Β-sheet Structure Research Articles

Development of “clean label” gluten-free breads fortified with flaxseed slurry and sesame cake: Implications on batter rheology, bread quality and shelf life

Following the recently emerged trends for products without additives and valorization of food processing by-products, “clean label” gluten-free (GF) breads were developed using a flaxseed slurry (FS) at 3% or 4.5% level (flaxseed basis), instead of the commonly used structurant in GF doughs, methylcellulose, and 3 or 6% (flour mixture basis) sesame cake (SC) for further product nutritional improvement. These alternative ingredients increased batter hardness and cohesiveness (back extrusion test) as well as elastic modulus, complex and steady shear viscosity (rheometry), compared to formulation containing only methylcellulose (control). The fortified breads had lower loaf specific volumes, than the control, but significantly higher than a GF bread made without any added hydrocolloid. FS and SC enhanced the formation of β-sheet structures (FTIR spectroscopy) in the batter and bread protein matrix. FS breads exhibited similar crumb hardness, cohesiveness, and springiness (TPA test) with the control, whereas inclusion of SC compromised these textural attributes. Nevertheless, the latter samples showed less pronounced textural changes upon storage (25°C-48 h), with similar extent of staling to control. For the fortified stored breads, there were no significant changes in protein conformation, compared to fresh products, whereas crumb starch retrogradation (calorimetry-DSC) was lower than the control. Additionally, the FS and SC increased the protein and dietary fiber contents and introduced nutty and sesame-like flavor notes to the breads, leading to improved overall acceptability scores. Overall, FS and SC seemed to be promising functional ingredients for the development of “clean label” GF breads with enhanced quality and shelf life.

ELECTROWRITING (CELL-LADEN) NATURAL-DERIVED POLYMER FIBRES

Orthopaedic soft tissues, such as tendons, ligaments, and articular cartilage, rely on their unique collagen fiber architectures for proper functionality. When these structures are disrupted in disease or fail to regenerate in engineered tissues, the tissues transform into dysfunctional fibrous tissues. Unfortunately, collagen synthesis in regenerating tissues is often slow, and in some cases, collagen fibers do not regenerate naturally after injury, limiting repair options. One of the research focuses of my team is to develop functional fiber replacements that can promote in vivo repair of musculoskeletal tissues throughout the body. In this presentation, I will discuss our recent advancements in electrowriting 3D printing of natural polymers for creating functional fiber replacements. This manufacturing process utilizes electrical signals to control the flow of polymeric materials through an extrusion nozzle, enabling precise deposition of polymeric fibers with sizes that cannot be achieved using conventional extrusion printing methods. Furthermore, it allows for the formation of fiber organizations that surpass the capabilities of conventional electrospinning processes. During the presentation, I will showcase examples of electrowritten microfiber scaffolds using various naturally-derived polymers, such as gelatin (a denatured form of collagen) and silk fibroin. I will discuss the functional properties of silk-based scaffolds and highlight how they exhibit restored β-sheet and α-helix structures [1]. This restoration results in an elastic response of up to 20% deformation and the ability to withstand cyclic loading without plastic deformation. Additionally, I will present our latest results on the compatibility of this technique with patterning cell-laden fiber structures [2]. This novel biofabrication process allows for the printing of biomimetic microscale architectures with high cell viability, and offers a promising approach to understanding how shear and elongation forces influence cell development of hierarchical (collagen) fibers.Acknowledgements: The author would like to thank the Reprint project (OCENW.XS5.161) and the program “Materials Driven Regeneration” (024.003.013) by the Netherlands Organization for Scientific Research for the financial support.

Tunable thermal diffusivity of silk protein assemblies based on their structural control and photo-induced chemical cross-linking.

Silk, which has excellent mechanical properties and is lightweight, serves as a structural material in natural systems. However, the structural and functional applications of silk in artificial systems have been limited due to the difficulty in controlling its properties. In this study, we demonstrate the tunable thermal diffusivity of silk-based assemblies (films) based on secondary structural control and subsequent cross-linking. We found that the thermal diffusivity of the silk film is increased by the formation of β-sheet structures and dityrosine between Tyr residues adjacent to the β-sheet structures. Our results demonstrate the applicability of silk proteins as material components for thermally conductive biopolymer-based materials.

Biotechnological applications of amyloid fibrils.

Protein aggregates and amyloid fibrils have special qualities and are used in a variety of biotechnological applications. They are extensively employed in bioremediation, biomaterials, and biocatalysis. Because of their capacity to encapsulate and release pharmaceuticals and their sensitivity to certain molecules, respectively, they are also used in drug delivery and biosensor applications. They have also demonstrated potential in the domains of food and bioremediation. Additionally, amyloid peptides have drawn interest in biological applications, especially in the investigation of illnesses like Parkinson's and Alzheimer's. The unique characteristics of amyloid fibrils, namely their mechanical strength and β-sheet structure, make them adaptable to a wide range of biotechnological uses. Even with their promise, one important factor to keep in mind before widely using modified amyloid materials is their potential toxicity. Thus, current research aims to overcome safety concerns while maximizing their potential.

Porous organic cages as inhibitors of Aβ42 peptide aggregation: a simulation study.

The aggregation of Aβ monomers into oligomers with β-sheet structures is an important cause of Alzheimer's disease (AD), while the Aβ42 peptide is more toxic and prone to aggregate. It is of great significance to study the inhibition mechanism of Aβ42 monomer aggregation and find excellent inhibitors for the treatment of AD. Research in recent years has focused on small molecule compounds and nanoparticles, but they all have certain limitations. As a new type of porous material, a porous organic cage (POC) has potential application feasibility in the biomedical field due to its unique physicochemical properties. In this work, molecular dynamics simulations were used for the first time to explore the interaction and conformational transformation of the Aβ42 peptide in CC3 crystals with different morphologies (planar and spherical). The results show that the adsorption of the Aβ42 peptide on different CC3 crystals is mainly achieved through strong van der Waals forces. During the simulations, the Aβ42 peptide undergoes various degrees of structural changes. Compared to that in water, this binding induces more irregular structures, such as turns and 3-helices, and inhibits the production of β-sheets, while enhancing the overall backbone rigidity of the Aβ42 peptide. The transformation analysis of peptide conformation is further complemented by free energy landscape and cluster analysis. These findings provide a strong basis for CC3 crystals as novel inhibitors to inhibit the toxicity and aggregation of the Aβ42 peptide.

Molecular mechanisms involved in the destabilization of two types of R3-R4 tau fibrils associated with chronic traumatic encephalopathy by Fisetin.

Chronic traumatic encephalopathy is a neurodegenerative tauopathy pathologically characterized by fibrillary tau aggregates in the depth of sulci. Clearing fibrous tau aggregates is considered a promising strategy in the treatment of CTE. Fisetin (FS), a natural polyphenolic small molecule, was confirmed to disassociate the tau filaments in vitro. However, the molecular mechanisms of FS in destabilizing the CTE-related R3-R4 tau fibrils remain largely unknown. In this study, we compared the atomic-level structural differences of the two types of CTE-related R3-R4 tau fibrils and explored the influence and molecular mechanisms of FS on the two types of fibrils by conducting multiple molecular dynamics (MD) simulations. The results reveal that the type 1 fibril displays higher structural stability than the type 2 fibril, with a lower root-mean-square-fluctuation value and higher β-sheet structure probability. FS can destabilize both types of fibrils by decreasing the β-sheet structure content, interrupting the mainchain H-bond network, and increasing the solvent accessible surface area and β7-β8 angle of the fibrils. H-bonding, π-π stacking and cation-π are the common interactions driving FS molecules binding on the two types of fibrils, while the hydrophobic interaction occurs only in the type 2 fibril. Due to the relatively short simulation time, our study captures the early molecular mechanisms. However, it does provide beneficial information for the design of drugs to prevent or treat CTE.

Self-assembled peptide hydrogel loaded with functional peptide Dentonin accelerates vascularized bone tissue regeneration in critical-size bone defects.

Regeneration of oral craniofacial bone defects is a complex process, and reconstruction of large bone defects without the use of exogenous cells or bioactive substances remains a major challenge. Hydrogels are highly hydrophilic polymer networks with the potential to promote bone tissue regeneration. In this study, functional peptide Dentonin was loaded onto self-assembled peptide hydrogels (RAD) to constitute functionally self-assembling peptide RAD/Dentonin hydrogel scaffolds with a view that RAD/Dentonin hydrogel could facilitate vascularized bone regeneration in critical-size calvarial defects. The functionalized peptide RAD/Dentonin forms highly ordered β-sheet supramolecular structures via non-covalent interactions like hydrogen bonding, ultimately assembling into nano-fiber network. RAD/Dentonin hydrogels exhibited desirable porosity and swelling properties, and appropriate biodegradability. RAD/Dentonin hydrogel supported the adhesion, proliferation and three-dimensional migration of bone marrow mesenchymal stem cells (BMSCs) and has the potential to induce differentiation of BMSCs towards osteogenesis through activation of the Wnt/β-catenin pathway. Moreover, RAD/Dentonin hydrogel modulated paracrine secretion of BMSCs and increased the migration, tube formation and angiogenic gene expression of human umbilical vein endothelial cells (HUVECs), which boosted the angiogenic capacity of HUVECs. In vivo, RAD/Dentonin hydrogel significantly strengthened vascularized bone formation in rat calvarial defect. Taken together, these results indicated that the functionalized self-assembling peptide RAD/Dentonin hydrogel effectively enhance osteogenic differentiation of BMSCs, indirectly induce angiogenic effects in HUVECs, and facilitate vascularized bone regeneration in vivo. Thus, it is a promising bioactive material for oral and maxillofacial regeneration.

Novel non-helical antimicrobial peptides insert into and fuse lipid model membranes.

This research addresses the growing menace of antibiotic resistance by exploring antimicrobial peptides (AMPs) as alternatives to conventional antibiotics. Specifically, we investigate two linear amphipathic AMPs, LE-53 (12-mer) and LE-55 (16-mer), finding that the shorter LE-53 exhibits greater bactericidal activity against both Gram-negative (G(-)) and Gram-positive (G(+)) bacteria. Remarkably, both AMPs are non-toxic to eukaryotic cells. The heightened effectiveness of LE-53 is attributed to its increased hydrophobicity (H) compared to LE-55. Circular dichroism (CD) reveals that LE-53 and LE-55 both adopt β-sheet and random coil structures in lipid model membranes (LMMs) mimicking G(-) and G(+) bacteria, so secondary structure is not the cause of the potency difference. X-ray diffuse scattering (XDS) reveals increased lipid chain order in LE-53, a potential key distinction. Additionally, XDS study uncovers a significant link between LE-53's upper hydrocarbon location in G(-) and G(+) LMMs and its efficacy. Neutron reflectometry (NR) confirms the AMP locations determined using XDS. Solution small angle X-ray scattering (SAXS) demonstrates LE-53's ability to induce vesicle fusion in bacterial LMMs without affecting eukaryotic LMMs, offering a promising strategy to combat antibiotic-resistant strains while preserving human cell integrity, whereas LE-55 has a smaller ability to induce fusion.

Cation effects and charge inversion contribute to the electrostatic stabilisation of protein bioconjugates in neat ionic liquids.

Dispersing and stabilising proteins in ionic liquids (ILs) provides significant opportunities for green solvent-based biocatalysis, especially in industrial processes at elevated temperatures. While unmodified proteins undergo denaturation, their polymer-conjugated counterparts have been stabilised in neat ILs. However, the nature of interactions and the generality of protein-bioconjugate stabilisation in neat ILs require further understanding. Using a combination of different physio-chemical experimental tools and molecular dynamics (MD) simulations, here we investigate the dispersion and driving force for the stabilisation of bioconjugates in neat ILs. Solvent-free bioconjugates of different proteins, viz. myoglobin, α-chymotrypsin, and regenerated silk fibroin having predominant α-helical, β-sheet, and random coiled secondary structures, respectively, were prepared by electrostatic coupling with polyethylene-glycol (PEG)-based polymer-surfactant (PS). Protic IL (PIL, N-methyl-2-pyrolidonium trifluoromethane sulfonate; [NMP][OTF]) and aprotic ionic liquid (AIL, 1-methyl-3-(4-sulfobutyl)-1H-imidazol-3-ium trifluoromethane sulfonate; [MEZ][OTF]) were synthesized to study the bioconjugate dispersion. Interestingly, time-dependent polarised optical microscopy combined with transmittance measurements showed complete dispersion of all bioconjugates only in AIL. MD simulations of the PS-cCT bioconjugate were carried out in the same ILs as the experiments. The surface electrostatic potential of PS-cCT reversed from positive in PIL to negative in AIL due to overcharging by the AIL anion and lower mobility of the AIL cation. Strong screening of electrostatic potentials between two PS-cCT complexes in PIL resulted in reduced dispersion stability. Lower diffusivity of long alkyl chain [MEZ] cations of AIL leads to a depletion zone of IL ions between the two PS-cCT complexes (separation <70 Å), thus resulting in a significant negative potential between the complexes. Hence, protein bioconjugates in AIL were stabilised by a combination of surface overcharging and steric exclusion of [MEZ] cations from the space between the approaching bioconjugate complexes.

Experimental techniques for detecting and evaluating the amyloid fibrils.

Amyloid fibrils are insoluble proteins with intricate β-sheet structures associated with various human diseases, including Parkinson's, Alzheimer's, and prion diseases. Proteins can form aggregates when their structure is misfolded, resulting in highly organized amyloid fibrils or amorphous aggregates. The formation of protein aggregates is a promising research field for mitigating diseases and the pharmaceutical and food industries. It is important to monitor and minimize the appearance of aggregates in these protein products. Several methods exist to assess protein aggregation, that includes from basic investigations to advanced biophysical techniques. Physicochemical parameters such as molecular weight, conformation, structure, and dimension are examined to study aggregation. There is an urgent need to develop methods for the detection of protein aggregation and amyloid fibril formation both in vitro and in vivo. This chapter focuses on a comprehensive discussion of the methods used to characterize and evaluate aggregates and amyloid fibrils.

Novel photocatalytic carbon dots: efficiently inhibiting amyloid aggregation and quickly disaggregating amyloid aggregates.

Amyloid aggregation is implicated in the pathogenesis of various neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD). It is critical to develop high-performance drugs to combat amyloid-related diseases. Most identified nanomaterials exhibit limited biocompatibility and therapeutic efficacy. In this work, we used a solvent-free carbonization process to prepare new photo-responsive carbon nanodots (CNDs). The surface of the CNDs is densely packed with chemical groups. CNDs with large, conjugated domains can interact with proteins through π-π stacking and hydrophobic interactions. Furthermore, CNDs possess the ability to generate singlet oxygen species (1O2) and can be used to oxidize amyloid. The hydrophobic interaction and photo-oxidation can both influence amyloid aggregation and disaggregation. Thioflavin T (ThT) fluorescence analysis and circular dichroism (CD) spectroscopy indicate that CNDs can block the transition of amyloid from an α-helix structure to a β-sheet structure. CNDs demonstrate efficacy in alleviating cytotoxicity induced by Aβ42 and exhibit promising blood-brain barrier (BBB) permeability. CNDs have small size, low biotoxicity, good fluorescence and photocatalytic properties, and provide new ideas for the diagnosis and treatment of amyloid-related diseases.

Biochemical and Biophysical Characterization of Tau and α-Linolenic Acid Vesicles In Vitro.

Alzheimer's disease (AD) is characterized by the abnormal accumulation of disordered protein, that is, extracellular senile plaques of amyloid-β (Aβ) and intracellular neurofibrillary tangles of Tau. Tau protein has gained the attention in recent years owing to the ability to propagate in a "prion-like" nature. The disordered protein Tau possesses a high positive charge, which allows its binding to anionic proteins and factors. The native disorder of proteins attends the β-sheet structure from its random-coiled conformation upon charge compensation by various polyanionic agents such as heparin, RNA, etc. Anionic lipids such as arachidonic acid (AA) and oleic acid (OA) are also one of the factors which can induce aggregation of Tau in physiological conditions. The free units of Tau protein can bind to lipid membranes through its repeat domain (RD), the anionic side chains of the membrane lipids induce aggregation of Tau by reducing the activation barrier. In this study, we investigated the role of α-linolenic acid (ALA) as an inducing agent for Tau aggregation in vitro conditions. Omega-3 fatty acids bear a capacity to reduce the pathology of Tau by downregulating the Tau phosphorylation pathway. We have studied by using various biochemical or biophysical methods the potency of ALA as an aggregating agent for Tau. We have implemented different techniques such as SDS-PAGE, transmission electron microscopy, CD spectroscopy to evaluated higher-order aggregates of Tau upon induction by ALA.

Pregnenolone derivatives for the treatment of Alzheimer's disease: synthesis, and in vitro inhibition of amyloid β1-42 peptide aggregation, acetylcholinesterase and carbonic anhydrase-II.

The amyloid state, which is a specific conformation of proteins, offers valuable information about both functional protein structures and the pathological assemblies associated with various diseases. One of the major hallmarks of Alzheimer's disease includes primarily the extracellular build-up of a peptide known as amyloid-β, which has a sequence consisting of 39 to 42 amino acid residues, and the formation of intracellular neurofibrillary tangles mostly consisting of hyperphosphorylated tau protein. Drugs that are expected to reduce Aβ production, prevent Aβ aggregation, and promote Aβ clearance are promising approaches for treating AD. Current work is focused on identifying the compounds that have balanced even mild biological activities against multiple targets instead of finding one-target compound with high potency. We synthesized pregnenolone derivatives and evaluated their potential against inhibition of eeAChE/eqBChE, hCA-II and self-mediated Aβ1-42 peptide aggregation. Our synthesized derivatives 23, and 25-27 exhibited concomitant inhibition of all the tested macromolecular targets. All the active compounds were found to be BBB penetrants in the PAMPA assay. Furthermore, these selected compounds were found to be non-neurotoxic in the MTT assay on neuroblastoma SH-SY5Y cells. Docking studies support dual binding site (PAS and CAS) inhibition of AChE which showed Aβ1-42 aggregation and AChE inhibition. Moreover, docking studies carried out on the 3D crystallographic structure of Aβ1-42 peptide (PDB ID = 1IYT) showed significant interactions with amino acid residues Asp 23 and Lys 28, and hydrophobic interactions with the Phe19, Phe20, and Ala 30 effectively impeding the formation of β-sheet structures.

Identification and taste characteristics of novel umami peptides from Yanjin black bone chicken hydrolysates and their binding mechanism with umami receptor.

This study aimed to obtain umami peptides from Yanjin black bone chicken and to investigate the formation mechanism of umami taste. The umami peptides were purified from the enzymatic hydrolysate of chicken using ultrafiltration (UF), gel filtration chromatography (GFC), and reversed-phase high-performance liquid chromatography (RP-HPLC). Potential novel umami peptides were then identified by nano-scale liquid chromatography-tandem mass spectrometry (nano-HPLC-MS/MS). Based on the predictions of iUmami-SCM and BIOPEP-UWM databases, five umami peptides (EELK, EEEIK, EELMK, LEEEIK, DELDKYS) with high umami scores were synthesized. Sensory evaluation revealed that these umami peptides exhibited a threshold ranging from 0.12 mg mL-1 to 0.36 mg mL-1. Circular dichroism (CD) analysis indicated the presence of β-sheet structures in the umami peptides that could be associated with taste formation. In addition, molecular docking and molecular dynamics (MD) were employed to investigate the binding mechanisms between umami peptides and the umami receptor T1R1/T1R3. The findings reveal that Lys155, Arg255, and Gln250 of T1R1/T1R3 potentially play critical roles in umami peptide binding. Taken together, our results lay a foundation for researching flavor substances and for developing flavor products from Yanjin black bone chicken.

Oncogenic R248W mutation induced conformational perturbation of the p53 core domain and the structural protection by proteomimetic amyloid inhibitor ADH-6.

The involvement of p53 aggregation in cancer pathogenesis emphasizes the importance of unraveling the mechanisms underlying mutation-induced p53 destabilization. And understanding how small molecule inhibitors prevent the conversion of p53 into aggregation-primed conformations is pivotal for the development of therapeutics targeting p53-aggregation-associated cancers. A recent experimental study highlights the efficacy of the proteomimetic amyloid inhibitor ADH-6 in stabilizing R248W p53 and inhibiting its aggregation in cancer cells by interacting with the p53 core domain (p53C). However, it remains mostly unclear how R248W mutation induces destabilization of p53C and how ADH-6 stabilizes this p53C mutant and inhibits its aggregation. Herein, we conducted all-atom molecular dynamics simulations of R248W p53C in the absence and presence of ADH-6, as well as that of wild-type (WT) p53C. Our simulations reveal that the R248W mutation results in a shift of helix H2 and β-hairpin S2-S2' towards the mutation site, leading to the destruction of their neighboring β-sheet structure. This further facilitates the formation of a cavity in the hydrophobic core, and reduces the stability of the β-sandwich. Importantly, two crucial aggregation-prone regions (APRs) S9 and S10 are disturbed and more exposed to solvent in R248W p53C, which is conducive to p53C aggregation. Intriguingly, ADH-6 dynamically binds to the mutation site and multiple destabilized regions in R248W p53C, partially inhibiting the shift of helix H2 and β-hairpin S2-S2', thus preventing the disruption of the β-sheets and the formation of the cavity. ADH-6 also reduces the solvent exposure of APRs S9 and S10, which disfavors the aggregation of R248W p53C. Moreover, ADH-6 can preserve the WT-like dynamical network of R248W p53C. Our study elucidates the mechanisms underlying the oncogenic R248W mutation induced p53C destabilization and the structural protection of p53C by ADH-6.

A novel approach to chiral separation: thermo-sensitive hydrogel membranes.

The application of membrane technology for separating chiral compounds is hindered due to the restricted availability of chiral recognition sites on the membrane surface. In this study, we propose a novel approach for chiral separation through a selector (bovine serum albumin, BSA) mediated thermo-sensitive membrane system. A thermo-sensitive hydrogel-coated membrane (termed PDTAN) was developed by anchoring poly(N-isopropylacrylamide) (PNIPAm) onto a polyethersulfone (PES) membrane through an adhesive and hydrophilic dopamine hydrochloride (PDA)/tannic acid (TA)/chitosan (Chi) intermediate layer. The results demonstrate outstanding chiral separation efficiency, achieving αL/D = 3.30 for D-phenylalanine (D-Phe) rejection at 40 °C on a BSA-mediated PDTAN membrane system, with significant stability and minimal fouling, surpassing previous findings. Moreover, the PDTAN membrane altered the selective properties of recognition sites in BSA, transitioning from rejecting L-Phe to rejecting D-Phe. Analysis using fourth-order derivative UV-vis, circular dichroism (CD), and in situ Fourier transform infrared spectroscopy (FTIR) techniques revealed a transition in the secondary structure of BSA from α-helix to β-sheet as the temperature increased. This transition, facilitated by hydrogen bonding between BSA and PNIPAm, enabled selective recognition of D-Phe, demonstrating a distinct shift in chiral recognition properties. Importantly, with D-Phe adsorbed onto β-sheet structures of BSA, hydrogen-bond interactions between BSA and the PDTAN membrane were significantly reduced, thereby minimizing membrane fouling and achieving the durability of membrane-based chiral separation.

Fusion of amyloid beta with ferritin yields an isolated oligomeric beta-sheet-rich aggregate inside the ferritin cage.

Alzheimer's disease is a severe brain condition caused by the formation of amyloid plaques composed of amyloid beta (Aβ) peptides. These peptides form oligomers, protofibrils, and fibrils before deposition into amyloid plaques. Among these intermediates, Aβ oligomers (AβOs) were found to be the most toxic and therefore an appealing target for drug development and understanding their role in the disease. However, precise isolation and characterization of AβOs have proven challenging because AβOs tend to aggregate and form heterogeneous mixtures in solution. As a solution, we genetically fused the Aβ peptide with a ferritin monomer. Such fusion allowed the encapsulation of precisely 24 Aβ peptides inside the 24-mer ferritin cage. Using high-speed atomic force microscopy (HS-AFM), we disassembled ferritin and directly visualized the Aβ core enclosed within the cage. The thioflavin-T assay (ThT) and attenuated total reflection infrared spectroscopy (ATR-IR) revealed the presence of a β-sheet structure in the encapsulated oligomeric aggregate. Gallic acid, an amyloid inhibitor, can inhibit the fluorescence of ThT bound AβOs. Our approach represents a significant advancement in the isolation and characterization of β-sheet rich AβOs and is expected to be useful for future studies of other disordered peptides such as α-synuclein and tau.

Dual-network DNA-silk fibroin hydrogels with controllable surface rigidity for regulating chondrogenic differentiation.

Osteoarthritis (OA) is a common joint disease known for cartilage degeneration, leading to a substantial burden on individuals and society due to its high disability rate. However, current clinical treatments for cartilage defects remain unsatisfactory due to the unclear mechanisms underlying cartilage regeneration. Tissue engineering hydrogels have emerged as an attractive approach in cartilage repair. Recent research studies have indicated that stem cells can sense the mechanical strength of hydrogels, thereby regulating their differentiation fate. In this study, we present the groundbreaking construction of dual-network DNA-silk fibroin (SF) hydrogels with controllable surface rigidity. The supramolecular networks, formed through DNA base-pairing, induce the development of β-sheet structures by constraining and aggregating SF molecules. Subsequently, SF was cross-linked via horseradish peroxidase (HRP)-mediated enzyme reactions to form the second network. Experimental results demonstrated a positive correlation between the surface rigidity of dual-network DNA-SF hydrogels and the DNA content. Interestingly, it was observed that dual-network DNA-SF hydrogels with moderate surface rigidity exhibited the highest effectiveness in facilitating the migration of bone marrow mesenchymal stem cells (BMSCs) and their chondrogenic differentiation. Transcriptome sequencing further confirmed that dual-network DNA-SF hydrogels primarily enhanced chondrogenic differentiation of BMSCs by upregulating the Wnt and TGF-β signaling pathways while accelerating collagen II synthesis. Furthermore, in vivo studies revealed that dual-network DNA-SF hydrogels with moderate surface rigidity significantly accelerated cartilage regeneration. In summary, the dual-network DNA-SF hydrogels represent a promising and novel therapeutic strategy for cartilage regeneration.

Valorizacija ljušture invazivnih rakova iz reke Dunav (Faxonius limosus) - ekstrakcija i karakterizacija proteina

In order to deal with invasive crayfish (Faxonius limosus) impact on the native crayfish, as well as fish biodiversity in the Danube River, a possible solution would be to find and adopt mechanisms for its utilization for novel valuable product production. Apart from utilizing edible parts for novel food products, the shell waste can also be considered a source of valuable compounds. The complex structure of the shell mainly consists of three basic compounds: chitin, protein and minerals-mainly calcium carbonate. In this study, shell waste proteins were extracted using three extraction methods. The first method involved using naturally present enzymes (proteases and lipases) in crayfish wastes to recover proteins through an autolysis process. To accelerate the process, UV radiation was used. The remaining two extraction methods were alkaline extraction of proteins, wherein one method alkaline extraction was applied directly to the shell waste, and the other method, alkaline extraction followed an acidic demineralization step of the shell waste. The obtained protein concentrates were analysed for yield, crude protein content, DPPH radical scavenging ability, amino acid content, and structure. The results have shown that a similar percentage of protein content was obtained by all three methods: 67.20-68.03 %, but the extraction yield was considerably different. Alkaline deproteinization with or without the step of demineralization resulted in a 9.20-10.98 % yield, while UV radiation accelerated autolysis resulted in only a 3.41 % yield. Although protein extraction without using exogenous enzymes or chemicals is a very interesting approach, a drawback of this approach is the low process yield. FTIR spectroscopy revealed a secondary structure that was similar in all three concentrates, according to peak deconvolution, with the autolytic concentrate differing to a lesser extent, showing a slightly higher share of b-sheet structures. The DPPH assay revealed high antioxidant activity of the concentrates (72.00-88.06 %), probably originating from active peptides derived from proteins and residual carotenoids, mainly astaxanthin.

Novel adhesive based on black soldier fly larvae flour for particleboard production

Protein-based adhesives are receiving considerable attention due to their potential to contribute to the development of environmentally friendly materials. Black soldier fly larvae (BSFL) can convert organic waste into high value-added products, such as proteins and fatty acids. The objective of this study is to formulate adhesives from BSFL flour and evaluate their suitability in the production of particleboards. To expose the functional groups of the proteins for reaction with the wood, denaturation strategies involving alkaline treatments were assessed. The effectiveness of these treatments was evaluated through Bradford solubility, SDS-PAGE, and FTIR analysis. Bradford analysis confirmed that the alkali treatment increased the protein solubility from 10.1 g/l for 0 M to 89.1 g/l for 1 M. FTIR analysis revealed changes in the relative intensity of bands attributable to the Amide I, II, and III regions, which are associated with the α-helix and β-sheet structures. The adhesive was applied in the manufacturing process via spraying, requiring an evaluation of the rheological properties of different formulations. Based on the rheological properties, the viscosities of 0.25 M and 1 M were measured at 0.082 Pa·s and 0.113 Pa·s, respectively, falling within the suggested range for particleboard manufacture. Subsequently, eucalyptus wood panels were manufactured using the BSFL-based adhesives, and their mechanical properties as well as water resistance were evaluated. Static bending and tensile strength perpendicular to the surface testing revealed that boards produced with BSFL adhesive exhibited mechanical values comparable to those of commercial resins. The performance of the manufactured materials was compared with the minimum standards outlined in international regulations.