A mutant of hydrophobin HGFI tuning the self-assembly behaviour and biosurfactant activity.

Hydrophobins are a series of low molecular weight proteins produced by filamentous fungi that play an important role in fungal growth. They have a globular structure and possess a unique hydrophobic patch on their surface that makes them amphiphilic, making them among the most surface-active proteins. Herein, the surface charge properties of HGFI, a class I hydrophobin from Grifola frondosa, were altered by replacing the negatively charged Glu24 with a positively charged Lys to generate the ME24 mutant. Pichia pastoris GS115 was used for recombinant expression of the ME24 mutant, which was purified by a two-step procedure. The function of the mutated residue in HGFI self-assembly was investigated. Reverse-phase high-performance liquid chromatography analysis revealed that the polarity of ME24 was enhanced compared with HGFI. Circular dichroism, thioflavin T assay, water contact angle and atomic force microscopy indicated that Glu24 participates in rodlet formation. Water solubility detection and dynamic light scattering showed that Glu24 affects the assembled state of HGFI in aqueous solution. The behaviour of the mutant in an emulsion, in the dispersion of insoluble materials and in large-scaled protein production suggests the functions of hydrophobins can be tuned for new applications.

A self-elastic chitosan sponge integrating active and passive hemostatic mechanisms for effectively managing uncontrolled coagulopathic hemorrhage.

Developing a self-elastic sponge integrating active and passive hemostatic mechanisms for the effective management of uncontrolled coagulopathic hemorrhage remains a challenge. We here developed a chitosan-based sponge by integrating freeze-drying, chemical decoration of alkyl chains and phosphate groups, and physical loading of thrombin. The sponge exhibited high mechanical strength, self-elasticity, and rapid shape recovery. The sponge facilitated blood cell adhesion, aggregation, and activation through hydrophobic and electrostatic interactions, as well as accelerated blood clotting. The sponge exhibited higher efficacy than commercial gauze and gelatin sponge in managing uncontrolled hemorrhage from heparinized rat tail amputation, liver superficial injury, and liver perforating wound models. In addition, the sponge exhibited favorable biodegradability and biocompatibility. These findings revealed that the developed sponge holds great potential as a novel hemostat for effectively managing uncontrolled coagulopathic hemorrhage from superficial and perforating wounds.

Liraglutide Protects Pancreatic Islet From Ischemic Injury by Reducing Oxidative Stress and Activating Akt Signaling During Cold Preservation to Improve Islet Transplantation Outcomes.

BACKGROUND: Islet transplantation is a promising therapy for patients with type 1 diabetes. However, ischemic injury to the donor islets during cold preservation leads to reduced islet quality and compromises transplant outcome. Several studies imply that liraglutide, a glucagon-like peptide-1 receptor agonist, has a positive effect on promoting islet survival, but its impact on islet cold-ischemic injury remains unexplored. Therefore, the aim of this study was to investigate whether liraglutide can improve islet transplantation efficacy by inhibiting cold-ischemic injury and to explore the underlying mechanisms. METHODS: Liraglutide was applied in a mouse pancreas preservation model and a human islets cold-preservation model, and islet viability, function, oxidative stress levels were evaluated. Furthermore, islet transplantation was performed in a syngeneic mouse model and a human-to-nude mouse islet xenotransplantation model. RESULTS: The supplementation of liraglutide in preservation solution improved islet viability, function, and reduced cell apoptosis. Liraglutide inhibited the oxidative stress of cold-preserved pancreas or islets through upregulating the antioxidant enzyme glutathione levels, inhibiting reactive oxygen species accumulation, and maintaining the mitochondrial membrane integrity, which is associated with the activation of Akt signaling. Furthermore, the addition of liraglutide during cold preservation of donor pancreas or donor islets significantly improved the subsequent transplant outcomes in both syngeneic mouse islet transplantation model and human-to-nude mouse islet xenotransplantation model. CONCLUSIONS: Liraglutide protects islets from cold ischemia-related oxidative stress during preservation and hence improved islet transplantation outcomes, and this protective effect of liraglutide in islets is associated with the activation of Akt signaling.

Identification and Characterization of a Predominant Hydrophobin in the Edible Mushroom Grifola frondosa.

Hydrophobins (HFBs) are a group of small, secreted amphipathic proteins of fungi with multiple physiological functions and potential commercial applications. In this study, HFB genes of the edible mushroom, Grifola frondosa, were systematically identified and characterized, and their transcriptional profiles during fungal development were determined. In total, 19 typical class I HFB genes were discovered and bioinformatically analyzed. Gene expression profile examination showed that Gf.hyd9954 was particularly highly upregulated during primordia formation, suggesting its major role as the predominant HFB in the lifecycle of G. frondosa. The wettability alteration profile and the surface modification ability of recombinant rGf.hyd9954 were greater than for the Grifola HFB HGFII-his. rGf.hyd9954 was also demonstrated to form the typical class I HFB characteristic-rodlet bundles. In addition, rGf.hyd9954 was shown to possess nanoparticle characteristics and emulsification activities. This research sheds light on the regulation of fungal development and its association with the expression of HFB genes.

A novel hydrophobin encoded by hgfII from Grifola frondosa exhibiting excellent self-assembly ability.

Hydrophobins are small proteins from filamentous fungi, which have remarkable self-assembly properties of great potential, e.g., as drug carriers and as anti-bacterial agents, but different hydrophobins, with improved properties, are needed. HGFI (a hydrophobin from Grifola frondosa) is a class I hydrophobin, which can self-assemble into rodlet structures with a length range 100-150 nm. In this study, we identified a new hydrophobin gene (hgfII) from the mycelium of G. frondosa with a much higher transcriptional level than hgfI. Heterologous expression of hgfII was accomplished in the Pichia pastoris. X-ray photoelectron spectroscopy and water contact angle assay measurements revealed that HGFII can self-assemble into a protein film at the air-solid interface, with circular dichroism and thioflavin T fluorescence studies showing that this effect was accompanied by a decrease in α-helix content and an increase in ß-sheet content. Using atomic force microscopy, it was shown that HGFII self-assembled into rodlet-like structures with a diameter of 15-30 nm, showing that it was a class I hydrophobin, with self-assembly behavior different from HGFI. The surface hydrophobicity of HGFII was stronger than that of HGFI, meanwhile, in emulsification trials, HGFII displayed better dispersive capacity to the soybean oil than HGFI, producing a more stable and durable emulsion.

The enhancement of surface activity and nanoparticle stability through the alteration of charged amino acids of HGFI.

HGFI (hydrophobin found in Grifola frondosa) is a small surface-active protein, belonging to class I hydrophobins, and can form amphipathic protein films at hydrophobic- hydrophilic material interfaces. HGFI has a low isoelectric point (pI) because no basic amino acids are present in its sequence. Here we constructed two HGFI mutants using a site-directed mutation method. The two mutants were named MGF3 (E24K/D35K/D37K) and MGF6 (E12K/D19K/E24K/D35K/D37K/D66K). MGF3 was generated by replacing three acidic amino acids, namely Glu24, Asp35 and Asp37, with the basic amino acid Lys. MGF6 was obtained by substituting Glu12, Asp19, Glu24, Asp35, Asp37 and Asp66 with six Lyses. Zeta-potential analysis showed that the pI values of MGF3 and MGF6 were between 5 and 6. The water contact angle (WCA) and quartz-crystal microbalance (QCM) analyses revealed enhanced adsorption capacity of these two mutants. Circular dichroism measurements (CD), Thioflavin T (THT) assays and atomic force microscopy (AFM) tests indicated that neither MGF3 nor MGF6 have the ability to form rodlets. An in vitro drug release assay showed the water solubility of the indometacin (IND) nano-particles (NPs) was improved by both MGF3 and MGF6 modification. Furthermore, the mutant hydrophobin-modified NPs were more stable and exhibited a lower release rate in solutions at pH 1.2 and 7 at 37 °C. The MGF6-IND NPs showed a particularly improved drug release behavior. These results suggest new applications of the mutated hydrophobins in sustained delivery of oral drugs in the gastrointestinal tract.

The DnaJ protein OsDjA6 negatively regulates rice innate immunity to the blast fungus Magnaporthe oryzae.

Rice blast, caused by Magnaporthe oryzae (synonym: Pyricularia oryzae), severely reduces rice production and grain quality. The molecular mechanism of rice resistance to M. oryzae is not fully understood. In this study, we identified a chaperone DnaJ protein, OsDjA6, which is involved in basal resistance to M. oryzae in rice. The OsDjA6 protein is distributed in the entire rice cell. The expression of OsDjA6 is significantly induced in rice after infection with a compatible isolate. Silencing of OsDjA6 in transgenic rice enhances resistance to M. oryzae and also results in an increased burst of reactive oxygen species after flg22 and chitin treatments. In addition, the expression levels of WRKY45, NPR1 and PR5 are increased in OsDjA6 RNAi plants, indicating that OsDjA6 may mediate resistance by affecting the salicylic acid pathway. Finally, we found that OsDjA6 interacts directly with the E3 ligase OsZFP1 in vitro and in vivo. These results suggest that the DnaJ protein OsDjA6 negatively regulates rice innate immunity, probably via the ubiquitination proteasome degradation pathway.

A high-throughput method for screening of L-tyrosine high-yield strains by Saccharomyces cerevisiae.

A biosensor screening assay based on the synthesis of betaxanthin was applied to relatively high throughput screening of the L-tyrosine mutant library. In the assays, fluorescence output showed a linear relationship between extracellular L-tyrosine content and yellow pigment formation. In addition, the yellow pigment accumulation of the L-tyrosine high-yield strain can be easily distinguished with the naked eye compared with the wild-type strain. As a result, numerous mutants that exhibited significantly increased coloration, were screened out after random mutagenesis, and p-coumaric acid production in mutants NK-A3 and NK-B4, were remarkably improved by 4-fold more than that of the wild-type strain. In general, this study provides a novel strategy for screening mutant libraries in the search for highly L-tyrosine-producing strains.