the emergence of mutations that could be involved in the evolutionary leaps that characterize variants of concern.

Solid-Phase Cell-Free Protein Synthesis and Its Applications in Biotechnology.

Cell-free systems for the in vitro production of proteins have revolutionized the synthetic biology field. In the last decade, this technology is gaining momentum in molecular biology, biotechnology, biomedicine and even education. Materials science has burst into the field of in vitro protein synthesis to empower the value of existing tools and expand its applications. In this sense, the combination of solid materials (normally functionalized with different biomacromolecules) together with cell-free components has made this technology more versatile and robust. In this chapter, we discuss the combination of solid materials with DNA and transcription-translation machinery to synthesize proteins within compartments, to immobilize and purify in situ the nascent protein, to transcribe and transduce DNAs immobilized on solid surfaces, and the combination of all or some of these strategies.

Expanding the Substrate Scope of Acyltransferase LovD9 for the Biosynthesis of Statin Analogues.

Invited for the cover of this issue are the groups of Gonzalo Jiménez-Osés and Fernando López-Gallego at CIC bioGUNE and CIC biomaGUNE, respectively. The image depicts the substrate scope of an engineered acyl transferases for the synthesis of statin derivatives. Read the full text of the article at 10.1002/chem.202300911.

Mechanistic studies of a lipase unveil effect of pH on hydrolysis products of small PET modules.

Biocatalysis is a key technology enabling plastic recycling. However, despite advances done in the development of plastic-degrading enzymes, the molecular mechanisms that govern their catalytic performance are poorly understood, hampering the engineering of more efficient enzyme-based technologies. In this work, we study the hydrolysis of PET-derived diesters and PET trimers catalyzed by the highly promiscuous lipase B from Candida antarctica (CALB) through QM/MM molecular dynamics simulations supported by experimental Michaelis-Menten kinetics. The computational studies reveal the role of the pH on the CALB regioselectivity toward the hydrolysis of bis-(hydroxyethyl) terephthalate (BHET). We exploit this insight to perform a pH-controlled biotransformation that selectively hydrolyzes BHET to either its corresponding diacid or monoesters using both soluble and immobilized CALB. The discoveries presented here can be exploited for the valorization of BHET resulting from the organocatalytic depolymerization of PET.

Immobilized biocatalyst engineering: Biocatalytic tool to obtain attractive enzymes for industry.

Biocatalysis can improve current bioprocesses by identifying or improving enzymes that withstand harsh and unnatural operating conditions. Immobilized Biocatalyst Engineering (IBE) is a novel strategy integrating protein engineering and enzyme immobilization as a single workflow. Using IBE, it is possible to obtain immobilized biocatalysts whose soluble performance would not be selected. In this work, Bacillus subtilis lipase A (BSLA) variants obtained through IBE were characterized as soluble and immobilized biocatalysts, and how the interactions with the support affect their structure and catalytic performance were analyzed using intrinsic protein fluorescence. Variant P5G3 (Asn89Asp, Gln121Arg) showed a 2.6-fold increased residual activity after incubation at 76 °C compared to immobilized wild-type (wt) BSLA. On the other hand, variant P6C2 (Val149Ile) showed 4.4 times higher activity after incubation in 75 % isopropyl alcohol (36 °C) compared to Wt_BSLA. Furthermore, we studied the advancement of the IBE platform by performing synthesis and immobilizing the BSLA variants using a cell-free protein synthesis (CFPS) approach. The observed differences in immobilization performance, high temperature, and solvent resistance between the in vivo-produced variants and Wt_BSLA were confirmed for the in vitro synthesized enzymes. These results open the door for designing strategies integrating IBE and CFPS to generate and screen improved immobilized enzymes from genetic diversity libraries. Furthermore, it was confirmed that IBE is a platform that can be used to obtain improved biocatalysts, especially those with an unremarkable performance as soluble biocatalysts, which wouldn't be selected for immobilization and further development for specific applications.

Engineered repeat proteins as scaffolds to assemble multi-enzyme systems for efficient cell-free biosynthesis.

Multi-enzymatic cascades with enzymes arranged in close-proximity through a protein scaffold can trigger a substrate channeling effect, allowing for efficient cofactor reuse with industrial potential. However, precise nanometric organization of enzymes challenges the design of scaffolds. In this study, we create a nanometrically organized multi-enzymatic system exploiting engineered Tetrapeptide Repeat Affinity Proteins (TRAPs) as scaffolding for biocatalysis. We genetically fuse TRAP domains and program them to selectively and orthogonally recognize peptide-tags fused to enzymes, which upon binding form spatially organized metabolomes. In addition, the scaffold encodes binding sites to selectively and reversibly sequester reaction intermediates like cofactors via electrostatic interactions, increasing their local concentration and, consequently, the catalytic efficiency. This concept is demonstrated for the biosynthesis of amino acids and amines using up to three enzymes. Scaffolded multi-enzyme systems present up to 5-fold higher specific productivity than the non-scaffolded ones. In-depth analysis suggests that channeling of NADH cofactor between the assembled enzymes enhances the overall cascade throughput and the product yield. Moreover, we immobilize this biomolecular scaffold on solid supports, creating reusable heterogeneous multi-functional biocatalysts for consecutive operational batch cycles. Our results demonstrate the potential of TRAP-scaffolding systems as spatial-organizing tools to increase the efficiency of cell-free biosynthetic pathways.

Expanding the Substrate Scope of Acyltransferase LovD9 for the Biosynthesis of Statin Analogues.

This study identifies new acyl donors for manufacturing statin analogues through the acylation of monacolin J acid by the laboratory evolved acyltransferase LovD9. Vinyl and p-nitrophenyl esters have emerged as alternate substrates for LovD9-catalyzed acylation. While vinyl esters can reach product yields as high as the ones obtained by α-dimethyl butyryl-S-methyl-3-mercaptopropionate (DMB-SMMP), the thioester for which LovD9 was evolved, p-nitrophenyl esters display a reactivity even higher than DMB-SMMP for the first acylation step yet the acylation product yield is lower. The reaction mechanisms were elucidated through quantum mechanics (QM) calculations.

Large Magnetoresistance of Isolated Domain Walls in La2/3 Sr1/3 MnO3 Nanowires.

Generation, manipulation, and sensing of magnetic domain walls are cornerstones in the design of efficient spintronic devices. Half-metals are amenable for this purpose as large low field magnetoresistance signals can be expected from spin accumulation at spin textures. Among half metals, La1- x Srx MnO3 (LSMO) manganites are considered as promising candidates for their robust half-metallic ground state, Curie temperature above room temperature (Tc = 360 K, for x = 1/3), and chemical stability. Yet domain wall magnetoresistance is poorly understood, with large discrepancies in the reported values and conflicting interpretation of experimental data due to the entanglement of various source of magnetoresistance, namely, spin accumulation, anisotropic magnetoresistance, and colossal magnetoresistance. In this work, the domain wall magnetoresistance is measured in LSMO cross-shape nanowires with single-domain walls nucleated across the current path. Magnetoresistance values above 10% are found to be originating at the spin accumulation caused by the mistracking effect of the spin texture of the domain wall by the conduction electrons. Fundamentally, this result shows the importance on non-adiabatic processes at spin textures despite the strong Hund coupling to the localized t2g electrons of the manganite. These large magnetoresistance values are high enough for encoding and reading magnetic bits in future oxide spintronic sensors.