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Physical unclonable functions (PUFs) are emerging as a cutting-edge technology for enhancing information security by providing robust security authentication and non-reproducible cryptographic keys. Incorporating renewable and biocompatible materials into PUFs ensures safety for handling, compatibility with biological systems, and reduced environmental impact. However, existing PUF platforms struggle to balance high encoding capacity, diversified encryption signatures, and versatile functionalities with sustainability and biocompatibility. Here, all-biomaterial-based unclonable anti-counterfeiting labels featuring multi-mode encoding, multi-level cryptographic keys, and multiple authentication operations are developed by imprinting biomimetic-grown calcites on versatile silk protein films. In this label, the inherent non-clonability comes from the randomized characteristics of calcites, mediated by silk protein during crystal growth. The successful embedding of photoluminescent molecules into calcite lattices, assisted by silk protein, allows the resulting platform to utilize fluorescence patterns alongside birefringence for high-capacity encoding. This design facilitates easy and rapid authentication through Hamming distance and convolutional neural networks using standard cameras and portable microscopes. Moreover, angle-dependent polarization patterns enable multi-level key generation, while multi-spectral fluorescence signals offer multi-channel keys. The developed anti-counterfeiting labels combine biodegradability, green manufacture, easy authentication, high-level complexity, low cost, robustness, patternability, and versatility, offering a practical and high-security solution to combat counterfeiting across various applications., (© 2024 Wiley‐VCH GmbH.)

The management of neurological disorders is profoundly complicated by spinal cord injury (SCI), which leads to the impairment of motor and sensory functions. A major challenge in the treatment of SCI is the formation of a dysfunctional pathological microenvironment characterized by an excessive inflammatory response, deposition of inhibitory molecules, glial scarring, and vascular dysfunction. A thorough understanding of the pathological and physiological changes following SCI is essential to elucidate the mechanisms underlying functional recovery and to develop effective therapeutic interventions. Recent research indicates that the adverse microenvironment associated with SCI can be modified through the implantation of functional biomaterials at the injury site, thereby facilitating axonal regeneration, myelin repair, and functional recovery. Silk fibroin, in particular, has demonstrated remarkable efficacy in SCI reconstruction due to its superior biocompatibility, biodegradability, and tunable mechanical properties. This review provides an overview of the pathological microenvironmental dysfunctions following SCI and explores the potential advantages of silk fibroin in enhancing axonal regeneration and neural circuit formation in SCI repair. The benefits and challenges associated with silk fibroin and its derivatives in facilitating effective SCI repair are examined. This review aims to offer significant insights into the application of silk-based biomaterials for SCI treatment., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nanopatterning on biomaterials has attracted significant attention as it can lead to the development of biomedical devices capable of performing diagnostic and therapeutic functions while being biocompatible. Among various nanopatterning techniques, electron-beam lithography (EBL) enables precise and versatile nanopatterning in desired shapes. Various biomaterials are successfully nanopatterned as bioresists by using EBL. However, the use of high-energy electron beams (e-beams) for high-resolutive patterning has incorporated functional materials and has caused adverse effects on biomaterials. Moreover, the scattering of electrons not absorbed by the bioresist leads to proximity effects, thus deteriorating pattern quality. Herein, EBL-based nanopatterning is reported by inducing molecular degradation of amorphous silk fibroin, followed by selectively inducing secondary structures. High-resolution EBL nanopatterning is achievable, even at low-energy e-beam (5 keV) and low doses, as it minimizes the proximity effect and enables precise 2.5D nanopatterning via grayscale lithography. Additionally, integrating nanophotonic structures into fluorescent material-containing silk allows for fluorescence amplification. Furthermore, this post-exposure cross-linking way indicates that the silk bioresist can maintain nanopatterned information stored in silk molecules in the amorphous state, utilizing for the secure storage of nanopatterned information as a security patch. Based on the fabrication technique, versatile biomaterial-based nanodevices for biomedical applications can be envisioned., (© 2024 Wiley‐VCH GmbH.)

Sericin-derived oligopeptides (SDOs) from yellow silk cocoons exhibit antihypertensive and hypoglycemic properties in both in vitro and in vivo studies. This study investigated the acute toxicity of SDOs as a novel food for human consumption using female ICR mice and Wistar rats, as well as the chronic toxicity test on both sexes of Wistar rats. Clinical chemistry, hematology, and histopathological studies revealed that SDOs were safe for a single dose of 2000 mg kg-1 body weight (BW) and daily oral administration of 50, 100, and 200 mg kg-1 BW for six months. The chronic toxicity study additionally measured the rats' systolic blood pressure (SBP) and blood sugar monthly as they slowly aged. In the 2nd month for male rats and the 4th month for both sexes, SDOs had a significant hypotensive effect on Wistar rats' blood pressure, lowering it from 130 mmHg to a plateau at 110-115 mmHg. In contrast, the blood pressure of the control rats exceeded 140 mmHg after five months. Nonetheless, the hypoglycemic effect was not observed. Measurements of SBP and blood glucose in aged rats during chronic toxicity tests yielded insights beyond ordinary toxicity, including the health and fitness of the lab rats, perhaps resulting in novel discoveries or areas of study that justify the sacrifice of the animals' lives.

Compared to conventional fibers, electrospun porous nanofibers with hierarchical structures often involve additional active sites, interfaces, and internal spaces which boost the performances of functional materials. Here in this study, coaxial composite cellulose acetate@silk fibroin (CA@SF) fibrous membranes are constructed through an electrostatic spinning technique combining solvent-induced phase separation. Hierarchical core-shell structures on the fibers are achieved, which significantly increases the surface area and benefits the mechanical property, flux, as well as the electroless deposition of Ag nanoparticles. The total electromagnetic shielding efficiency of the sandwiched hierarchical CA@SF@Ag composite membrane with a thickness of only 100 μm reaches up to 100 dB, surpassing around 82% beyond nonhierarchical ones. To be noticed, when post-treated by ethanol, the membrane enables an enhanced tensile strength of up to 10 MPa with a thickness of only 50 μm. Our findings pave the way to the application of electrospun fiber membranes in the field of ultrathin electromagnetic shielding films.

The efficient synthesis of silk protein is heavily reliant on the ingestion of massive nutrients during the peak growth phase in the silkworm. However, the molecular mechanism of nutritional regulation of silk protein synthesis remains unknown. In this study, we investigated the impact of nutrient deficiency on the synthesis of silk protein. Nutritional deficiency led to a reduction in silk yield, accompanied by decreased levels of silk proteins and fibroin heavy chain (FibH)-activating transcription factors SGF1 and Dimm. Furthermore, insulin enhanced the protein levels of SGF1 and Dimm, which can be attenuated by specific inhibitors of PI3K. Co-immunoprecipitation analysis showed that the nutrient pathway factor protein kinase B (Akt) could interact with SGF1 protein. Knockdown of Akt reduced the phosphorylation level of SGF1 and impedes its nuclear translocation. Further studies revealed that SGF1 was directly bound to Fkh site in the 22-43 region upstream of ATG of Dimm gene to activate its transcription. In conclusion, during the peak growth phase, nutrition promotes the massive synthesis of silk protein through the PI3K-Akt-SGF1-Dimm pathway. This study offers valuable insights into the efficient synthesis of silk proteins and establishes a theoretical foundation for improving silk yield., Competing Interests: Declaration of competing interest The authors declare they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Developing hybrid hydrogel dressings with anti-inflammatory, antioxidant, angiogenetic, and antibiofilm activities with higher bone tissue penetrability to accelerate diabetic wound healing and prevent diabetic foot osteomyelitis (DFO) is highly desirable in managing diabetic wounds. Herein, the glycopeptide teicoplanin is used for the first time as a green reductant to chemically reduce graphene oxide (GO). The resulting teicoplanin-decorated reduced graphene oxide (rGO) is incorporated into a mixture of silk proteins (SP) and crosslinked with genipin to yield a physicochemically crosslinked rGO-SP hybrid hydrogel. This hybrid hydrogel exhibits high porosity, self-healing, shear-induced thinning, increased cell proliferation and migration, and mechanical properties suitable for tissue engineering. Moreover, the hybrid hydrogel eradicates bacterial biofilms with a high penetrability index in agar and hydroxyapatite disks covered with biofilms, mimicking bone tissue. In vivo, the hybrid hydrogel accelerates the healing of noninfected wounds in a diabetic rat and infected wounds in a diabetic mouse by upregulating anti-inflammatory cytokines and downregulating matrix metalloproteinase-9, promoting M2 macrophage polarization and angiogenesis. The implantation of hybrid hydrogel into the infected site of mouse tibia improves bone regeneration. Hence, the rGO-SP hybrid hydrogel can be a promising wound dressing for treating infectious diabetic wounds, providing a further advantage in preventing DFO., (© 2024 Wiley‐VCH GmbH.)

Aqueous zinc-iodine (Zn-I 2 ) batteries hold potential for large-scale energy storage but struggle with shuttle effects of I 2 cathodes and poor reversibility of Zn anodes. Here, an interfacial gelation strategy is proposed to suppress the shuttle effects and improve the Zn reversibility simultaneously by introducing silk protein (SP) additive. The SP can migrate bidirectionally toward cathode and anode interfaces driven by the periodically switched electric field direction during charging/discharging. For I 2 cathodes, the interaction between SP and polyiodides forms gelatinous precipitate to avoid the polyiodide dissolution, evidenced by excellent electrochemical performance, including high specific capacity and Coulombic efficiency (CE) (215 mAh g -1 and 99.5% at 1 C), excellent rate performance (≈170 mAh g -1 at 50 C), and extended durability (6000 cycles at 10 C). For Zn anodes, gelatinous SP serves as protective layer to boost the Zn reversibility (99.7% average CE at 2 mA cm -2 ) and suppress dendrites. Consequently, a 500 mAh Zn-I 2 pouch cell with high-loading cathode (37.5 mg iodine cm -2 ) and high-utilization Zn anode (20%) achieves remarkable energy density (80 Wh kg -1 ) and long-term durability (>1000 cycles). These findings underscore the simultaneous modulation of both cathode and anode and demonstrate the potential for practical applications of Zn-I 2 batteries., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)

Creating photonic crystals that can integrate and switch between multiple structural color images will greatly advance their utility in dynamic information transformation, high-capacity storage, and advanced encryption, but has proven to be highly challenging. Here, it is reported that by programmably integrating newly developed 1D quasi-periodic folding structures into a 3D photonic crystal, the generated photonic superstructure exhibits distinctive optical effects that combine independently manipulatable specular and anisotropic diffuse reflections within a versatile protein-based platform, thus creating different optical channels for structural color imaging. The polymorphic transition of the protein format allows for the facile modulation of both folding patterns and photonic lattices and, therefore, the superstructure's spectral response within each channel. The capacity to manipulate the structural assembly of the superstructure enables the programmable encoding of multiple independent patterns into a single system, which can be decoded by the simple adjustment of lighting directions. The multifunctional utility of the photonic platform is demonstrated in information processing, showcasing its ability to achieve multimode transformation of information codes, multi-code high-capacity storage, and high-level numerical information encryption. The present strategy opens new pathways for achieving multichannel transformable imaging, thereby facilitating the development of emerging information conversion, storage, and encryption media using photonic crystals., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Polyurethane/silk protein-bismuth halide oxide composite films were fabricated using a blending-wet phase transformationin situsynthesis method. The crystal structure, micromorphology, and optical properties were conducted using XRD, SEM, and UV-Vis DRS characterize techniques. The results indicated that loaded silk protein enhanced the hydrophilicity and pore structure of the polyurethane composite films. The active species BiOX were observed to grow as nanosheets with high dispersion on the internal skeleton and silk protein surface of the polyurethane-silk protein film. The photocatalytic efficiency of BiOX/PU-SF composite films was assessed through the degradation of Rhodamine B under visible light irradiation. Among the tested films, the BiOBr/PU-SF composite exhibited the highest removal rate of RhB at 98.9%, surpassing the removal rates of 93.7% for the BiOCl/PU-SF composite and 85.6% for the BiOI/PU-SF composite. Furthermore, an active species capture test indicated that superoxide radical (•O 2 - ) and hole (h + ) species played a predominant role in the photodegradation process.

The improper and inadequate treatment of industrial, agricultural, and household wastewater exerts substantial pressure on the existing ecosystem and poses a serious threat to the health of both humans and animals. To address these issues, different types of materials have been employed to eradicate detrimental pollutants from wastewater and facilitate the reuse of water resources. Nevertheless, owing to the challenges associated with the degradation of these traditional materials post-use and their incompatibility with the environment, natural biopolymers have garnered considerable interest. Silk protein, as a biomacromolecule, exhibits advantageous characteristics including environmental friendliness, low carbon emissions, biodegradability, sustainability, and biocompatibility. Considering recent research findings, this comprehensive review outlines the structure and properties of silk proteins and offers a detailed overview of the manufacturing techniques employed in the production of silk-based materials (SBMs) spanning different forms. Furthermore, it conducts an in-depth analysis of the state-of-the-art SBMs for water treatment purposes, encompassing adsorption, catalysis, water disinfection, desalination, and biosensing. The review highlights the potential of SBMs in addressing the challenges of wastewater treatment and provides valuable insights into prospective avenues for further research., (© 2024 Wiley-VCH GmbH.)

In transdermal drug delivery system (TDDS) patches, achieving prolonged adhesion, high drug loading, and rapid drug release simultaneously presented a significant challenge. In this study, a PHT-SP-Cu 2+ adhesive was synthesized using polyethylene glycol (PEG), hexamethylene diisocyanate (HDI), trimethylolpropane (TMP), and silk protein (SP) as functional monomers which were combined with Cu 2+ to improve the adhesion, drug loading, and drug release of the patch. The structure of the adhesion chains and the formation of Cu 2+ -p-π conjugated network in PHT-SP-Cu 2+ were characterized and elucidated using different characterization methods including FT-IR, 13 C NMR, XPS, SEM imaging and thermodynamic evaluation. The formulation of pressure-sensitive adhesive (PSA) was optimized through comprehensive research on adhesion, mechanics, rheology, and surface energy. The formulation of 3 wt.% SP and 3 wt.% Cu 2+ provided superior adhesion properties compared to commercial standards. Subsequently, the peel strength of PHT-SP-Cu 2+ was 7.6 times higher than that of the commercially available adhesive DURO-TAK® 87-4098 in the porcine skin peel test. The adhesion test on human skin confirmed that PHT-SP-Cu 2+ could adhere to the human body for more than six days. Moreover, the drug loading, in vitro release test and skin permeation test were investigated using ketoprofen as a model drug, and the results showed that PHT-SP-Cu 2+ had the efficacy of improving drug compatibility, promoting drug release and enhancing skin permeation as a TDDS. Among them, the drug loading of PHT-SP-Cu 2+ was increased by 6.25-fold compared with PHT, and in the in vivo pharmacokinetic analysis, the AUC was similarly increased by 19.22-fold. The mechanism of α-helix facilitated drug release was demonstrated by Flori-Hawkins interaction parameters, molecular dynamics simulations and FT-IR. Biosafety evaluations highlighted the superior skin cytocompatibility and safety of PHT-SP-Cu 2+ for transdermal applications. These results would contribute to the development of TDDS patch adhesives with outstanding adhesion, drug loading and release efficiency. STATEMENT OF SIGNIFICANCE: A new adhesive, PHT-SP-Cu 2+ , was created for transdermal drug delivery patches. Polyethylene glycol, hexamethylene diisocyanate, trimethylolpropane, silk protein, and Cu 2+ were used in synthesis. Characterization techniques confirmed the structure and Cu 2+ -p-π conjugated networks. Optimal formulation included 3 wt.% SP and 3 wt.% Cu 2+ , exhibiting superior adhesion. PHT-SP-Cu 2+ showed 7.6 times higher peel strength than DURO-TAK® 87-4098 on porcine skin and adhered to human skin for over six days. It demonstrated a 6.25-fold increase in drug loading compared to PHT, with 19.22-fold higher AUC in vivo studies. α-helix facilitated drug release, proven by various analyses. PHT-SP-Cu 2+ showed excellent cytocompatibility and safety for transdermal applications. This study contributes to developing efficient TDDS patches., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Biology resolves design requirements toward functional materials by creating nanostructured composites, where individual components are combined to maximize the macroscale material performance. A major challenge in utilizing such design principles is the trade-off between the preservation of individual component properties and emerging composite functionalities. Here, polysaccharide pectin and silk fibroin were investigated in their composite form with pectin as a thermal-responsive ion conductor and fibroin with exceptional mechanical strength. We show that segregative phase separation occurs upon mixing, and within a limited compositional range, domains ∼50 nm in size are formed and distributed homogeneously so that decent matrix collective properties are established. The composite is characterized by slight conformational changes in the silk domains, sequestering the hydrogen-bonded β-sheets as well as the emergence of randomized pectin orientations. However, most dominant in the composite's properties is the introduction of dense domain interfaces, leading to increased hydration, surface hydrophilicity, and increased strain of the composite material. Using controlled surface charging in X-ray photoelectron spectroscopy, we further demonstrate Ca ions (Ca 2+ ) diffusion in the pectin domains, with which the fingerprints of interactions at domain interfaces are revealed. Both the thermal response and the electrical conductance were found to be strongly dependent on the degree of composite hydration. Our results provide a fundamental understanding of the role of interfacial interactions and their potential applications in the design of material properties, polysaccharide-protein composites in particular.

Juvenile hormone esterase (JHE) is the specific enzyme that degrades juvenile hormone (JH) and regulates the JH titer in insects. JH also regulates the development of the silk gland and the synthesis and secretion of silk proteins in Bombyx mori . Here, we identified nine possible JHE family members, Bmjhe1-9. Notably, Bmjhe6 is specifically expressed in the silk gland. Using semi-quantitative, quantitative real-time RT-PCR and Western blot, it was confirmed that Bmjhe6 was specifically expressed in the middle silk gland (MSG) with high levels in the anterior region of the MSG (A-MSG). The immunofluorescence localization analysis revealed that Bmjhe6 is produced within cells, secreted into the gland lumen, and co-transported with silk proteins into the anterior silk gland (ASG). In vitro hormone induction experiments demonstrated that Bmjhe6 responds to a JH analog, increasing its expression after 12-24 h, whereas 20-hydroxyecdysone inhibited it. In addition, Bmjhe6 knockdown using dsBmjhe6 injections accelerated larval development, resulting in increased larval body and silk gland weight. This induced disordered sericin genes ( Ser2 , Ser3 ) expression, and key genes in the JH synthesis pathway ( BmKr-h1 and BmMet1 ) were significantly upregulated along with the transcription factors ( SGF-1 and Sage ). These results indicate that Bmjhe6 plays an important role in silk gland growth and silk protein synthesis by modulating JH signal.

Precise patterning of metallic micro/nanostructures enables application of silk protein in biomedical devices with a seamless human-machine interface. However, high-quality, expensive equipment and facilities involved in micro/nanofabrication hinder rapid prototyping for explorative laboratory-based research. Here, we report cost-effective and high-resolution light-emitting diode-based projection lithography methods for fabricating a Cr photomask and metallic microstructures on a silk protein layer. After two-step photolithography performed using a commercial camera and microscopic objective lens, inkjet-printed patterns are successfully projected on the silk layers with 100× and 500× demagnification ratios. A lift-off process is conducted to integrate Au patterns on the lithographic-patterned resist/silk layer, and various Au microstructures with sizes <2 μm are generated. In all the processes, the silk protein exhibits a high resistance to chemicals for resist solvent, development, resist strip, and lift-off, as well as a strong adhesion to gold, along with low cytotoxicity. Dopamine sensing and transistor operating capabilities are proved by measuring the changes in the electrical signals through the Au patterns. The proposed method is a cost-effective and simple approach for rapid prototyping of silk-based biomedical devices.

It is crucial to develop a three-dimensional scaffold with tunable physical properties for the biomedical application of silk fibroin (SF). The crystallization of polymers dictates their bulk properties. The presence of two unique crystal types, silk I and silk II, provides a mechanism for controlling the properties of SF biomaterials. However, it remains challenging to manipulate silk I crystallization. In this study, we demonstrate the stability and tunability of SF scaffolds with silk I structure prepared by a freezing-annealing processing. The porous structure and mechanical properties of the scaffolds can be readily regulated by SF concentration. XRD results show that the typical peaks representing silk I do not shift when subjected to various post-treatments, such as ethanol soaking, heating, water vapor annealing, UV irradiation, and high-temperature/high-pressure, indicating the stability of silk I crystal type. Moreover, the crystallization kinetics can be regulated by changing annealing time. This physical process can regulate the transition from non-crystalline to silk I, in turn controlling the mechanical properties and degradation rate of the SF scaffolds. Our result show that this green, all-aqueous strategy provides new directions for the design of SF-based biomaterials with controllable properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The increasingly pervasive problem of counterfeiting affects both individuals and industry. In particular, public health and medical fields face threats to device authenticity and patient privacy, especially in the post-pandemic era. Physical unclonable functions (PUFs) present a modern solution using counterfeit-proof security labels to securely authenticate and identify physical objects. PUFs harness innately entropic information generators to create a unique fingerprint for an authentication protocol. This paper proposes a facile protein self-assembly process as an entropy generator for a unique biological PUF. The posited image digitization process applies a deep learning model to extract a feature vector from the self-assembly image. This is then binarized and debiased to produce a cryptographic key. The NIST SP 800-22 Statistical Test Suite was used to evaluate the randomness of the generated keys, which proved sufficiently stochastic. To facilitate deployment on physical objects, the PUF images were printed on flexible silk-fibroin-based biodegradable labels using functional protein bioinks. Images from the labels were captured using a cellphone camera and referenced against the source image for error rate comparison. The deep-learning-based biological PUF has potential as a low-cost, scalable, highly randomized strategy for anti-counterfeiting technology.

The efficient production of silkworm silk is crucial to the silk industry. Silk protein synthesis is regulated by the juvenile hormone (JH) and 20-Hydroxyecdysone (20E). Therefore, the genetic regulation of silk production is a priority. JH binding protein (JHBP) transports JH from the hemolymph to target organs and cells and protects it. In a previous study, we identified 41 genes containing a JHBP domain in the Bombyx mori genome. Only one JHBP gene, BmJHBPd2 , is highly expressed in the posterior silk gland (PSG), and its function remains unknown. In the present study, we investigated the expression levels of BmJHBPd2 and the major silk protein genes in the high-silk-producing practical strain 872 ( S872 ) and the low-silk-producing local strain Dazao . We found that BmJHBPd2 was more highly expressed in S872 than in the Dazao strain, which is consistent with the expression pattern of fibroin genes. A subcellular localization assay indicated that BmJHBPd2 is located in the cytoplasm. In vitro hormone induction experiments showed that BmJHBPd2 was upregulated by juvenile hormone analogue (JHA) treatment. BmKr-h1 upregulation was significantly inhibited by the overexpression of BmJHBPd2 (BmJHBPd2OE) at the cell level when induced by JHA. However, overexpression of BmJHBPd2 in the PSG by transgenic methods led to the inhibition of silk fibroin gene expression, resulting in a reduction in silk yield. Further investigation showed that in the transgenic BmJHBPd2OE silkworm, the key transcription factor of the JH signaling pathway, Krüppel homolog 1 (Kr-h1), was inhibited, and 20E signaling pathway genes, such as broad complex (Brc), E74A, and ultraspiracle protein (USP), were upregulated. Our results indicate that BmJHBPd2 plays an important role in the JH signaling pathway and is important for silk protein synthesis. Furthermore, our findings help to elucidate the mechanisms by which JH regulates silk protein synthesis.

Biomineralization refers to the process through which minerals nucleate in a structured manner to form specific crystal structures by the regulating of biomacromolecules. Biomineralization occurs in bones and teeth within the human body, where collagen acts as a template for the nucleation of hydroxyapatite (HA) crystals. Similar to collagen, silk proteins spun by silkworms can also serve as templates for the nucleation and growth of inorganic substances at interfaces. By enabling the binding of silk proteins to inorganic minerals, the process of biomineralization enhances the properties of silk-based materials and broadens their potential applications, rendering them highly promising for use in biomedical applications. In recent years, the development of biomineralized materials using silk proteins has garnered considerable attention in the biomedical field. This comprehensive review outlines the mechanism of biomineral formation mediated by silk proteins, as well as various biomineralization methods used to prepare silk-based biomineralized materials (SBBMs). Additionally, we discuss the physicochemical properties and biological functions of SBBMs, and their potential applications in various fields such as bioimaging, cancer therapy, antibacterial treatments, tissue engineering, and drug delivery. In conclusion, this review highlights the significant role that SBBMs can play in the biomedical field.

The current investigation aimed at bimetallic gold-silver nanoparticles (Au/Ag NPs), here called BM-GS NPs, synthesis using sericin protein as the reducing agent in an easy, cost-effective, and sustainable way. The obtained BM-GS NPs were characterized by UV-Visible spectroscopy, Transmission electron microscopy (TEM), energy dispersive X-ray analysis (EDS), atomic force microscopy (AFM), Dynamic light scattering (DLS) and Zeta potential, X-ray Powder Diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), and Thermogravimetric analysis followed by evaluation of its multitherapeutic and photocatalytic degradation potentials. The TEM analysis revealed its spherical nature and the EDS result displayed the presence of both Ag and Au elements, confirming the synthesis of BM-GS NPs. The XRD pattern verified the crystalline nature of the nanoparticles (NPs). The DLS analysis showed an average size of 86.08 d nm and the zeta potential showed a highly negative value (-26.3 mV) which specifies that the generated bimetallic NPs are stable. The BM-GS NPs exhibited positive wound healing potential (with 63.38% of wound closure rate at 25 μg/ml, as compared to 54.42% by the untreated control) with very negligible toxicity effect on the cell viability of the normal keratinocyte cells. It also demonstrated promising antioxidant properties with 65.00%, 69.23%, and 63.03% activity at 100 μg/ml concentration for ABTS (2, 2-azinobis) (3-ethylbenzothiazoline-6-sulfonic acid)), DPPH (1, 1 diphenyl-2-picrylhydrazyl) and SOD (superoxide dismutase enzyme) assays respectively, antidiabetic potential (with a significantly high α-glucosidase inhibition potential of 99.69% at 10μg/ml concentration and 62.11% of α-amylase enzyme inhibition at 100 μg/ml concentration) and moderate tyrosinase inhibitory potential (with 17.09% at 100 μg/ml concentration). Besides, it displayed reasonable antibacterial potential with the diameter of zone of inhibition ranging between 10.89 and 12.39 mm. Further, its antibacterial mode of action reveals that its effects could be due to being very smaller, the NPs could have penetrated inside the cellular membrane thereby causing rupture and damage to the interior materials leading to cellular lysis. The photocatalytic evaluation showed that synthesized BM-GS NPs have the efficiency of degrading methylene blue dye by 34.70% within 3 h of treatment. The above findings revealed the multi-therapeutic efficacy of the sericin globular protein-mediated BM-GS NPs and its potential future applications in the cosmetics and food sector and environmental contamination management industries., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jayanta Kumar Patra reports financial support was provided by National Research Foundation of Korea., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Skin wound healing in dynamic environments remains challenging. Conventional gels are not ideal dressing materials for wound healing due to difficulties in completely sealing wounds and the inability to deliver drugs quickly and precisely to the injury. To tackle these issues, we propose a multifunctional silk gel that rapidly forms strong adhesions to tissue, has excellent mechanical properties, and delivers growth factors to the wound. Specifically, the presence of Ca 2+ in the silk protein leads to a solid adhesion to the wet tissue through a chelation reaction with water-trapping behavior; the integrated chitosan fabric and CaCO 3 particles ensure enhanced mechanical strength of the silk gel for better adhesion and robustness during wound repair; and the preloaded growth factor further promoted wound healing. The results showed the adhesion and tensile breaking strength were as high as 93.79 kPa and 47.20 kPa, respectively. MSCCA@CaCO 3 -aFGF could remedy the wound model in 13 days, with 99.41 % wound shrinkage without severe inflammatory responses. Due to strong adhesion properties and mechanical strength, MSCCA@CaCO 3 -aFGF can be a promising alternative to conventional sutures and tissue closure staples for wound closure and healing. Therefore, MSCCA@CaCO 3 -aFGF is expected to be a strong candidate for the next generation of adhesives., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The management of diabetic wounds remains a critical therapeutic challenge. Platelet-rich plasma (PRP) gel, PRP-derived exosomes (PRP-Exos), and mesenchymal stem cell-derived exosomes (MSC-Exos) have demonstrated therapeutic potential in wound treatment. Unfortunately, their poor mechanical properties, the short half-lives of growth factors (GFs), and the burst release of GFs and exosomes have limited their clinical applications. Furthermore, proteases in diabetic wounds degrade GFs, which hampers wound repair. Silk fibroin is an enzyme-immobilization biomaterial that could protect GFs from proteases. Herein, we developed novel dual-crosslinked hydrogels based on silk protein (SP) (sericin and fibroin), including SP@PRP, SP@MSC-Exos, and SP@PRP-Exos, to promote diabetic wound healing synergistically. SP@PRP was prepared from PRP and SP using calcium gluconate/thrombin as agonist, while SP@PRP-Exos and SP@MSC-Exos were derived from exosomes and SP with genipin as crosslinker. SP provided improved mechanical properties and enabled the sustained release of GFs and exosomes, thereby overcoming the limitations of PRP and exosomes in wound healing. The dual-crosslinked hydrogels displayed shear-induced thinning, self-healing, and eradication of microbial biofilms in a bone-mimicking environment. In vivo , the dual-crosslinked hydrogels contributed to faster diabetic wound healing than PRP and SP by upregulating GFs expression, down-regulating matrix metalloproteinase-9 expression, and by promoting an anti-NETotic effect, angiogenesis, and re-epithelialization. Hence, these dual-crosslinked hydrogels have the potential to be translated into a new generation of diabetic wound dressings., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

With the development of science and technology, human-machine interaction has brought great benefits to the society. Here, we design a voice and gesture signal translator (VGST), which can translate natural actions into electrical signals and realize efficient communication in human-machine interface. By spraying silk protein on the copper of the device, the VGST can achieve improved output and a wide frequency response of 20-2000 Hz with a high sensitivity of 167 mV/dB, and the resolution of frequency detection can reach 0.1 Hz. By designing its internal structure, its resonant frequency and output voltage can be adjusted. The VGST can be used as a high-fidelity platform to effectively recover recorded music and can also be combined with machine learning algorithms to realize the function of speech recognition with a high accuracy rate of 97%. It also has good antinoise performance to recognize speech correctly even in noisy environments. Meanwhile, in gesture recognition, the triboelectric translator is able to recognize simple hand gestures and to judge the distance between hand and the VGST based on the principle of electrostatic induction. This work demonstrates that triboelectric nanogenerator (TENG) technology can have great application prospects and significant advantages in human-machine interaction and high-fidelity platforms.

Sericin protein is a type of protein derived from silk cocoons. Sericin hydrogen bonds cause adhesion to the silk cocoon. This substance contains a large amount of serine amino acids in its structure. At first, the medicinal properties of this substance were unknown, but today many properties have been discovered for this substance. The unique properties of this substance have made it widely used in the pharmaceutical and cosmetic industries. The applications of Sericin in pharmacy are as follows. Sericin is used to repair wounds by producing collagen. Other uses for the drug include anti-diabetic, anti-cholesterol, metabolic modulator, anti-tumor, heart protection, antioxidant, antibacterial, wound healing, cell proliferation, UV protection, freezing, and skin moisturizing. The physicochemical properties of Sericin have attracted the attention of pharmacists and their widespread use in the production of drugs and treatment of diseases. One of the critical and unique properties of Sericin is its anti-inflammatory property. In this article, this property of Sericin is discussed in detail, and according to the experiments performed by pharmacists, this substance has shown a significant effect in eliminating inflammation. This study aimed to evaluate the impact of Sericin protein in relieving inflammation., Competing Interests: Conflict of interest None., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Background: People have used many natural materials such as plant leaves, roots, liquids derived from plants, and animal products to treat wounds throughout history. It can be said that the research on wound care in recent years have focused on traditional and natural products again. This study aimed to investigate the effects of sweetgum oil, propolis, silk protein, and Ankaferd Blood Stopper (ABS) on wound healing in an experimental excisional wound model., Methods: : Including 36 Balb/c inbreed mice in the study were divided equally into four groups. Two circular excisional wounds were created on the dorsal skin of mice under anesthesia using a punch biopsy device. The wounds of the first group of mice were topically dressed with sweetgum oil, the second group mice with propolis, the third group mice with silk protein, and the fourth group mice with ABS daily. Tissue samples were taken from the wounds of mice on the 7th and 14th day of wound formation, and histological examinations were performed. On the 14th day, the wounds created in all mice were healed, and the experiment was terminated., Results: Mice in the silk protein group had faster wound healing. There was no statistical difference between the groups in immunohistochemical examinations. In the ABS group, the findings of the inflammatory process were more prominent., Discussion: In conclusions, propolis, sweetgum oil, silk protein, and ABS positively affect different parameters in wound healing and support wound healing.

Introduction: A number of biological wastes and factory waste materials have been tested recently for the eco-friendly biosynthesis of nanoparticles. Sericin protein (SSP) is usually removed from the silk cocoon during the degumming process in the process of making the silk, and this sericin protein is normally thrown away by the sericulture industries as waste materials. It is found that this sericin protein possesses a number of biological properties., Methods: Considering this, in the present study, an effort has been made to biosynthesize gold nanoparticles (SSP-AuNPs) using the waste sericin solution as the reducing and capping agent and investigate its biopotential in terms of its wound healing, antioxidant and antibacterial activities., Results: The synthesis of SSP-AuNPs was perceived by the visual color change and confirmed by UV-Vis spectroscopy with absorption maxima at 522 nm. Further characterization of SSP-AuNPs was done by TEM, EDS, XRD, FTIR, DLS, zeta potential, TGA, AFM, etc. The size of SSP-AuNPs was found out to be 54.82 nm as per the particle size analyzer and the zeta potential is -19.8 mV. The SSP-AuNPs displayed promising wound healing potential of 70.96 and 69.76% wound closure rate at 5 and 10 µg/mL respectively as compared to 74.91% by the Centella asiatica taken as a positive control. It also exhibited promising antioxidant potential in terms of the DPPH, ABTS free radical scavenging, reducing power potential, and total antioxidant capacity. Besides, the SSP-AuNPs also displayed significant antibacterial activities against the tested pathogenic bacterial with the diameter of inhibition zones ranging between 12.10 and 14.96 mm as compared to the positive control cephalexin that displayed inhibition zones ranging between 12.08 and 13.24 mm., Discussion: Taken together, SSP-AuNPs could serve as an interesting candidate for food, cosmetics, and biomedical fields in the applications of wound healing, cosmetics, antibacterial bandages, and ointments, etc., Competing Interests: The authors declare that there is no conflict of interest exists with this manuscript., (© 2023 Das et al.)

Growth hormone deficiency (GHD) has become a serious healthcare burden, and presents a huge impact on the physical and mental health of patients. Here, we developed an actively separated microneedle patch (PAA/NaHCO 3 -Silk MN) based on silk protein for sustained release of recombinant human growth hormone (rhGH). Silk protein, as a friendly carrier material for proteins, could be constructed in mild full-water conditions and ensure the activity of rhGH. After manually pressing PAA/NaHCO 3 -Silk MN patch to skin for 1 min, active separation is achieved by absorbing the interstitial fluid (ISF) to trigger HCO 3 ‒ in the active backing layer to produce carbon dioxide gas (CO 2 ). In rats, the MN patch could maintain the sustained release of rhGH for more than 7 days, and produce similar effects as daily subcutaneous (S.C.) injections of rhGH in promoting height and weight with well tolerated. Moreover, the PAA/NaHCO 3 -Silk MN patch with the potential of painless self-administration, does not require cold chain transportation and storage possess great economic benefits. Overall, the PAA/NaHCO 3 -Silk MN patch can significantly improve patient compliance and increase the availability of drugs, meet current unmet clinical needs, improve clinical treatment effects of GHD patients., (© 2022 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

Traditional Chinese medicine (TCM) has a good curative effect, but its disadvantages include complex components, poor drug stability, potential drug interaction, etc. Therefore, it is particularly important to construct a novel drug delivery system that can load Chinese medicine monomers to solve this problem. Silk fibroin is a kind of natural polymer material with unique properties. It can be used as a carrier material to load Chinese medicine monomers to prepare novel drug delivery systems that significantly affect treating diseases without toxic and side effects. However, there is still a lack of a review on silk fibroin as a carrier material to load Chinese medicine monomers to explore and analyze the current research results and progress. Here, our article focuses on the in-depth excavation and analysis of the recent research on novel drug delivery systems prepared by silk fibroin and TCM monomers. Besides, the characteristics, existing problems, and prospects of silk fibroin are discussed and explained. It is hoped that this research can provide a reference and basis for the modernization of TCM, the design of novel drug delivery systems, the research and development of new drugs in the future, and contribute to the innovation of silk protein., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Silk protein is being increasingly introduced as a prospective material for biomedical devices. However, a limited locus to intervene in nature-oriented silk protein makes it challenging to implement on-demand functions to silk. Here, we report how polymorphic transitions are related with molecular structures of artificially synthesized silk protein and design principles to construct a green-lithographic and high-performative protein resist. The repetition number and ratio of two major building blocks in synthesized silk protein are essential to determine the size and content of β-sheet crystallites, and radicals resulting from tyrosine cleavages by the 193 nm laser irradiation induce the β-sheet to α-helix transition. Synthesized silk is designed to exclusively comprise homogeneous building blocks and exhibit high crystallization and tyrosine-richness, thus constituting an excellent basis for developing a high-performance deep-UV photoresist. Additionally, our findings can be conjugated to design an electron-beam resist governed by the different irradiation-protein interaction mechanisms. All synthesis and lithography processes are fully water-based, promising green lithography. Using the engineered silk, a nanopatterned planar color filter showing the reduced angle dependence can be obtained. Our study provides insights into the industrial scale production of silk protein with on-demand functions.

Animals widely use minerals and organic components to construct biomaterials with excellent properties, such as teeth, bones, molluscan shells and eggshells. The larvae of the oriental moth, Monema (Cnidocampa) flavescens Walker, secrete silk proteins that combine closely with calcareous minerals to construct a hard cocoon, which is completely different from the mineral-free Bombyx mori cocoon. The cocoons of oriental moths are likely to be the hardest among the cocoons constructed by insect species. The cocoons of oriental moths were found to be mainly composed of calcium oxalates and Asx/Ser/Gly-rich cocoon proteins, but the types of calcium oxalates and cocoon proteins remain to be elucidated. In this study, we provide an in-depth explanation of the inorganic and organic components in the oriental moth cocoon. Microscopy and imaging technologies revealed that the cocoon is composed of mineral crystals, silk fibers and other organic matter. X-ray diffraction and infrared spectral analyses showed that the mineral crystals in the oriental moth cocoon were mainly CaC 2 H 2 O 4 ·H 2 O. ICP-OES analysis suggested that the mineral crystals in the cocoons were mainly CaC 2 H 2 O 4 ·H 2 O. LC-MS/MS-based proteomics allowed us to identify 467 proteins from the oriental moth cocoon, including 252 uncharacterized proteins, 87 enzymes, 36 small molecule binding proteins, and 5 silk proteins. Among the uncharacterized proteins, 25 of which were Asn-rich proteins because they contained a high proportion of Asn residues (19.1%-41.4%). Among the top 20 cocoon proteins with the highest abundance, 9 of which were Asn-rich proteins. The qPCR was used to investigate the expression patterns of the major cocoon protein-coding genes. Three fibroins and three Asn-rich proteins were expressed only in the silk gland but not in other tissues. The expression of Asn-rich proteins in the silk gland gradually increased from the anterior silk gland to the posterior silk gland. These findings provide important references for understanding the formation mechanism and mechanical properties of mineralized hard cocoons constructed by oriental moths., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Naturally derived protein-based biopolymers are considered potential biomaterials in biomedical applications and eco-friendly materials for replacing current petroleum-based polymers due to their good biocompatibility, low environmental impact, and tunable degradability. However, current strategies for fabricating protein-based materials with superior properties and tailored functionality in a scalable manner are still lacking. Here, we demonstrate an aqueous-based scalable approach for fabricating silk protein-based films through controlled molecular self-assembly (CMS) of silk proteins with plasticizers and salt ions. The films fabricated using this method can achieve a toughness of up to 64±5 MJ/m 3 with a stretchability of up to 574±31%. We also demonstrate the tunable enzymatic degradability, low in vitro cytotoxicity, and good in vivo biocompatibility of the films. Furthermore, the films can be patterned with predesigned complex structures through laser cutting and functionalized with bioactive components. The functional silk protein-based films show great potential in various applications, including flexible electronics, bioelectronics, tissue engineering, and bioplastic packaging. STATEMENT OF SIGNIFICANCE: Inspired by the naturally optimized multi-scale self-assembly of silk proteins in natural silks, we develop an aqueous-based approach for scalable production of superior protein-based films through controlled molecular self-assembly (CMS) of silk proteins with glycerol and calcium ions. The prepared silk films present outstanding mechanical properties, controlled enzymatic biodegradability, low in vitro cytotoxicity, and good in vivo biocompatibility. Notably, the films fabricated using this method can achieve a high toughness of 64±5 MJ/m 3 with a stretchability of 594±31%. The approach introduced in this work provides a facile route toward making silk-based materials with superior properties. It also paves new avenues for developing functional protein-based materials with precisely controlled structures and properties for various applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Protein-based soft ionic conductors have attracted considerable research interest in recent years with great potential in applications at the human-machine interfaces. However, a fundamental mechanistic understanding of the ionic conductivity of silk-based ionic conductors is still unclear. Here, we first developed an environmental-friendly and scalable method to fabricate silk-based soft ionic conductors using silk proteins and calcium chloride. The mechanistic understanding of the ion transport and molecular interactions between calcium ions and silk proteins at variable water contents was investigated in-depth by combining experimental and simulation approaches. The results show that calcium ions primarily interact with amide groups in proteins at a low water content. The ionic conductivity is low since the calcium ions are confined around silk proteins within 2.0-2.6 Å. As water content increases, the calcium ions are hydrated with the formation of water shells, leading to the increased distance between calcium ions and silk proteins (3.3-6.0 Å). As a result, the motion of the calcium ions increased to achieve a higher ionic conductivity. By optimizing the ratio of the silk proteins, calcium ions, and water, silk-based soft ionic conductors with good stretchability and self-healing properties can be obtained. Such protein-based soft ionic conductors can be further used to fabricate smart devices such as electrochromic devices.

A eutectogel (ETG) based on immobilizing a zinc salt deep eutectic solvent (DES) in a silk protein backbone is prepared by a coagulating bath method as a solid electrolyte for Zn-ion hybrid supercapacitors (ZHSCs). The Zn salt DES is composed by ethylene glycol (EG), urea, choline chloride (ChCl), and zinc chloride (ZnCl 2 ) with a molar ratio of 6:10:3:3. A strong bonding of the DES liquid to the silk protein backbone is formed between protein macromolecules and the DES due to plenty of hydrogen bonds in both materials. The as-prepared ETG membrane is dense and has no obvious void defects, which possesses a fracture strength of 7.58 MPa and environmental stability. As a solid electrolyte, the ETG membrane exhibits a higher Zn 2+ transference number of about 0.60 and a high ionic conductivity (12.31 mS cm -1 at room temperature and 3.63 mS cm -1 at -20 °C). A ZHSC (Zn∥ETG∥C) with the silk protein-based ETG electrolyte is assembled by Zn and active carbon as the anode and the cathode, respectively, which delivers a specific capacitance of 342.8 F g -1 at a current density of 0.2 A g -1 and maintains excellent cycling stability with 80% capacitance retention after 20,000 cycles at a high current rate (5 A g -1 ) at room temperature. Moreover, the Zn∥ETG∥C device can safely work under a lower temperature of about -18 °C and damaging situations, such as folding states and even cutting tests. The interface evolutions between the Zn anode and the ETG electrolyte are explored, and it was found that a ZnCO 3 /Zn(CH 2 OCO 2 ) 2 solid electrolyte interphase is in situ formed on the Zn anode, which can inhibit the growth of Zn dendrites. This work provides a new way to fabricate advanced electrolytes for applications in Zn-ion hybrid supercapacitors.

Electrospinning-based wound dressings have multiple functions such as antibacterial, anti-inflammatory, and therapeutic, and are important in skin wound care. Herein, we designed a phlorizin (PHL)-loaded silk protein/polyvinylpyrrolidone (SF/PVP) composite nanofibrous membrane, which can be used as multiple wound dressings. In particular, SF/PVP/PHL scaffolds have high porosity and mechanical properties, exhibiting suitable permeability and hydrophilicity. The SF/PVP/PHL scaffolds containing PHL also have excellent antibacterial and antioxidant activities. Furthermore, the nanofiber significantly accelerated the wound healing process in a full-thickness skin injury model by enhancing wound re-epithelialization and collagen deposition density, increasing the content of macrophage antigen (CD68), platelet-endothelial cell adhesion molecule (CD31), proliferating cell nuclear antigen (PCNA) and inhibiting the expression of α-smooth muscle actin (α-SMA) at the wound site. The mechanism may be related to the inhibition of activation of phosphatidylinositol 3-kinase/serine-threonine kinase/ target of rapamycin (PI3K/AKT/mTOR) signaling pathway to enhance autophagy. Therefore, SF/PVP/PHL nanofibers can ideally meet the various requirements of the wound healing process and are promising wound dressing candidates for future clinical applications., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Oral wounds are among the most troublesome injuries which easily affect the patients' quality of life. To date, the development of functional antibacterial dressings for oral wound healing remains a challenge. In this regard, we investigated antibacterial silk protein-based membranes for the application as wound dressings in oral and maxillofacial surgery. The present study includes five variants of casted membranes, i.e., i) membranes-silver nanoparticles (CM-Ag), ii) membranes-gentamicin (CM-G), iii) membranes-control (without functionalization) (CM-C), iv) membranes-silk sericin control (CM-SSC), and v) membranes-silk fibroin/silk sericin (CM-SF/SS), and three variants of nonwovens, i.e., i) silver nanoparticles (NW-Ag), ii) gentamicin (NW-G), iii) control (without functionalization) (NW-C). The surface structure of the samples was visualized with scanning electron microscopy. In addition, antibacterial testing was accomplished using agar diffusion assay, colony forming unit (CFU) analysis, and qrt-PCR. Following antibacterial assays, biocompatibility was evaluated by cell proliferation assay (XTT), cytotoxicity assay (LDH), and live-dead assay on L929 mouse fibroblasts. Findings indicated significantly lower bacterial colony growth and DNA counts for CM-Ag with a reduction of bacterial counts by 3log levels (99.9% reduction) in CFU and qrt-PCR assay compared to untreated control membranes (CM-C and CM-SSC) and membranes functionalized with gentamicin (CM-G and NW-G) (p < 0.001). Similarly, NW-G yielded significantly lower DNA and colony growth counts compared to NW-Ag and NW-C (p < 0.001). In conclusion, CM-Ag represented 1log level better antibacterial activity compared to NW-G, whereas NW-G showed better cytocompatibility for L929 cells. As data suggest, these two membranes have the potential of application in the field of bacteria-free oral wound healing. However, provided that loading strategy and cytocompatibility are adjusted according to the antibacterial agents' characteristic and fabrication technique of the membranes., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Bombyx mori is an important economic insect and an animal model in pharmacomedical research. Although its physiology has been studied for many years, the mechanism via which silk protein genes are regulated remains unclear. In this study, we performed two-step expression screening, namely co-expression network and time-course expression analyses to screen silk protein regulation factors. A co-expression network analysis using RNA-seq data that were obtained from various tissues, including the silk glands of B. mori , was performed to identify novel silk protein regulatory factors. Overall, 91 transcription factors, including some known ones, were found to be co-expressed with silk protein genes. Furthermore, time-course expression analysis during the fifth instar larvae stage revealed that the expression pattern of 13 novel transcription factors was highly relevant to that of silk protein genes and their known regulatory factor genes. In particular, the expression peak of several transcription factors (TFs) was detected before the expression of silk protein genes peak. These results indicated that a larger number of genes than expected may be involved in silk protein regulation in B. mori . Functional analyses of function-unknown transcription factors should enhance our understanding of this system.

This paper describes the use of silk protein, including fibroin and sericin, from an alkaline solution of Ca(OH) 2 for the clean degumming of silk, which is neutralized by sulfuric acid to create calcium salt precipitation. The whole sericin (WS) can not only be recycled, but completely degummed silk fibroin (SF) is also obtained in this process. The inner layers of sericin (ILS) were also prepared from the degummed silk in boiling water by 120 °C water treatment. When the three silk proteins (SPs) were individually grafted with glycidyl methacrylate (GMA), three grafted silk proteins (G-SF, G-WS, G-ILS) were obtained. After adding I2959 (a photoinitiator), the SP bioinks were prepared with phosphate buffer (PBS) and subsequently bioprinted into various SP scaffolds with a 3D network structure. The compressive strength of the SF/ILS (20%) scaffold added to G-ILS was 45% higher than that of the SF scaffold alone. The thermal decomposition temperatures of the SF/WS (10%) and SF/ILS (20%) scaffolds, mainly composed of a β-sheet structures, were 3 °C and 2 °C higher than that of the SF scaffold alone, respectively. The swelling properties and resistance to protease hydrolysis of the SP scaffolds containing sericin were improved. The bovine insulin release rates reached 61% and 56% after 5 days. The L929 cells adhered, stretched, and proliferated well on the SP composite scaffold. Thus, the SP bioinks obtained could be used to print different types of SP composite scaffolds adapted to a variety of applications, including cells, drugs, tissues, etc. The techniques described here provide potential new applications for the recycling and utilization of sericin, which is a waste product of silk processing.

Organisms develop unique systems in a given environment. In the process of adaptation, they employ materials in a clever way, which has inspired mankind extensively. Understanding the behavior and material properties of living organisms provides a way to emulate these natural systems and engineer various materials. Silk is a material that has been with human for over 5000 years, and the success of mass production of silkworm silk has realized its applications to medical, pharmaceutical, optical, and even electronic fields. Spider silk, which was characterized later, has expanded the application sectors to textile and military materials based on its tough mechanical properties. Because silk proteins are main components of these materials and there are abundant creatures producing silks that have not been studied, the introduction of new silk proteins would be a breakthrough of engineering materials to open innovative industry fields. Therefore, in this review, we present diverse silk and silk-like proteins and how they are utilized with respect to organism's survival. Here, the range of organisms are not constrained to silkworms and spiders but expanded to other insects, and even marine creatures which produce silk-like proteins that are not observed in terrestrial silks. This viewpoint broadening of silk and silk-like proteins would suggest diverse targets of engineering to design promising silk-based materials. STATEMENT OF SIGNIFICANCE: Silk has been developed as a biomedical material due to unique mechanical and chemical properties. For decades, silks from various silkworm and spider species have been intensively studied. More recently, other silk and silk-like proteins with different sequences and structures have been reported, not only limited to terrestrial organisms (honeybee, green lacewing, caddisfly, and ant), but also from marine creatures (mussel, squid, sea anemone, and pearl oyster). Nevertheless, there has hardly been well-organized literature on silks from such organisms. Regarding the relationship among sequence-structure-properties, this review addresses how silks have been utilized with respect to organism's survival. Finally, this information aims to improve the understanding of diverse silk and silk-like proteins which can offer a significant interest to engineering fields., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

We compared the efficacy for protein extraction of water versus enzymatic extraction. The amino-acid composition, inhibitory activity against enzymes α-amylase and α-glucosidase, and anti-glycation activities of silk protein extract (SPE) were determined. We used water extraction (100 °C, six hours) and protease-enzymatic extraction. The microstructure of silk fibers was obviously different after extraction. The results showed that enzymatic extraction gave the greater values of protein content, amino acids, total phenolic content (TPC), and total flavonoid content (TFC), as well as all biological activities parameters tested, but it also provided a more bitter taste in the extract-contributing amino acids of 51% (arginine, phenylalanine, histidine, valine, tryptophan, isoleucine, and leucine) and less sweet and umami taste contributing amino acids than did water extraction, which could be more suitable to be used as concentrated nutraceuticals.

Background: The advances in products based on nanotechnology have directed extensive research on low-cost, biologically compatible, and easily degradable materials., Main Body: Sericin (SER) is a protein mainly composed of glycine, serine, aspartic acid, and threonine amino acids removed from the silkworm cocoon (particularly Bombyx mori and other species). SER is a biocompatible material with economic viability, which can be easily functionalized due to its potential crosslink reactions. Also, SER has inherent biological properties, which makes possible its use as a component of pharmaceutical formulations with several biomedical applications, such as anti-tumor, antimicrobials, antioxidants and as scaffolds for tissue repair as well as participating in molecular mechanisms attributed to the regulation of transcription factors, reduction of inflammatory signaling molecules, stimulation of apoptosis, migration, and proliferation of mesenchymal cells., Conclusion: In this review, the recent innovations on SER-based nano-medicines (nanoparticles, micelles, films, hydrogels, and their hybrid systems) and their contributions for non-conventional therapies are discussed considering different molecular mechanisms for promoting their therapeutic applications.

The secondary structures of silk fibroin (SF) are critical in the determination of the mechanical properties of the animal silks. Different characterization techniques, such as X-ray diffraction, nuclear magnetic resonance, Raman spectroscopy, and Fourier transform infrared (FTIR) technique, have been applied to study the secondary structure of animal silks. Among these techniques, FTIR is most widely used as it is sensitive to all secondary structures of proteins. Especially with the development of FTIR imaging, it is now possible to image the secondary structures of proteins at the micrometer scale, so as to understand the spatial distribution of proteins and the interaction of proteins with other materials at specific locations of interest. In this chapter, we present the methods and protocols of FTIR imaging to silk protein-based materials. We primarily introduce how to set up the instruments and accessories, as well as how to choose the appropriate imaging methods and sample preparation methods according to sample morphologies. The critical protocols for data analysis are also introduced in the last section., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Seroin 1 and seroin 2 are abundant in silkworm cocoon silk and show strong antibacterial activities, and thus are thought to protect cocoon silk from damage by bacteria. In this study, we characterized the expression pattern of silkworm seroin 3, and found that seroin 3 is synthesized in the female ovary and secreted into egg to play its roles. After being infected, seroin 1, 2, and 3 were significantly up-regulated in the silkworm. We synthesized the full-length protein of seroin 1, 2, and 3 and their N/C-terminal domain (seroin-N/C), and compared the antimicrobial activities in vitro. All three seroins showed higher antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. Seroin 2 showed better antibacterial effect than seroin 1 and 3, whereas seroin 1/2/3-N was better than seroin 1/2/3-C. We found that seroin 2-C has stronger peptidoglycan binding ability than seroin 2-N per the ELISA test. The binding sites of seroin 2 with bacteria were blocked by peptidoglycan, which resulted in the loss of the antibacterial activity of seroin 2. Collectively, these findings suggest that seroin 1 and 2 play antibacterial roles in cocoon silk, whereas seroin 3 functions in the eggs. The three silkworm seroins have the same antibacterial mechanism, that is, binding to bacterial peptidoglycan by the C-terminal domain and inhibiting bacterial growth by the N-terminal domain.

Silk protein builds up one of the strongest fibers superior to most synthetic and natural polymers. However, the strengthening mechanisms of the silk proteins remain largely elusive because of their complex nanocomposite structures. Here, we report an unusual behavior of this kind of material that is distinctively different from those of metals and other polymers. We find that there are multiple interface microcracks nucleating and stacking under the shear loading, dividing the interchain interface into small segments, by which the silk protein can achieve a high strength even with the ultralong chains. This is a new strategy of microstructure design of soft matter that could avoid the "larger is weaker" fate due to the increase of the chain length. This novel mechanism is crucial for building strong polymer materials with long chain molecules and at the same time retaining their complex functional and structural properties.

Due to the poor repair ability of cartilage tissue, regenerative medicine still faces great challenges in the repair of large articular cartilage defects. Quercetin is widely applied as a traditional Chinese medicine in tissue regeneration including liver, bone and skin tissues. However, the evidence for its effects and internal mechanisms for cartilage regeneration are limited. In the present study, the effects of quercetin on chondrocyte function were systematically evaluated by CCK8 assay, PCR assay, cartilaginous matrix staining assays, immunofluorescence assay, and western blotting. The results showed that quercetin significantly up-regulated the expression of chondrogenesis genes and stimulated the secretion of GAG (glycosaminoglycan) through activating the ERK, P38 and AKT signalling pathways in a dose-dependent manner. Furthermore, in vivo experiments revealed that quercetin-loaded silk protein scaffolds dramatically stimulated the formation of new cartilage-like tissue with higher histological scores in rat femoral cartilage defects. These data suggest that quercetin can effectively stimulate chondrogenesis in vitro and in vivo, demonstrating the potential application of quercetin in the regeneration of cartilage defects., (Copyright © 2020 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Tailored surface coatings have been used for decades to improve material performance in blood. Among different approaches, heparin based biomedical coatings have found great success in the commercial catheter market. However, they have their own limitations. Coating of a vascular device with a heparin binding peptide (HBP), which can sequester the circulating heparin, presents numerous advantages over both systemic heparin therapy and direct heparin bound surfaces. Embedding HBP in a silk biopolymer provides the mechanical integrity necessary under dynamic flow conditions to both insert the catheter and maintain proper blood flow. Furthermore, due to the similarity in structure of HBP with antimicrobial peptides, it is predicted that the fusion protein will also show antimicrobial property, a critical and unique aspect to combat catheter related blood stream infections and extend the longevity of hemodialysis catheters. To assess this hypothesis, a recombinant fusion protein (S 4 H 4 ) containing both silk amino acid motifs and HBP was assessed as a coating on a silicone surface. After validating that, the protein was deposited on the surface via XPS, Raman spectroscopy, ATR and SEM imaging, antimicrobial and anticoagulant activities were evaluated. The coating was able to prevent not only planktonic bacterial growth but also prevented the growth of a biofilm. Finally, the coating had both antibacterial and anticoagulant effect simultaneously. This study proves the successful production of a silk-based biopolymer that can be embedded with a heparin-binding functionality to create a dual functional device coating that can prevent infection and thrombosis together., Competing Interests: Declaration of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Silk biomaterials with tunable mechanical properties and biological properties are of special importance for tissue engineering. Here, we fabricated silk fibroin (SF, from Bombyx mori silk) scaffolds from cryogelation under controlled temperature and catalytic cross-linking conditions. Structurally, the cryogelled scaffolds demonstrated a greater β-sheet content but significantly smaller β-sheet domains compared to that without chemical cross-linking and catalyst. Mechanically, the cryogelled scaffolds were softer and highly elastic under tension and compression. The 120% tensile elongation and >85% recoverable compressive strain were among the best properties reported for SF scaffolds. Cyclic compression tests proved the robustness of such scaffolds to resist fatigue. The mechanical properties, as well as the degradation rate of the scaffolds, can be fine-tuned by varying the concentrations of the catalyst and the cross-linker. For biological responses, in vitro rat bone mesenchymal stem cell (rBMSC) culture studies demonstrated that cryogelled SF scaffolds supported better cell attachment and proliferation than the routine freeze-thawed scaffolds. The in vivo subcutaneous implantation results showed excellent histocompatibility and tissue ingrowth for the cryogelled SF scaffolds. This straightforward approach of enhanced elasticity of SF scaffolds and fine-tunability in mechanical performances, suggests a promising strategy to develop novel SF biomaterials for soft tissue engineering and regenerative medicine.

Combining the properties of natural protein and polysaccharide is a promising strategy to generate bioactive biomaterials with controlled structure. Here, a new method of preparing water-insoluble silk fibroin/hyaluronic acid (SF/HA) scaffolds with tunable performances using an all-aqueous process is reported. Freezing-induced assembly was used to form silk I crystallization in the SF/HA blends. Silk I crystallization enhanced the stability of SF/HA scaffolds in water by forming silk I crystal networks to entrap blended HA without chemical cross-linking. Increasing HA content significantly enhanced the flexibility and water binding capacity of porous scaffolds, but high amount of HA reduced the water-stability of porous scaffolds due to insufficient silk I crystal cross-links. The enzymatic degradation behavior of the SF/HA scaffolds was investigated, revealing that the regulation ability of HA in the SF scaffolds. This novel nonchemically cross-linked protein/polysaccharide scaffold may be useful for soft tissue engineering due to excellent biocompatibility and tunable performances., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The infected diabetic wound ulcer is a significant problem for the diabetic patients, which leads to removal of affected foot site due to its delayed/non-healing tissues. The poly-microbial infections and active matrix metalloproteinases (MMP) are the significant influencing factors to delayed healing in diabetic mice. The main purposes of present investigation are to evaluation the targeted inactivation of MMP and avoid polymicrobial infections by using a combined therapeutic effect of metal chelating dipeptide (L-carnosine) and curcumin with the biocompatible silk protein hydrogel (L-car@cur/SF) dressing in the infected diabetic wound ulcer. The in vitro biological assay methods, such as, cell viability, anti-oxidant activity, anti-inflammatory macrophage cells and inhibition collagenase exhibited that the designed hydrogel matrix to be human cell compatible and could be accelerate for significant diabetic healing potential. The activation of cur/SF matrix by the L-carnosine was persuading the inactivation of matrix metalloproteinase-9 (MMP-9) through its potent chelating effects of Zn 2+ ions from the MMP-9 active center. The L-car@cur/SF hydrogel was demonstrated for the effective MMP-9 inactivation and bacterial inhibition via in vivo mice wound site, which indorsed the diabetic wound healing efficiency in streptozotocin-tempted diabetic mice., (Copyright © 2019 Elsevier B.V. All rights reserved.)

To understand the natural silk spinning mechanism, synchrotron Fourier transform infrared (S-FTIR) microspectroscopy was employed in this study to monitor the conformation changes of silk protein in the silk gland of Bombyx mori silkworm. The ultrahigh brightness of S-FTIR microspectroscopy allowed the imaging of the silk gland with micrometer-scale spatial resolution. Herein, tissue sections of a silk gland, including cross-section slices and longitudinal-section slices, were characterized. The results obtained clearly confirm that the conformation of the silk fibroin changes gradually along the silk gland from the tail to the spinneret. In the middle silk gland, silk fibroin mainly contains random coil/helix conformation. When it comes to the spinneret through the anterior silk gland, the content of β-sheet increases, but the content of random coil/helix instead reduces gradually. Further, the β-sheet distribution in the cross-section of the anterior silk gland was imaged using S-FTIR mapping technique. The results show that the structural distribution of the silk fibroin in cross-section is uniform without significant shell-core structure, which implies that the primary driving force to induce the conformation transition of silk fibroin from random coil/helix to β-sheet during the spinning process is elongational flow of silk fibroin in the silk gland and not the shear force between the silk fibroin and the lumen wall of silk gland. These direct pieces of evidence of silk fibroin structure in the silk gland would definitely promote a deeper understanding of the natural spinning process.

Sericin is a protein component of the silkworm cocoon, and contains a high proportion of L-serine, but it has been mostly disposed of as an industrial waste. However, recent studies have revealed its unique biological functionalities beneficial to human health. This study aimed to evaluate the effect of acute oral intake of sericin on amino acid and neurotransmitter metabolism in the mouse brain. Acute administration of chemically modified sericin (0.26 g/30 g body weight) increased L-serine and L-tyrosine levels in the serum and brain, although the L-tyrosine content in the sericin was less than 3% (w/w). In addition, sericin administration led to a significant facilitation of noradrenergic turnover via enhancement of 3-methoxy-4-hydroxyphenylethyleneglycol, a principal metabolite of noradrenaline, in several of the brain regions examined. These present findings suggest that oral intake of sericin efficiently delivers L-serine and L-tyrosine to the brain, thus stimulating noradrenergic activity in the brain. Abbreviations : DA: dopamine; 5-HIAA: 5-hydroxyindoleicetic acid; 5-HT: 5-hydroxytryptamine; HVA: homovanillic acid; MHPG: 3-methoxy-4-hydroxyphenylethyleneglycol; 3-MT: 3-methoxytyramine; NA: noradrenaline; NM: normetanephrine; Veh: vehicle.