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The development of protein-based in situ forming hydrogel remains a big challenge due to the limited chemical groups in proteins. Keratins are a group of cysteine-rich structural protein found abundant in skin and skin appendant. Recently, our lab has established a disulfide shuffling strategy to prepare keratin hydrogels via oxygen (O2) oxidation. However, such hydrogel still needed to be molded in advance. In this work, inspired by the fact that glucose commonly exists in body fluids, a glucose-triggered in situ forming keratin hydrogel was developed based on the disulfide shuffling strategy via a higher oxidation force of hydrogen peroxide (H2O2). The hydrogel precursor solution consisted of keratin, cysteine and glucose oxidase (GOD), which could generate H2O2 in an indirect and mild way via GOD-catalyzed oxidation of glucose in body fluids. Our findings demonstrated that the GOD-catalyzed oxidation method not only shortened the gelation time but improved the mechanical strength of the hydrogel by comparison with O2 oxidation and direct addition of H2O2 solution methods, and realized in situ gelation within 3 min on a full-thickness wound bed in normal mice. Moreover, the in situ forming keratin hydrogel was applied as a drug depot for wound repair, and the deferoxamine-loaded one accelerated healing in the full-thickness wounds of streptozotocin-induced diabetic rats, notably by promoting angiogenesis and neovascularization in wounds. STATEMENT OF SIGNIFICANCE: Studies show that keratin hydrogels possess tissue regeneration capacity, especially in skin wound repair. However, most of the reported keratin hydrogels needed to be molded in advance and cannot fit irregular wound shape. This work describes a glucose-triggered in situ forming keratin hydrogel via a disulfide shuffling strategy under the oxidation of hydrogen peroxide. Of note, the hydrogen peroxide was supplied indirectly by glucose oxidase-catalyzed oxidation of glucose in wound fluids, and this method needed no additional crosslinking agents or chemical modifications on keratins. Our findings showed that this hydrogel realized in situ gelation within 3 min on a full-thickness wound bed and enabled an injectable mode with good filling ability toward irregular wounds. Moreover, this hydrogel could be applied as a drug depot for the treatment of diabetic wounds.

Succinoglycan is an industrially important exopolysaccharide biosynthesized by bacteria. In this study, mutant strain 18052 N-11 was obtained from the wild type strain Rhizobium radiobacter ATCC 19358 by NTG mutagenesis. It has a high yield succinoglycan of 32.5 g/L cultured in a 15 L-fementer for 72 h. Succinoglycan SG-A from the wild type strain has two components, and the molecular weights were 1.55 × 107 Da and 1.26 × 106 Da, respectively. While, succinoglycan SG-N from the mutant strain was a homogeneous polysaccharide, and the molecular weight was 1.01 × 107 Da. The molecular weight of both succinoglycan was higher than those reported in literatures. DSC thermogram of SG-A showed a higher endothermic peak than that of SG-N due to the higher crystallinity of SG-A. The dynamic frequency sweep test of SG-A and SG-N showed that the elastic modulus G' and viscosity modulus G" curves intersected at 65 °C, indicating the thermally induced order-disorder conformation. The results of effect of concentrations (2.5–15%) and temperatures (25–75 °C) on apparent viscosity of SG-A and SG-N showed that the succinoglycan solutions exhibited non-Newtonian, shear-thinning behavior. Both SG-A and SG-N showed an excellent emulsification activity. The characterizations and rheological properties make SG-A and SG-N prominent candidates in food, cosmetics, pharmaceutical and petroleum industries.

Curdlan is a neutral linear exopolysaccharide produced by Agrobacterium spp. under nitrogen-limiting conditions. In this study, we explored the role of glnA in curdlan biosynthesis in Agrobacterium sp. CGMCC 11546. The curdlan production of the ΔglnA strain was impaired, decreasing by 93% compared with that of the wild-type strain after 96 h fermentation. Analysis of fermentation profiles revealed that cell growth and utilization of carbon and nitrogen sources were impaired in the ΔglnA strain. Transcriptome analysis indicated that various of genes involved in curdlan biosynthesis were downregulated after 24 h fermentation in the ΔglnA strain, particularly genes involved in heme synthesis and the electron transport chain, which are essential for energy generation. Metabolomics analysis revealed flavin adenine dinucleotide (FAD) and adenosine diphosphate (ADP) accumulation in the ΔglnA strain, suggesting insufficient energy supply. Furthermore, glnA overexpression led to an 18% increase in the curdlan yield of the ΔglnA mutant compared with that of the wild-type strain after 96 h fermentation. Taken together, the findings demonstrate that glnA plays a vital role in curdlan biosynthesis by supplying ATP via regulating the expression of genes involved in heme synthesis and the electron transport chain.

Protein-templated gold nanoclusters have attracted attention in fluorescence imaging due to their simple synthesis and good biocompatibility. However, limitations still exist such as poor colloid stability and undesirable fluorescence intensity. Here we describe the self-assembly of keratin-templated gold nanoclusters via a simple and mild preparation process, including keratin-templated synthesis of gold nanoclusters (AuNCs@Keratin), silver ions modification of AuNCs@Keratin (AuNCs-Ag@Keratin), and gadolinium ions-induced aggregation of AuNCs-Ag@Keratin (AuNCs-Ag@Keratin-Gd). It was demonstrated that the AuNCs-Ag@Keratin-Gd obtained an enhanced fluorescence intensity (6.5 times that of AuNCs@Keratin), high colloid stability for more than 4 months, and good biocompatibility. Moreover, the AuNCs-Ag@Keratin-Gd holds promise in multifunctional applications such as near-infrared (NIR) fluorescence imaging, magnetic resonance (MR) imaging, and redox-responsive drug delivery, extending the applicability of fluorescent gold nanoclusters, especially in biomedical fields. Statement of significance Assembly-induced fluorescence enhancement has been rarely reported on as it relates to the protein-templated gold nanoclusters (AuNCs). In this work, self-assembly of protein-templated AuNCs was developed for enhanced fluorescence intensity and multifunctional applications, including bioimaging and responsive drug delivery. A cysteine-rich protein, keratin, was utilized as the template to synthesize AuNCs, which underwent silver ion modification and gadolinium ion-induced aggregation. The silver modification of the keratin-templated AuNCs facilitated the formation of a dense aggregate after gadolinium ion-induced assembly, thus generating an enhanced fluorescence intensity. Such a mechanism was confirmed by fluorescence correlation spectroscopy analysis. We believe that this work will extend the applicability of the fluorescent gold nanoclusters, especially in biomedical fields, and provided an effective approach for the mechanism analysis of the assembly-induced fluorescence enhancement via fluorescence correlation spectroscopy.

Curdlan is a water-insoluble exopolysaccharide produced by Agrobacterium species under nitrogen starvation. The curdlan production in the ΔmdeA, ΔmetA, ΔmetH, and ΔmetZ mutants of methionine biosynthesis pathway of Agrobacterium sp. CGMCC 11546 were significantly impaired. Fermentation profiles of four mutants showed that the consumption of ammonia and sucrose was impaired. Transcriptome analysis of the ΔmetH and ΔmetZ mutants showed that numerous differentially expressed genes involved in the electron transfer chain (ETC) were significantly down-regulated, suggesting that methionine biosynthesis pathway affected the production of energy ATP during the curdlan biosynthesis. Furthermore, metabolomics analysis of the ΔmetH and ΔmetZ mutants showed that ADP and FAD were significantly accumulated, while acetyl-CoA was diminished, suggesting that the impaired curdlan production in the ΔmetH and ΔmetZ mutants might be caused by the insufficient supply of energy ATP. Finally, the addition of both dibasic sodium succinate as a substrate of FAD recycling and methionine significantly restored the curdlan production of four mutants. In conclusion, methionine biosynthesis pathway plays an important role in curdlan biosynthesis in Agrobacterium sp. CGMCC 11546, which affected the sufficient supply of energy ATP from the ETC during the curdlan biosynthesis.

Cured isotropic conductive adhesive (ICA) with different conductive fillers (micro silver flakes) contents were prepared. Based on the loading mode of “rapid loading-holding-rapid unloading” and semi-empirical method from the generalized Kelvin model, micro-indentation tests were carried out to investigate the effect of hygrothermal aging on the mechanical properties of cured ICA. On the whole, the elastic modulus and hardness of cured ICA with high silver content are relatively larger. As the hygrothermal aging time increases, both elastic modulus and hardness decrease at first and then stabilize, and the creep deformation is more evident. Indentation creep displacement–time curves for cured ICA were characterized by the generalized Kelvin model. During the holding stage, creep compliance increases slowly at first and then increases quickly with modulus loss. With the aggravated aging creep compliance increases, and creep compliance of cured ICA with high silver content increases faster at the later stage of hygrothermal aging. The widened retardation peaks with increasing retardation time reflect the different retardation processes during the holding stage. With increasing aging time, the creep rate sensitivity exponent increases and creep resistance reduces accordingly.

Curdlan is a bacterial, water-insoluble, linear homopolysaccharide that has been widely used in the food industry. In this study, genome information of strain CGMCC 11546, a UV-induced high-yield mutant of the model curdlan-producing strain Agrobacterium sp. ATCC 31749, was used to investigate the molecular mechanism of curdlan biosynthesis. The maximum curdlan yield of 47.97 ± 0.57 g/L was obtained from strain CGMCC 11546 by using optimal media containing 60 g/L sucrose, 6 g/L yeast, 2 g/L KH2PO4, 0.4 g/L MgSO4·7H2O, 2 g/L CaCO3, 0.1 g/L FeSO4·7H2O, 0.04 g/L MnSO4, and 0.02 g/L ZnCl2 at 30 °C and 280 rpm after 96 h of fermentation. The gel strength of curdlan was improved by 41 % by knocking out the β-1,3-glucanase genes exoK and exsH of strain CGMCC 11546. Furthermore, the application of curdlan from the ΔexoK-exsH strain in noodles significantly improved the eating quality of both raw and cooked noodles.

In this work, different concentrations of Se-incorporated mesoporous silica nanospheres (MSNs) (5Se/MSNs and 10Se/MSNs) were successfully synthesized via an in-situ one-pot method. Their physicochemical properties were characterized by X-ray diffraction (XRD), transmission electron microscopy, and X-ray photoelectron spectroscopy (XPS). The release behaviors of Se and Si were investigated in a phosphate-buffered saline (pH = 5.5, 7.4) solution (PBS). In vitro antibacterial properties of the prepared samples were evaluated with Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). The cytocompatibilities of the samples were then assessed using L929 cells. Se nanoparticles were successfully loaded onto the outer and inner surfaces of hierarchical mesoporous silica. The sizes of the Se/MSNs nanoparticles were approximately 120 nm for 5Se/MSNs and 210 nm for 10Se/MSNs. The XRD and XPS results showed that Se mainly existed in the form of Se0 in the samples. The Se/MSNs exhibited stable and sustained release of both Si and Se in PBS solution. In vitro antibactericidal tests indicated that the Se/MSNs could exhibit better antibacterial activity against S. aureus than pure Se nanoparticles after 6 and 24 h of culturing. The minimal inhibitory concentration (MIC) of 10Se/MSN was 100 μg mL-1. However, the Se/MSNs exhibited no inhibitory effect on E. coli bacteria. Furthermore, all the samples exhibited excellent cell viability. These studies demonstrate initial in vitro antibacterial activity and good cytocompatibility of Se/MSNs and their potential application in antibacterial nanomedicine.

A novel ordered TiO2 nanodot array on 316LSS (NTA/316LSS) was first prepared by sol-gel in situ growth method. The surface microstructure, chemical components, antibacterial activity and cytocompatibility of the samples were characterized. The results showed that anatase TiO2 nanogranules (45 ± 5 nm) were embedded into nanopits (50 ± 5 nm) and arrayed in an ordered manner. The in vitro antibacterial test showed that there was a 96.3 ± 2.4% reduction in E. coli on the NTA/316LSS surface and a 98.1 ± 1.3% reduction in S. aureus compared to that on untreated 316LSS controls. Furthermore, NTA/316LSS showed good cytocompatibility in vitro. Our study revealed that such TiO2 nanodot surface can be designed to improve the use of 316LSS for various implant application.

Protein-based hydrogels that possess tunable properties have long been a challenge in tissue engineering. Keratin is a group of natural proteins derived from skin and skin appendant, and features a rich content of cysteine residue which exists in the form of disulfide bonds. Inspired by this, in this work, a simple disulfide shuffling strategy was utilized to develop keratin hydrogels by converting the intramolecular disulfide bonds into the intermolecular disulfide bonds. To achieve this, the intramolecular disulfide bonds were first cleaved by the reductive reagent such as cysteine, to liberate free thiol group, which formed intermolecular disulfide bonds through thiol oxidation. It was demonstrated that control of the cysteine level led to a tunable disulfide crosslinking density, and thus an altered network structure, gel degradation, and drug release rate. Also, this strategy enables good biocompatibility of the material owing to avoiding extra chemical crosslinkers in the preparation procedure. Moreover, this keratin hydrogel had redox-responsive capacity in both gel degradation and drug release due to the disulfide-bond based network structure, providing extensive applicability in tissue engineering and drug release.

Based on the linear rheological constitutive model that is used to describe viscoelastic-plastic behavior of viscoelastic materials, a formula of the Poisson’s ratio was deduced according to the relationship between the shear creep compliance and tensile compliance. Instrumented indentation under various loading conditions and universal creep tests were performed on polyamide 12 samples to obtain the relevant rheological parameters. Results show that the Poisson’s ratio for a step load indentation can obtain a constant but overrated value. However, the Poisson’s ratio approaches an asymptotic value and an accurate value can be gained at a certain loading rate.

The accurate acquisition of the elastoplastic mechanical parameters of metals through in situ indentation tests is an important topic, especially for the technological domains of micro-nano manufacturing. In view of existing uniqueness and effectiveness problems of the current indentation reverse analyses, a method for extracting the power hardening elastoplastic constitutive relation using plural indenters is proposed. Elastic modulus E and Poisson's ratio υ are obtained on the basis of the test result in cylindrical flat punch indenter, and the loading curves are corrected by eliminating the indentation size effects of two pyramid indenters with different equivalent semi-cone angles. The yielding stress σy and strain hardening exponent n can be uniquely determined by combining the representative stress σr under two pyramid indenters that are obtained from the numerical inversion analyses with corresponding representative plastic strain ɛr of certain pyramid indenters. Specific implementing procedures are carried out on Cu and Al. Results show the effectiveness and accuracy of this analytical method.