Kirigami triboelectric spider fibroin microneedle patches for comprehensive joint management.

Joint injuries are among the leading causes of disability. Present concentrations were focused on oral drugs and surgical treatment, which brings severe and unnecessary difficulties for patients. Smart patches with high flexibility and intelligent drug control-release capacity are greatly desirable for efficient joint management. Herein, we present a novel kirigami spider fibroin-based microneedle triboelectric nanogenerator (KSM-TENG) patch with distinctive features for comprehensive joint management. The microneedle patch consists of two parts: the superfine tips and the flexible backing base, which endow it with great mechanical strength to penetrate the skin and enough flexibility to fit different bends. Besides, the spider fibroin-based MNs served as a positive triboelectric material to generate electrical stimulation, thereby forcing drug release from needles within 720 min. Especially, kirigami structures could also transform the flat patch into three dimensions, which could impart the patch with flexible properties to accommodate the complicated processes produced by joint motion. Benefiting from these traits, the KSM-TENG patch presents excellent performance in inhibiting the inflammatory response and promoting wound healing in mice models. The results indicated that the mice possessed only 2% wound area and the paw thickness was reduced from 10.5 mm to 6.2 mm after treatment with the KSM-TENG patch, which further demonstrates the therapeutic effect of joints in vivo. Thus, it is believed that the proposed novel KSM-TENG patch is valuable in the field of comprehensive treatments and personalized clinical applications.

[Primary Structure Characterization and Biosynthesis of Spider Silk Proteins for Multifunctional Biomaterials].

Spider silk is a natural fiber known as "biosteel" with the strongest composite performance, such as high tensile strength and toughness. It is also equipped with excellent biocompatibility and shape memory ability, thus shows great potential in many fields such as biomedicine and tissue engineering. Spider silk is composed of macromolecular spidroin with rich structural diversity. The characteristics of the primary structure of natural spidroin, such as the high repeatability of amino acids in the core repetitive region, the high content of specific amino acids, the large molecular weight, and the high GC content of the spidroin gene, have brought great difficulties in heterologous expression. This review discusses focuses on the relationship between the featured motifs of the microcrystalline region in the repetitive unit of spidroin and its structure, as well as the spinning performance and the heterologous expression. The optimization design for the sequence of spidroin combined with heterologous expression strategy has greatly promoted the development of the biosynthesis of spider silk proteins. This review may facilitate the rational design and efficient synthesis of recombinant spidroin.

Efficient Biosynthetic Fabrication of Spidroins with High Spinning Performance.

The unique 3D structure of spider silk protein (spidroin) determines the excellent mechanical properties of spidroin fiber, but the difficulty of heterologous expression and poor spinning performance of recombinant spider silk protein limit its application. A high-yield low-molecular-weight biomimetic spidroin (Amy-6rep) is obtained by sequence modification, and its excellent spinning performance is verified by electrospinning it for use as a nanogenerator. Amy-6rep increases the highly fibrogenic microcrystalline region in the core repeat region of natural spidroin with limited sequence length and replaces the polyalanine sequence with an amyloid polypeptide through structural similarity. Due to sequence modification, the expression of Amy-6rep increased by ≈200%, and the self-assembly performance of Amy-6rep significantly increased. After electrospinning with Amy-6rep, the nanofibers exhibit good tribopower generation capacity. In this paper, a biomimetic spidroin sequence design with high yield and good spinning performance is reported, and a strategy for electrospinning to produce an artificial nanogenerator is explored.

In situ formed depot of elastin-like polypeptide-hirudin fusion protein for long-acting antithrombotic therapy.

Thrombosis, induced by abnormal coagulation or fibrinolytic systems, is the most common pathology associated with many life-threatening cardio-cerebrovascular diseases. However, first-line anticoagulant drugs suffer from rapid drug elimination and risk of hemorrhagic complications. Here, we developed an in situ formed depot of elastin-like polypeptide (ELP)-hirudin fusion protein with a prodrug-like feature for long-term antithrombotic therapy. Highly secretory expression of the fusion protein was achieved with the assistance of the Ffu312 tag. Integration of hirudin, ELP, and responsive moiety can customize fusion proteins with properties of adjustable in vivo retention and controllable recovery of drug bioactivity. After subcutaneous injection, the fusion protein can form a reservoir through temperature-induced coacervation of ELP and slowly diffuse into the blood circulation. The biological activity of hirudin is shielded due to the N-terminal modification, while the activated key proteases upon thrombus occurrence trigger the cleavage of fusion protein together with the release of hirudin, which has antithrombotic activity to counteract thrombosis. We substantiated that the optimized fusion protein produced long-term antithrombotic effects without the risk of bleeding in multiple animal thrombosis models.

Engineering Substrate Promiscuity of Nucleoside Phosphorylase Via an Insertions-Deletions Strategy.

Nucleoside phosphorylases (NPs) are the key enzymes in the nucleoside metabolism pathway and are widely employed for the synthesis of nucleoside analogs, which are difficult to access via conventional synthetic methods. NPs are generally classified as purine nucleoside phosphorylase (PNP) and pyrimidine or uridine nucleoside phosphorylase (PyNP/UP), based on their substrate preference. Here, based on the evolutionary information on the NP-I family, we adopted an insertions-deletions (InDels) strategy to engineer the substrate promiscuity of nucleoside phosphorylase AmPNPΔS2V102 K, which exhibits both PNP and UP activities from a trimeric PNP (AmPNP) of Aneurinibacillus migulanus. Furthermore, the AmPNPΔS2V102 K exerted phosphorylation activities toward arabinose nucleoside, fluorosyl nucleoside, and dideoxyribose, thereby broadening the unnatural-ribose nucleoside substrate spectrum of AmPNP. Finally, six purine nucleoside analogues were successfully synthesized, using the engineered AmPNPΔS2V102 K instead of the traditional "two-enzymes PNP/UP" approach. These results provide deep insights into the catalytic mechanisms of the PNP and demonstrate the benefits of using the InDels strategy to achieve substrate promiscuity in an enzyme, as well as broadening the substrate spectrum of the enzyme.

Immobilization and Characterization of a Processive Endoglucanase EG5C-1 from Bacillus subtilis on Melamine-Glutaraldehyde Dendrimer-Functionalized Magnetic Nanoparticles.

Exploring an appropriate immobilization approach to enhance catalytic activity and reusability of cellulase is of great importance to reduce the price of enzymes and promote the industrialization of cellulose-derived biochemicals. In this study, Fe3O4 magnetic nanoparticles (MNPs) were functionalized with meso-2,3-dimercaptosuccinic acid to introduce carboxyl groups on the surface (DMNPs). Then, melamine-glutaraldehyde dendrimer-like polymers were grafted on DMNPs to increase protein binding sites for the immobilization of processive endoglucanase EG5C-1. Moreover, this dendrimer-like structure was beneficial to protect the conformation of EG5C-1 and facilitate the interaction between substrate and active center. The loading capacity of the functionalized copolymers (MG-DMNPs) for EG5C-1 was about 195 mg/g, where more than 90% of the activity was recovered. Immobilized EG5C-1 exhibited improved thermal stability and increased tolerability over a broad pH range compared with the free one. Additionally, MG-DMNP/EG5C-1 biocomposite maintained approximately 80% of its initial hydrolysis productivity after five cycles of usage using filter paper as the substrate. Our results provided a promising approach for the functionalization of MNPs, enabling the immobilization of cellulases with a high loading capacity and excellent activity recovery.

Discovery and Mechanistic Understanding of a Lipase from Rhizorhabdus dicambivorans for Efficient Ester Aminolysis in Aromatic Amines.

The formation of amide bonds via aminolysis of esters by lipases generates a diverse range of amide frameworks in biosynthetic chemistry. Few lipases have satisfactory activity towards bulky aromatic amines despite numerous attempts to improve the efficiency of this transformation. Here, we report the discovery of a new intracellular lipase (Ndbn) with a broad substrate scope. Ndbn turns over a range of esters and aromatic amines in the presence of water (2 %; v/v), producing a high yield of multiple valuable amides. Remarkably, a higher conversion rate was observed for the synthesis of amides from substrates with aromatic amine rather than aliphatic amines. Molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) studies showcase the mechanism for the preference for aromatic amines, including a more suitable orientation, shorter catalytic distances in the active site pocket and a lower reaction barrier for aromatic than for aliphatic amines. This unique lipase is thus a promising biocatalyst for the efficient synthesis of aromatic amides.

Evolving the 3-O/6-O regiospecificity of a microbial glycosyltransferase for efficient production of ginsenoside Rh1 and unnatural ginsenoside.

Glycosyltransferase is a popular and promising enzyme to produce high-value-added natural products. Rare ginsenoside Rh1 and unnatural ginsenoside 3ß-O-Glc-PPT are promising candidates for drugs. Herein, the microbial glycosyltransferase UGTBL1 was able to catalyze the 20(S)-protopanaxatriol (PPT) 3-O/6-O-glycosylation with poor 6-O-regiospecificity. A structure-guided strategy of mutations involving loop engineering, PSPG motif evolution, and access tunnel engineering was proposed to engineer the enzyme UGTBL1. The variant I62R/M320H/P321Y/N170A from protein engineering achieved a great improvement in 6-O regioselectivity which increased from 10.98 % (WT) to 96.26 % and a booming conversion of 95.57 % for ginsenoside Rh1. A single mutant M320W showed an improved 3-O regioselectivity of 84.83 % and an increased conversion of 98.13 % for the 3ß-O-glc-PPT product. Molecular docking and molecular dynamics (MD) simulations were performed to elucidate the possible molecular basis of the regiospecificity and catalytic activity. The unprecedented high titer of ginsenoside Rh1 (20.48 g/L) and 3ß-O-Glc-PPT (18.04 g/L) was attained with high regioselectivity and yields using fed-batch cascade reactions from UDPG recycle, which was the highest yield reported to date. This work could provide an efficient and cost-effective approach to the valuable ginsenosides.

Structure-guided engineering of branched-chain α-keto acid decarboxylase for improved 1,2,4-butanetriol production by in vitro synthetic enzymatic biosystem.

Efficient synthetic routes for biomanufacturing chemicals often require the overcoming of pathway bottlenecks by tailoring enzymes to improve the catalytic efficiency or even implement non-native activities. 1,2,4-butanetriol (BTO), a valuable commodity chemical, is currently biosynthesized from D-xylose via a four-enzyme reaction cascade, with the ThDP-dependent α-keto acid decarboxylase (KdcA) identified as the potential bottleneck. Here, to further enhance the catalytic activity of KdcA toward the non-native substrate α-keto-3-deoxy-xylonate (KDX), in silico screening and structure-guided evolution were performed. The best mutants, S286L/G402P and V461K, exhibited a 1.8- and 2.5-fold higher enzymatic activity in the conversion of KDX to 3,4-dihydroxybutanal when compared to KdcA, respectively. MD simulations revealed that the two sets of mutations reshaped the substrate binding pocket, thereby increasing the binding affinity for KDX and promoting interactions between KDX and cofactor ThDP. Then, when the V461K mutant instead of wild type KdcA was integrated into the enzyme cascade, a 1.9-fold increase in BTO titer was observed. After optimization of the reaction conditions, the enzyme cocktail contained V461K converted 60 g/L D-xylose to 22.1 g/L BTO with a yield of 52.1 %. This work illustrated that protein engineering is a powerful tool for modifying the output of metabolic pathway.

Combination of On-Line and Off-Line Two-Dimensional Liquid Chromatography-Mass Spectrometry for Comprehensive Characterization of mAb Charge Variants and Precise Instructions for Rapid Process Development.

Charge variants, as an important quality attribute of mAbs, must be comprehensively characterized and monitored during development. However, due to their complex structure, the characterization of charge variants is challenging, labor-intensive, and time-consuming when using traditional approaches. This work combines on-line and off-line 2D-LC-MS to comprehensively characterize mAb charge variants and quickly offer precise instructions for process development. Six charge variant peaks of mAb 1 were identified using the developed platform. Off-line 2D-LC-MS analysis at the peptide level showed that the acidic peak P1 and the basic peaks P4 and P5 were caused by the deamidation of asparagine, the oxidation of methionine, and incomplete C-terminal K loss, respectively. On-line 2D-LC-MS at the intact protein level was used to identify the root causes, and it was found that the acidic peak P2 and the basic peak P6 were due to the glutathionylation of cysteine and succinimidation of aspartic acid, respectively, which were not found in off-line 2D-LC-MS because of the loss occurring during pre-treatment. These results suggest that process development could focus on cell culture for adjustment of glutathionylation. In this paper, we propose the concept of precision process development based on on-line 2D-LC-MS, which could quickly offer useful data with only 0.6 mg mAb within 6 h for precise instructions for process development. Overall, the combination of on-line and off-line 2D-LC-MS can characterize mAb charge variants more comprehensively, precisely, and quickly than other approaches. This is a very effective platform with routine operations that provides precise instructions for process development within hours, and will help to accelerate the development of innovative therapeutics.

Identification and Characterization of a Novel α-L-Fucosidase from Enterococcus gallinarum and Its Application for Production of 2'-Fucosyllactose.

2'-fucosyllactose (2'FL) is an important nutrient in human milk that stimulates beneficial microbiota and prevents infection. α-L-fucosidase is a promising component for 2'FL synthesis. In this study, a soil-oriented α-L-fucosidase-producing strain from Enterococcus gallinarum ZS1 was isolated. Escherichia coli was employed as a host for cloning and expressing the α-L-fucosidase gene (entfuc). The EntFuc was predicted as a member of the GH29 family with a molecular mass of 58 kDa. The optimal pH and temperature for the activity of EntFuc were pH 7.0 and 30 °C, respectively. The enzyme exhibited a strictly specific activity for 4-Nitrophenyl-α-L-fucopyranoside (pNP-Fuc) and had a negligible effect on hydrolyzing 2'FL. EntFuc could catalyze the synthesis of 2'FL via transfucosylation action from pNP-Fuc and lactose. The yield of 2'FL reached 35% under optimal conditions. This study indicated that EntFuc with a high conversion rate is a promising enzyme source for the biosynthesis of 2'FL.

Advanced Silk Fibroin Biomaterials-Based Microneedles for Healthcare.

Microneedles are a promising transdermal drug delivery system that has the advantages of minimal invasiveness, painlessness, and on-demand drug delivery compared with commonly used medical techniques. Natural resources are developed as next-generation materials for microneedles with varying degrees of success. Among them, silk fibroin is a natural polymer obtained from silkworms with good biocompatibility, high hardness, and controllable biodegradability. These properties provide many opportunities for integrating silk fibroin with implantable microneedle systems. In this review, the research progress of silk fibroin microneedles in recent years is summarized, including their materials, processing technology, detection, drug release methods, and applications. Besides, the research and development of silk fibroin in a multidimensional way are analyzed. Finally, it is expected that silk fibroin microneedles will have excellent development prospects in various fields.

Intestine-inspired wrinkled MXene microneedle dressings for smart wound management.

Composite MXene-based materials are prone to crack propagation, thus limiting their tensile properties. Numerous efforts have been devoted to removing material constraints and fabricating unitary MXene elastic films. Here, for the first time, inspired by the intestinal wrinkles and villi structure, we presented a ductile, biologically friendly, and highly conductive MXene-based microneedle (MMN) dressing composed of stacked MXene film and superfine microneedle arrays through a simple stretching and laser engraving strategy for wound healing. By utilizing photothermal responsive MXene, periodic porous structures, and a temperature-responsive polymer to construct the MMN dressing, the system can act as an effective route for facilitating controllable drug delivery controlled by near-infrared (NIR) irradiation. In addition, superior conductivity imparts them with the capacity to realize continuous and steady monitoring of motion sensing. The practical performance further demonstrated that the versatile MMN dressing showed obvious therapeutic efficacy in vivo animal wound models. Thus, it is believed that MMN dressings with biomimetic structures, controllable drug release, and conductive pathways will open a new chapter for wound management and widen other practical applications in biomedical fields, such as artificial tendons and soft robotics. STATEMENT OF SIGNIFICANCE: MXene-based materials have been demonstrated as critical tools in advancing our understanding of wound healing. However, the rapid crack propagation is a constraint on their tensile properties. Here, inspired by the intestinal wrinkles and villi structure, a single-step method has also been discussed to present a MXene-based microneedle dressing composed of unitary MXene elastic film and superfine microneedle arrays. At the same time, the dressing with biomimetic structures, controllable drug release, and conductive pathways has prospects in intelligent wound management and varieties of related biomedical fields.

Target-catalyzed self-assembled spherical G-quadruplex/hemin DNAzymes for highly sensitive colorimetric detection of microRNA in serum.

The accurate and visual detection of circulating microRNA (miRNA) has attracted increasing interest due to its pivotal role in clinical disease diagnosis. Taking advantages of nucleic acid isothermal amplification and enzyme-catalyzed chromogenic reaction, here, a colorimetric sensing strategy was proposed for sensitive miRNA analysis. When the target miRNA was present, local catalytic hairpin assembly (CHA) would be triggered and proceed continuously to form dozens of double-stranded oligonucleotides with G-rich sticky ends on the gold nanoparticle, which could self-assemble into a spherical G-quadruplex (GQ)/hemin DNAzyme by binding with hemin and potassium ions. As a horseradish peroxidase-mimic, GQ/hemin DNAzyme could catalyze the redox reaction and color change of the substrates. Taking miRNA-21 as an example, the developed method exhibited satisfactory specificity as well as high sensitivity with a detection limit of 90.3 fM. Furthermore, the sensing platform has been successfully employed to detect miRNA-21 in spiked serum, providing a promising tool for early diagnosis of cancers.

[Engineering and process management-based design of Comprehensive Biotechnology Experiment course].

Based on the demand of enterprise talents and the characteristics of manufacturing process management in biotechnology, in order to make the students acquire the ability to solve complex engineering problems in the production process, we developed a "Comprehensive Biotechnology Experiment" course, where two-step enzymatic production of l-aspartate and l-alanine were the key processes. In this course, we drew lessons from the site management of the production enterprise, performed the experimental operation mode of four shifts and three operations. The content of this course includes principles, methods and experimental techniques of several core curricula and the site management mode of enterprises. As to the evaluation, the summary of the experimental staff's handover records and the content of teamwork were examined and scored. Through teaching practice and continuous improvement, we developed a complete experimental teaching process and assessment mechanism. Overall, the Comprehensive Biotechnology Experiment course achieved good teaching effect, which may serve as a reference to promote the development of experimental teaching of biotechnology.

Toward Efficient Wound Management: Bioinspired Microfluidic and Microneedle Patch.

Microneedle (MN) patches hold demonstrated prospects in intelligent wound management. Herein, inspired by the highly folded structure of insect wings, a three-dimensional (3D) origami MN patch with superfine miniature needle structures, microfluidic channels, and multiple functions was reported to detect biomarkers, release drugs controllably and monitor motions to facilitate wound healing. By simply replicating the pre-stretched silicone rubber (Ecoflex) molds patterned by a laser engraving machine, the superfine structure MN patch with microfluidic channels was obtained from the restored molds. The bioinspired origami structure endows the MN patch with a high degree of functional integration, including microfluidic channels and electrocircuits. The microfluidic channels combined with the pH value and glucose concentration indicators enable the patch with the capability of biomarker sensing detection. Porous structures, a temperature-responsive hydrogel, and a photothermal-sensitive agent are utilized to form a controllable drug release system on the MN patch. Meanwhile, MXene electrocircuits were printed on the MN patch for motion sensing. In addition, the ability of the MN patch to accelerate wound healing was demonstrated by a mouse model experiment with full-thickness skin wounds. These results indicate that the multifunctional 3D origami MN patch is a valuable intelligent strategy for wound management.

Patterned Duplex Fabric Based on Genetically Modified Spidroin for Smart Wound Management.

The treatment of diabetic wounds remains a great challenge for the medical community. Here, a smart patterned DNA double helix (duplex)-like fabric based on genetically modified spider silk protein (PDF-S) which is inspired by soft plant tendrils, is proposed for diabetic wound treatment. Benefiting from spider silk protein (spidroin); PDF-S is equipped with high strength; high toughness, and excellent biocompatibility. Notably, the fabric crimped through the biomimetic DNA double-helix-like structure can effectively adapt to tensile impact and the maximum stretch rate reaches 1500%. A pattern-based microfluidic channel of PDF-S allowed wound secretion to flow spontaneously through the channel. Meanwhile; due to the optical properties of the introduced photonic crystal structure; PDF-S is equipped with fluorescence enhancement properties; enabling PDF-S to display color-sensitive behavior suitable for wound monitoring and guiding clinical treatment. In addition, to enable sensitive motion monitoring, microelectronic circuits are integrated on the surface of the PDF-S. These unique material features suggest that this study will lead to a new generation of biomimetic artificial spider silk materials for design and application in the biomedical field.

Bioinspired 3D-Printed MXene and Spidroin-Based Near-Infrared Light-Responsive Microneedle Scaffolds for Efficient Wound Management.

Biomedical dressings have been comprehensively explored for wound healing; however, the complicated manufacturing process and mono-function of the dressing remain critical challenges for further applications. Here, a versatile extrusion three-dimensional (3D) printing strategy to prepare MXene and spidroin-incorporated microneedle scaffolds with photothermal responsive and self-healing properties for promoting wound healing is proposed. Inspired by the cactus, the microneedle scaffold is composed of a top porous scaffold, and microneedles whose inverse opal (IO) photonic crystal (PC) structure and the ample space between the scaffold gaps endow the microneedle scaffold with high drug-carrying capacity. Furthermore, the excellent electrical and photothermal properties of MXene allow the microneedle scaffold to perform sensitive wound movement monitoring and controlled drug release under near-infrared irradiation. Moreover, the extensive hydrogen bonding and Schiff base between the spidroin, polyurethane (PU), and aloe vera gel (avGel) molecules conferred high self-healing and mechanical performance to the microneedle scaffold. In vivo experiments with rat models of wounds have shown that drug-laden microneedle scaffolds under near-infrared (NIR) light can promote the recovery of full-skin wounds. These unique characteristics suggest that 3D-printed multifunctional microneedle scaffolds show great potential for applications in facilitating wound healing and will find widespread applications in wound management.

Structural design strategies of microneedle-based vaccines for transdermal immunity augmentation.

As microneedle-based vaccines possess advantages of high compliance, moderate invasiveness and convenience that are highly relevant to their unique design, they are becoming an indispensable piece of the puzzle in the field of medical applications. By selecting appropriate materials and methods convenient for precise control over the structure and morphology, MN-based vaccines with strong mechanical properties and variable forms can be fabricated, and specific biomolecules can be used for monitoring or augmenting human immunity. The structural design strategies of MN-based vaccines are highlighted in this review, following a brief discussion of the mechanism of skin immunity and the classification and fabrication approaches of MNs. The biomedical applications of MN-based vaccines, including sampling from interstitial fluid and therapy in infectious diseases and cancers, have also been demonstrated. Finally, the central challenges in this field and opportunities for future developments are also deliberated.