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With the aim of effectively improving the performance of bio-friendly food packaging and circumventing the hazards associated with petroleum-based plastic food packaging, composite films of corn starch and polyvinyl alcohol were prepared using a new method that involved chemical cross-linking of glutaraldehyde and blending with cinnamon essential oil nanoemulsion (CNE). Glutaraldehyde and CNE enhance the film's network structure by chemical bonding and hydrogen bonding, respectively. This results in improved surface smoothness, mechanical properties, and UV shielding ability of the film. However, the films' surface hydrophilicity increased as a result of CNE, which is harmful for food preservation in high humidity. Overall, glutaraldehyde and CNE have a synergistic effect on some of the properties of the film which is mainly attributed to the films' structure improvement. The films have great potential for preparing flexible and UV-shielding films and offer new ideas for developing biodegradable films., 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 © 2024 Elsevier Ltd. All rights reserved.)

Cellulose aerogel, recognized for its eco-friendliness, bio-sustainability and excellent thermal insulation property, holds immense potential as an insulation material. However, the application of cellulose aerogel has been hindered by its inherent drawbacks, particularly its low strength and flammability. In this study, a reactive P/N-rich curdlan derivative (NT) was synthesized and characterized. Lightweight, ultrahigh-strength and flame-retardant composite aerogels were then prepared by slow freezing, freeze-drying and chemical crosslinking methods. Composite aerogel crosslinked with 30% NT exhibited exceptional thermal insulation property with a low thermal conductivity of 33.9 mW/(m·K), which rivaled the value of petroleum-based thermal insulation materials. It possessed an ultrahigh compressive modulus (0.786 MPa) at low density (21.6 mg/cm 3 ), which supported >12,000 times its weight. It also displayed superior flame-retardant property, with a limiting oxygen index of up to 33.1% and a V-0 rating in the UL-94 test. This study provides a new insight into the high-value utilization of natural polysaccharide and an innovative solution to the problem of low strength and flammability of cellulose aerogel. Cellulose aerogels with ultrahigh strength, superior flame retardancy and thermal insulation property possess a promising application in the fields of construction, aerospace and thermal protective clothing as high-performance thermal insulation materials., 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 © 2024 Elsevier B.V. All rights reserved.)

Considering that fruits are vulnerable to damage and waste during stockpiling, transport and marketing. Given this, an innovative curcumin inclusion compound (Cur@β-CD) was devised in this study to introduce oil-soluble curcumin (Cur) into water-soluble polyvinyl alcohol (PVA) materials, thereby fabricating food packaging films endowed with excellent properties. DPPH test manifested that the oxidation resistance for PCOMC-Cur@β-CD film was 95 % above PVA material. It was ascribed to the fact that the Cur@β-CD elevated the water solubility of Cur while the increase of water solubility heightened the antioxidant effect for Cur in the film. Additionally, the chitosan (CS) was crosslinked with malic acid (MA), which elevated the barrier property of the film, reduced the amount of oxygen transmission and further retarded the oxidation reaction of the fruits for packaging. The antibacterial test demonstrated that the antibacterial rates of PCOMC-Cur@β-CD film against E. coli and S. aureus reached 92 % and 95 %, respectively, which was attributed to the slow release of Cur when Cur@β-CD was dissolved in PVA material and the Schiff base reaction between Cur and amino groups on CS. These findings indicate that the PCOMC-Cur@β-CD film developed in this work can provide certain insights into the field of food packaging., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Poly(glycerol adipate) (PGA) is one of the aliphatic polyesters of glycerol. The most studied biomedical application of poly(glycerol adipate) is the use of its nanoparticles as drug delivery carriers. The PGA prepolymer can be crosslinked to network materials. The biomedical application of PGA-based network materials has largely remained unexplored till recently. The PGA-based network materials, such as poly(glycerol sebacate) elastomers, can be used in soft tissue regeneration due to their mechanical properties. The modulus of elasticity of PGA elastomers is within the range of MPa, which corresponds to the mechanical properties of human soft tissues. This short review aims at briefly summarizing the possible applications of PGA-based elastomers in tissue engineering, as indicated in recent years in research publications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kłusak and Gazińska.)

Porous starch materials are promising in several applications as renewable natural biomaterials. This study reports an approach combining methacrylation of starch and chemical crosslinked cryogelation to fabricate highly elastic macroporous starch (ST-MA) cryogels with impressed water/oil absorption capacity and wet thermal stability among starch based porous materials. Five different types of starch, including pea, normal corn, high amylose corn, tapioca, and waxy maize starch with different amylose content, have been studied. The methacrylation degree is not related with amylose content. All cryogels exhibited excellent compressive elasticity enduring 90 % deformation without failure and good robustness in cyclic tests. The ST-MA cryogels from pea starch exhibited the highest Young's modulus and compressive strength among five types of starch. These covalent cryogels exhibit high wet-thermal stability and enzymatic hydrolysis stability, while still are biodegradable. The dry ST-MA sponges (2 wt%) showed outstanding liquid absorption capacity, absorbing ~40 folds (g/g) of water or ~ 36 folds (g/g) of oil respectively. All types of starch have similar liquid absorption performance. This study provides a universal approach to fabricate highly elastic covalent starch macroporous materials with impressed liquid absorption capacity and outstanding stability, especially wet-thermal stability, and may expand their 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 © 2024. Published by Elsevier Ltd.)

Nanocomposite hydrogels are suitable in bone tissue engineering due to their resemblance with the extracellular matrix, ability to match complex geometries, and ability to provide a framework for cell attachment and proliferation. The nanocomposite hydrogel comprises organic and inorganic counterparts. Gelatin methacrylate (GELMA) is an extensively used organic biomaterial in tissue engineering due to its excellent biocompatibility, biodegradability, and bioactivity. The photo-crosslinking of GELMA presents a challenge when aiming to create thicker nanocomposite hydrogels due to opacity induced by fillers, which obstructs the penetration of ultraviolet (UV) light. Therefore, using a chemical crosslinking approach, we have developed nanocomposite GELMA hydrogel in this study by incorporating citrate-containing amorphous calcium phosphate (ACP_CIT). Ammonium persulfate (APS) and Tetramethylethylenediamine (TEMED) were deployed to crosslink the methacrylate group of GELMA. The oscillatory shear tests have confirmed that crosslinking enhances both storage (G') and loss modulus (G″) of GELMA. Subsequently, incorporation of ACP_CIT in GELMA hydrogel shows further enhancement in G' and G″ values. In vitro analysis of the developed hydrogels revealed that chemical crosslinking and incorporation of ACP_CIT do not compromise the cytocompatibility of the GELMA. Hence, for developing nanocomposite GELMA hydrogels employing APS/TEMED crosslinking emerges as a promising alternative to photo-crosslinking., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Indurkar, Rubenis, Boccaccini and Locs.)

Collagen nanofibrous materials have become integral to tissue engineering due to their exceptional properties and biocompatibility. Dehydrothermal crosslinking (DHT) enhances stability and maintains structural integrity without the formation of toxic residues. The study involved the crosslinking of electrospun collagen, applying DHT with access to air and under vacuum conditions. Various DHT exposure times of up to 72 h were applied to examine the time dependance of the DHT process. The DHT crosslinked collagen was subsequently chemically crosslinked using carbodiimides. The material crosslinked in this way evinced elevated Young's modulus values and ultimate tensile strength values, a lower swelling rate and lower shrinkage ratio during crosslinking, and a higher degree of resistance to degradation than the material crosslinked solely with DHT or carbodiimides. It was shown that the crosslinking mechanism using DHT occupies different binding sites than those using chemical crosslinking. Access to air for 12 h or less did not exert a significant impact on the material properties compared to DHT under vacuum conditions. However, concerning longer exposure times, it was determined that access to air results in the deterioration of the properties of the material and that reactions take place that occupy the free bonding sites, which subsequently reduces the effectiveness of chemical crosslinking using carbodiimides.

In the realization of the goal of circular economy, cellulose as one of sustainable biomass resources, have attracted much attention because of their abundant sources, biodegradability and renewability. However, the mechanical and waterproof performance of cellulose-based materials are usually not satisfying, which limits their high-value utilization. In this study, cellulose membrane with high-performance from the aspects of mechanical properties, water-resistance ability, oxygen barrier capacity and biodegradability, was prepared from bleached hardwood pulp (HBKP) in a AlCl 3 /ZnCl 2 /H 2 O solution. The AlCl 3 /ZnCl 2 /H 2 O acted as both solvent and catalyst to dissolve cellulose and facilitate the chemical crosslinking of epichlorohydrin (EPI) with cellulose, thus improved the overall performance of the obtained cellulose membrane. The addition sequence, amount and crosslinking time of EPI during chemical crosslinking had important effects on the properties of the membranes. When 7 wt% EPI was crosslinked for 24 h, the tensile stress reached 133 MPa and the strain reached 17 %. Moreover, the membrane had excellent oxygen insulation down to (1.1 ± 0.31) × 10 -4  cm 3 /m 2 ·d·Pa, and good water-resistance ability, no obvious swelling behavior after 450 days of immersion in distilled water. Furthermore, the membrane could be degraded by microorganisms in about 20 days. This cellulose-based membrane offers a sustainable and biodegradable packaging material., 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 © 2024 Elsevier B.V. All rights reserved.)

The photosystem I (PSI) of the green alga Chlamydomonas reinhardtii associates with 10 light-harvesting proteins (LHCIs) to form the PSI-LHCI complex. In the context of state transitions, two LHCII trimers bind to the PSAL, PSAH and PSAO side of PSI to produce the PSI-LHCI-LHCII complex. In this work, we took advantage of chemical crosslinking of proteins in conjunction with mass spectrometry to identify protein-protein interactions between the light-harvesting proteins of PSI and PSII. We detected crosslinks suggesting the binding of LHCBM proteins to the LHCA1-PSAG side of PSI as well as protein-protein interactions of LHCSR3 with LHCA5 and LHCA3. Our data indicate that the binding of LHCII to PSI is more versatile than anticipated and imply that LHCSR3 might be involved in the regulation of excitation energy transfer to the PSI core via LHCA5/LHCA3.

Tissue engineering (TE) demands scaffolds that have the necessary resistance to withstand the mechanical stresses once implanted in our body, as well as excellent biocompatibility. Hydrogels are postulated as interesting materials for this purpose, especially those made from biopolymers. In this study, the microstructure and rheological performance, as well as functional and biological properties of chitosan and collagen hydrogels (CH/CG) crosslinked with different coupling agents, both natural such as d-Fructose (F), genipin (G) and transglutaminase (T) and synthetic, using a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride with N-hydroxysuccinimide (EDC/NHS) will be assessed. FTIR tests were carried out to determine if the proposed crosslinking reactions for each crosslinking agent occurred as expected, obtaining positive results in this aspect. Regarding the characterization of the properties of each system, two main trends were observed, from which it could be established that crosslinking with G and EDC-NHS turned out to be more effective and beneficial than with the other two crosslinking agents, producing significant improvements with respect to the base CH/CG hydrogel. In addition, in vitro tests demonstrated the potential application in TE of these systems, especially for those crosslinked with G, T and EDC-NHS., 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 © 2024. Published by Elsevier B.V.)

Misfolding of mutant Cu/Zn-superoxide dismutase (SOD1) has been implicated in familial form of amyotrophic lateral sclerosis (ALS). A natively folded SOD1 forms a tight homodimer, and the dimer dissociation has been proposed to trigger the oligomerization/aggregation of SOD1. Besides increasing demand for probes allowing the detection of monomerized forms of SOD1 in various applications, the development of probes has been limited to conventional antibodies. Here, we have developed Mb(S4) monobody, a small synthetic binding protein based on the fibronectin type III scaffold, that recognizes a monomeric but not dimeric form of SOD1 by performing combinatorial library selections using phage and yeast-surface display methods. Although Mb(S4) was characterized by its excellent selectivity to the monomeric conformation of SOD1, the monomeric SOD1/Mb(S4) complex was not so stable (apparent K d  ~ μM) as to be detected in conventional pull-down experiments. Instead, the complex of Mb(S4) with monomeric but not dimeric SOD1 was successfully trapped by proximity-enabled chemical crosslinking even when reacted in the cell lysates. We thus anticipate that Mb(S4) binding followed by chemical crosslinking would be a useful strategy for in vitro and also ex vivo detection of the monomeric SOD1 proteins., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The widespread use of starch-based films is hindered by inadequate tensile strength and high water sensitivity. To address these limitations, a novel starch film with a dynamic network structure was produced via the dehydration-condensation reaction of N, N'-methylene diacrylamide (MBA) and microcrystalline cellulose (MCC). The improvement in mechanical properties was enhanced by the incorporation of MCC, which was achieved through intermolecular hydrogen bonding and chemical crosslinking. To verify the interactions among MCC, MBA, and starch, x-ray photoelectron spectroscopy (XPS), fourier transform infrared spectroscopy (FTIR), and x-ray diffraction (XRD) were conducted. The results established the predicted interactions. The dynamic network structure of the film reduced the water absorption capacity (WAC) of starch and MCC hydroxyl groups, as confirmed by differential scanning calorimeter (DSC) and dynamic mechanical thermal analysis (DMTA). These analyses showed a restriction in the mobility of starch chains, resulting in a higher glass transition temperature (Tg) of 69.26 °C. The modified starch films exhibited excellent potential for packaging applications, demonstrating a higher contact angle (CA) of 89.63°, the lowest WAC of 4.73 g/g, and the lowest water vapor transmission rate (WVTR) of 13.13 g/m 2 /d, along with improved mechanical properties and identical light transmittance compared to pure starch films., 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 © 2024 Elsevier B.V. All rights reserved.)

Chemical crosslinking-mass spectrometry (XL-MS) is an established tool that can be used to study the architecture and dynamics of proteins and protein assemblies. Here the application of XL-MS to study outer membrane proteins (OMPs) and their interactions with periplasmic chaperones is described, to inform on the molecular mechanisms underpinning OMP assembly. XL-MS data are especially powerful when used to complement high-resolution structural data, data from structural prediction or to drive molecular modeling of proteins and protein assemblies. The approach described here could be applied to the study of any protein assembly (including other membrane proteins)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The usage of natural polysaccharides is attractive to researchers around the world. At the same time, non-/low-toxic crosslinkers prepared by polysaccharides are expected to fabricate protein-based films in many fields. Herein, different dialdehyde polysaccharides (DPs) were successfully synthesized and applied to prepare gliadin-films under alkaline conditions. The functional properties and formation mechanisms of the films were fully investigated., Results: The results showed that the mechanical properties, water-resistant properties, thermal stability, and antibacterial properties of the gliadin-films were improved by DPs and alkali treatment. Particularly dialdehyde dextrin (DAD) crosslinked gliadin-films showed the highest tensile strength, but no additional effect on their elongation, or advancement on the other functional properties. The film-forming mechanisms indicated that Schiff base bonds, hydrophobic interactions, electrostatic interactions, and hydrogen bonds were the main forces in the films, supporting their improvement in functional properties., Conclusion: DPs, especially DAD, can be a promising crosslinker in fabricating gliadin-films. These findings have shown great promise to seek an effective crosslinker for fabricating gliadin/protein-based packaging. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Chemical crosslinking (XL) of non-covalent antigen-antibody complexes followed by mass spectrometric identification (MS) of inter-protein crosslinks can provide spatial constraints between relevant residues, which are valuable structural information associated with the molecular binding interface. To highlight the potential of XL/MS in the biopharmaceutical industry, we herein developed and validated an XL/MS workflow that employed a zero-length linker, 1,1'‑carbonyldiimidazole (CDI), and a widely used medium-length linker, disuccinimidyl sulfoxide (DSSO), for fast, accurate determination of antigen domains targeted by therapeutic antibodies. To avoid false identification, system suitability samples and negative samples were designed for all experiments, and all tandem mass spectra were manually examined. To validate the proposed XL/MS workflow, two complexes involving human epidermal growth factor receptor 2 Fc fusion protein (HER2Fc) with known crystal structures, including HER2Fc-pertuzumab and HER2Fc-trastuzumab, have been subjected to CDI and DSSO crosslinking. Crosslinks established by CDI and DSSO between HER2Fc and pertuzumab accurately revealed their interaction interface. CDI crosslinking contributes more than DSSO because of its short spacer arm and high reactivity towards hydroxyl groups, demonstrating its capacity in protein interaction analysis. The correct binding domain cannot be revealed solely based on DSSO in the HER2Fc-trastuzumab complex, because domain proximity revealed by this 7-atom spacer linker cannot be directly translated as binding interfaces. As the first successful XL/MS application in early-stage therapeutic antibody discovery, we analyzed the molecular binding interface between HER2Fc and H-mab, an innovant drug candidate whose paratopes have not been studied yet. We predict that H-mab probably targets HER2 Domain I. The proposed XL/MS workflow can serve as an accurate, fast, and low-cost method to study the interaction between antibodies and large multi-domain antigens. SIGNIFICANCE: This article described a fast, low-consumption approach based on chemical crosslinking mass spectrometry (XL/MS) using two linkers for binding domain determination in multidomain antigen-antibody complexes. Our results highlighted the higher importance of zero-length crosslinks established by CDI than 7-atom DSSO crosslinks, as residue proximity revealed by zero-length crosslinks is closely related to epitope-paratope interaction surfaces. Furthermore, the higher reactivity of CDI towards hydroxyl groups broadens the ranges of possible crosslinks, despite the necessity of delicate operation in CDI crosslinking. We suggest that all established CDI and DSSO crosslinks should be comprehensively considered for correct binding domain analysis because predictions solely based on DSSO might be ambiguous. We have determined the binding interface in the HER2-H-mab using CDI and DSSO, which is the first successful application of XL/MS in real-world early-stage biopharmaceutical development., Competing Interests: Declaration of Competing Interest Lingyu Wang, Liqi Xie, and Zhongli Zhang are employees of Henlius Biologics, Inc. (Shanghai, China). The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Photo- and chemical crosslinking of proteins have offered various avenues for studying protein structure and protein interactions with biomolecules. Conventional photoactivatable groups generally lack reaction selectivity toward amino acid residues. New photoactivatable groups reacting with selected residues have emerged recently, increasing crosslinking efficiency and facilitating crosslink identification. Traditional chemical crosslinking usually employs highly reactive functional groups, while recent advance has developed latent reactive groups with reactivity triggered by proximity, which reduce spurious crosslinks and improve biocompatibility. The employment of these residue selective chemical functional groups, activated by light or proximity, in small molecule crosslinkers and in genetically encoded unnatural amino acids is summarized. Together with new software development in identifying protein crosslinks, residue selective crosslinking has enhanced the research of elusive protein-protein interactions in vitro, in cell lysate, and in live cells. Residue selective crosslinking is expected to expand to other methods for the investigation of various protein-biomolecule interactions., 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 Ltd. All rights reserved.)

Hydrogels a three-dimensional network structure of hydrophilic polymers have gained significant interest in the field of biomedicine due to its high-water absorption properties and its resemblance to the native extracellular matrix. However, the hydrogel's physicochemical properties are important in its ability to serve as a matrix in biomedical applications. The variations in the molecular weight of polymers in the preparation of crosslinked hydrogels may alter the properties. Different molecular weight carboxymethyl cellulose polymers were employed in this work to determine the effect of molecular weight on the physicochemical parameters of the hydrogel's crosslinking reaction. For this study, two distinct molecular weight carboxymethyl cellulose (CMC) polymers (M w , 250,000 and 700,000) and various concentrations of crosslinker solution were used. The hydrogels were prepared through a chemical crosslinking reaction combining CMC and citric acid, which results in the formation of an ester bond between the two polymer chains. The crosslinking reaction is confirmed by Fourier transform infrared spectroscopy and total carboxyl content analysis. According to the physicochemical, thermal, and mechanical analysis, we have identified that 7 %, 9 % and 10 % citric acid showed the most promising hydrogels and found 7CMC hydrogel had superior quality. In vitro results demonstrated that the citric acid crosslinked CMC had excellent hemocompatibility and cytocompatibility., 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. Published by Elsevier B.V.)

As vaccine adjuvants, polyacrylate materials can induce a specific immune response in the body and have been widely studied in recent years due to their advantages, such as their safety, effectiveness, and low required dosage. In this study, a series of polyacrylates with hydrophobic physical crosslinking and chemical crosslinking were prepared using precipitation polymerization, and their structures were characterized by nuclear magnetic resonance spectroscopy and Fourier-transform infrared spectroscopy. The optimal reaction conditions were determined according to the effect of reaction time, azodiisobutyronitrile, Span 60, allyl pentaerythritol, and octadecyl methacrylate (OMA) contents on the viscosity of the polyacrylate microgel, combined with the effects of allyl pentaerythritol and OMA contents on the subcutaneous immune safety of the polyacrylate microgel in BALB/c mice. The polyacrylate microgels with different OMA contents showed good biological safety. In addition, in vivo immunity experiments were carried out in mice to analyze the adjuvant properties of ovalbumin as a model antigen. Based on the titer results of the IgG1 and IgG2a antibodies, with 1 wt % OMA content, the polyacrylate microgel vaccine could optimally induce the body to produce an immune response type dominated by Th2-type humoral immune response and supplemented by Th1-type cellular immune response., (© 2023 The Authors. Published by Wiley-VCH GmbH.)

VAR2CSA, a multidomain Plasmodium falciparum protein, mediates the adherence of parasite-infected red blood cells to chondroitin 4-sulfate (C4S) in the placenta, contributing to placental malaria. Therefore, detailed understanding of VAR2CSA structure likely help developing strategies to treat placental malaria. The VAR2CSA ectodomain consists of an N-terminal segment (NTS), six Duffy binding-like (DBL) domains, and three interdomains (IDs) present in sequence NTS-DBL1x-ID1-DBL2x-ID2-DBL3x-DBL4ε-ID3-DBL5ε-DBL6ε. Recent electron microscopy studies showed that VAR2CSA is compactly organized into a globular structure containing C4S-binding channel, and that DBL5ε-DBL6ε arm is attached to the NTS-ID3 core structure. However, the structural elements involved in inter-domain interactions that stabilize the VAR2CSA structure remain largely not understood. Here, limited proteolysis and peptide mapping by mass spectrometry showed that VAR2CSA contains several inter-domain disulfide bonds that stabilize its compact structure. Chemical crosslinking-mass spectrometry showed that all IDs interact with DBL4ε; additionally, IDs interact with other DBL domains, demonstrating that IDs are the key structural scaffolds that shape the functional NTS-ID3 core. Ligand binding analysis suggested that NTS considerably restricts the C4S binding. Overall, our study revealed that inter-domain disulfide bonds and interactions between IDs and DBL domains contribute to the stability of VAR2CSA structural architecture and formation of C4S-binding channel., Competing Interests: Declaration of competing interest D. Channe Gowda reports financial support was provided by NIAID, National Institutes of Health, USA., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study, we developed a method to fabricate chitosan-based network polysaccharides via the condensation between amino groups in water-soluble chitosan (WSCS) and a carboxylate-terminated maltooligosaccharide crosslinker. We previously reported on the fabrication of network-polysaccharide-based macroscopic hydrogels via the chemical crosslinking of water-soluble chitin (WSCh) with the crosslinker. Because the molecular weight of the WSCS was much smaller than that of the WSCh, in the present investigation, the chemical crosslinking of the WSCS with the crosslinker was observed at the nanoscale upon the condensation between amino and carboxylate groups in the presence of a condensing agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N -hydroxysuccinimide, affording nano-sized chitosan-based network polysaccharides. The occurrence of the crosslinking via the formation of amido linkages was supported by the IR analysis and 1 H NMR measurements after the dissolution via acid hydrolysis in DCl/D 2 O. The products formed nanogels, whose sizes depended on the amino/carboxylate feed ratio. The nanoscale morphology and size of the products were evaluated via scanning electron microscopy, dynamic light scattering analyses, and transition electron microscopy. In the present study, we successfully developed the method to fabricate nanogel materials based on network polysaccharide structures, which can practically be applied as new polysaccharide-based 3D bionanomaterials.

For the effective clinical antibacterial application of biomaterials, such as for wound management and tissue repair, the biomaterials need to show proper antibacterial capability as well as non-cytotoxicity. Furthermore, the material needs to have suitable mechanical characteristics for further medical use. Chitosan hydrogel is a potential candidate for various antibacterial biomedical applications due to its amine functionalities that lead to antimicrobial characteristics. Nevertheless, its antimicrobial capability is dependent upon the degree of protonation of amine groups caused by the pH value. Moreover, its mechanical compressive strength may not be high enough for clinical use if not chemically or physically crosslinked. This study utilized a novel chemical crosslinker, mercaptosuccinic acid, to improve its mechanical characteristics. The natural antibacterial agent, cinnamaldehyde, was grafted onto the crosslinked chitosan to improve its antimicrobial capability. Meanwhile, to take advantage of the thiol functionality in the mercaptosuccinic acid, the bactericidal silver nanoparticles were incorporated through silver-thiol covalent bounding. NMR analyses indicated the chitosan was successfully mercaptosuccinic acid-crosslinked and grafted with cinnamaldehyde at different ratios. Combined the results from the mechanical assessment, swelling experiments, antimicrobial assessment, and cytotoxicity assay, the chitosan hydrogel with the highest crosslinked degree and grafted with cinnamaldehyde and silver nanoparticles is of great promise for further clinical uses.

Skeletal muscles exhibit excellent properties due to their well-developed microstructures. Taking inspiration from nature that thick filaments and thin filaments are linked by "cross-bridges", leading to good stability and ion transport performance of muscles. In this work, extracted poplar lignin and microcrystalline cellulose (MCC) were connected by biomimetic covalent bonds, akin to biological muscle tissue, in which isophorone diisocyanate was used as the chemical crosslinking agent. Then, poplar lignin-MCC was mixed with polyacrylonitrile to serve as the precursor for electrospinning. The results show that due to the effective covalent-bond connection, the precursor fibers possess excellent morphology, smooth surface, good thermal stability, and high flexibility and toughness (average elongation-at-break is 51.84%). Therefore, after thermal stabilization and carbonization, derived lignocellulose-based carbon fibers (CFs) with a reduced cost, complete fiber morphology with a uniform diameter (0.48 ± 0.22 μm), and high graphitization degree were obtained. Finally, the electrodes fabrication and electrochemical testing were carried out. The results of electrochemical impedance spectroscopy (EIS) indicate that the Rs and Rct values of CFs supercapacitors are 1.18 Ω and 0.14 Ω, respectively. Results of cyclic voltammetry (CV) and galvanostatic charge-discharge (GCD) suggest that these CFs demonstrate great application potential in electrochemical materials.

Siderophores are high-affinity small-molecule chelators employed by bacteria to acquire iron from the extracellular environment. The Gram-negative bacterium Escherichia coli synthesizes and secretes enterobactin, a tris-catechol siderophore. Enterobactin is synthesized by six cytoplasmic enzyme activities: EntC, EntB (isochorismatase (IC) domain), EntA, EntE, EntB (aryl carrier protein (ArCP) domain), and EntF. While various pairwise protein-protein interactions have been reported between EntB, EntA, EntE, and EntF, evidence for an interaction between EntC and EntB has remained elusive. We have employed bacterial two-hybrid assays and in vivo crosslinking to demonstrate an intracellular EntC-EntB interaction. A T18-EntC/T25-EntB co-transformant exhibited a positive two-hybrid signal compared to a control T18-EntC/T25 co-transformant. In vivo formaldehyde crosslinking of E. coli cells co-expressing HA-tagged EntB and H6-tagged EntC resulted in an observable ∼80 kDa band on Western blots that cross-reacted with anti-HA and anti-H6, corresponding to one HA-EntB monomer (33 kDa) crosslinked with one H6-EntC monomer (45 kDa). This band disappeared upon sample boiling, confirming it to be a formaldehyde-crosslinked species. Bands of molecular masses greater than 80 kDa that cross-reacted with both antibodies were also observed. Automated docking of the crystal structures of monomeric EntC and dimeric EntB resulted in a top-ranked candidate docked ensemble in which the active sites of EntC and EntB were oriented in apposition and connected by an electropositive surface potentially capable of channeling negatively charged isochorismate. These research outcomes provide the first reported evidence of an EntC-EntB interaction, as well as the first experimental evidence of higher-order complexes containing EntC and EntB., Competing Interests: Declaration of competing interest The authors declare no conflict of interest for this manuscript., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Currently, the world faces difficulties related to the quantity and quality of water because of industrial expansion, population growth, and urbanization intensification. Biosorption is considered a promising technology that can be applied to remove toxic metals (TMs) and rare-earth metals (REMs) in wastewater at low concentrations, due to its efficiency and low cost. In this work, we investigated different non-conventional biosorbents to remove metallic ions (TMs and REMs) in biosorptive affinity tests. Metallic affinity assays among lanthanum and different biosorbents showed that greater affinities were found for sericin-alginate beads crosslinked with polyvinyl alcohol (SAPVA) (0.280 mmol/g) and polyethylene glycol diglycidyl ether (SAPEG) (0.277 mmol/g), expanded vermiculite (0.281 mmol/g), Sargassum filipendula seaweed (0.287 mmol/g), and seaweed biomass waste (0.289 mmol/g). Among the biosorbents evaluated, SAPVA and SAPEG beads, besides to sericin-alginate beads crosslinked with proanthocyanidins (SAPAs) were selected for affinity assays with other REMs and TMs. Compared to other particles, SAPVA beads showed higher potential for biosorption by REMs with the following order of affinity: Yb 3+ > Dy 3+ > Nd 3+ > Ce 3+ > La 3+ . Additionally, the biosorptive affinity of TMs by SAPVA beads followed the order: Al 3+ > Cr 3+ > Pb 2+ > Cu 2+ > Cd 2+ > Zn 2+ > Ni 2+ ., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Chemically crosslinked acellular bovine pericardium (ABP) has been widely used in clinical practice as bioprostheses. To ensure its consistency and durability, crosslinkers are used in excess, with stability guided by indicators including the hydrothermal denaturation temperature, the enzymatic resistance and the degree of crosslinking. Yet, understanding of the intermolecular structure in collagen fibrils which imparts the intrinsic stability of the ABPs is lacking, and the discrepancies in the stability criteria in varied conditions are poorly explained. In this study, synchrotron small-angle X-ray scattering (SAXS) in combination with thermal and colorimetric methods are employed to investigate the changes in the structure and the stability of ABPs during crosslinking using glutaraldehyde (GA) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) at different concentrations. Based on the findings, a mechanism is proposed to explicate the crosslinking effects on collagen structure and the relationship between the structure and each stability indicator. At low crosslinker concentrations, the telopeptidyl-helical linkages are preferred, which cause rearrangements in the intermolecular structure of collagen, and efficiently contribute to its stabilities. Excess crosslinking is revealed by a revert trend in structural changes and the plateauing of the stabilities, assigning to the helical-helical linkages and monovalent bindings. The former would improve thermal stability but not collagenase resistance, whereas the latter have negligible effects. Overall, this study provides a mechanistic understanding of the chemical crosslinking of ABPs, which will contribute to the future development of more efficient and economically viable strategies to produce bioprostheses. STATEMENT OF SIGNIFICANCE: Chemical crosslinking imparts suitable properties to acellular bovine pericardium (ABP) for clinical applications, yet the understanding is lacking on the structure-stability relationship especially under different crosslinking conditions. Structural evidence in this study differentiates the binding sites during crosslinking in collagen fibrils at different crosslinker concentrations, highlighting the excess usage in the conventional crosslinking treatments. The mechanism based on the structure of collagen also successfully explains the dissimilarity in hydrothermal and enzymatic stabilities with varied crosslinking conditions. Future researches focusing on developing biomaterials via chemical crosslinking of ABPs would benefit from this study, for its contribution to the better understanding of the relationship of collagen structure and functions., 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.)

Sargasso cellulose nanofibers (CNFs) and polyvinyl alcohol (PVA) components were composed to generate novel KCNF/PVA films by a simple and benign crosslinking strategy. Sargasso CNFs are a potential reinforcement material due to its extremely long length (15-20 μm) and natural nanostructure. The low water resistance of PVA limits their use in humid and watery environments. However, the chemical cross-linking between sargasso CNFs and PVA consumes a large number of hydrophilic hydroxyl groups, the resulting KCNF/PVA films exhibited outstanding water resistance (water contact angle 119°). Especially, the wet strength of KCNF/PVA films (50 MPa) was raised by 92 times when compared to that of pristine PVA films (0.54 MPa). Furthermore, these KCNF/PVA films could be dissolved and recycled easily. The regenerated films also show outstanding mechanical performance., (Copyright © 2022. Published by Elsevier Ltd.)

Although synthetic polymer hydrogels have excellent mechanical properties, they are not easily degraded, are toxic, and easily cause environmental pollution. Therefore, starch-based hydrogels have attracted great interest due to their biodegradability, biocompatibility, and non-toxicity. First of all, this review describes in detail the preparation of starch-based hydrogels by physical retrogradation method and chemical crosslinking methods, as well as the intrinsic and extrinsic factors that affect the performance of the hydrogels. Moreover, the commonly used methods to characterize starch-based hydrogels and the properties of responsive (thermo-, pH-, and enzyme-responsive) starch-based hydrogels have also attracted wide attention from researchers. In addition, the applications of starch-based hydrogels in starch noodles, treatment of sewage from the food industry, and food bioactive ingredient carriers are discussed. Furthermore, prospects and future directions of starch-based hydrogels are discussed. We believe that this review provides a valuable reference for the efficient preparation and application of starch-based hydrogels., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The small ubiquitin-like modifier (SUMO) is involved in various cellular processes and mediates known non-covalent protein-protein interactions by three distinct binding surfaces, whose interactions are termed class I to class III. While interactors for the class I interaction, which involves binding of a SUMO-interacting motif (SIM) to a hydrophobic groove in SUMO-1 and SUMO-2/3, are widely abundant, only a couple of examples have been reported for the other two types of interactions. Class II binding is conveyed by the E67 loop region on SUMO-1. Many previous studies to identify SUMO binders using pull-down or microarray approaches did not strategize on the SUMO binding mode. Identification of SUMO binding partners is further complicated due to the typically transient and low affinity interactions with the modifier. Here we aimed to identify SUMO-1 binders selectively enriched for class II binding. Using a genetically encoded photo-crosslinker approach, we have designed SUMO-1 probes to covalently capture class II SUMO-1 interactors by strategically positioning the photo-crosslinking moiety on the SUMO-1 surface. The probes were validated using known class II and class I binding partners. We utilized the probe with p-benzoyl-phenylalanine (BzF, also termed BpF or Bpa) at the position of Gln69 to identify binding proteins from mammalian cell extracts using mass spectrometry. By comparison with results obtained with a similarly designed SUMO-1 probe to target SIM-mediated binders of the class I type, we identified 192 and 96 proteins specifically enriched by either probe, respectively. The implicated preferential class I or class II binding modes of these proteins will further contribute to unveiling the complex interplay of SUMO-1-mediated interactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Brüninghoff, Wulff, Dörner, Geiss-Friedlander and Mootz.)

In order to promote the application of membrane technology in the treatment of textile wastewater containing small molecule dye, fabricating a hollow fiber loose nanofiltration (LNF) with a thin and compact separation layer and deepening the understanding of compactness-tailoring mechanism in chemical crosslinking are essential. Firstly, the mechanisms of synergistic crosslinking of PEI-70K and PEI-10K, along with a weakening of the PEI hydration by ethanol, were expounded in primary crosslinking. Then, some LNF separation layers with different compactness were prepared through crosslinking with different crosslinkers to further reduce pore size, which resulted in the efficient removal (~100%) of a small molecular dye (methyl orange (MO), M = 327 g mol -1 ). The removal of methyl orange is mainly caused by size sieving. The relationship among the pore size, the M w of the secondary crosslinkers, and the pore size reduction rate was interpreted by comparing the pore size reduction rate of three secondary crosslinkers with different molecular weights. In addition, the as-prepared separation layer exhibited excellent dimensional stability and solvent resistance. This paper not only provides a reference for fabricating hollow fiber LNF with better purification performance, but also shows their potential in developing solvent resistant nanofiltration., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Polysaccharide-based hydrogels are particularly attractive materials for biomedical applications. However, their use is restricted due to their brittleness and poor mechanical properties. Here, to overcome such limitations, we report an original, green, simple, and efficient strategy to synthesize a polysaccharide-based hydrogel of chitosan (Cht) and a vinyl-functionalized PVA (PVA-MA), a non-toxic synthetic polymer that is widely known to improve the mechanical properties and stability of materials containing polysaccharides. The hydrogel was crosslinked through an aza-Michael addition among the amino groups of Cht with the vinyl moieties of PVA-MA catalyzed by boric acid (B(OH) 3 ), an eco-friendly inorganic compound. Characterization analyses revealed that the prepared hydrogel has a porous-like morphology, an outstanding liquid uptake capacity (>665%), and improved stability in a physiological fluid for long periods. In summary, this original and simple strategy showed to be efficient in the synthesis of hydrogels with attractive properties for the biomedical field application., (Copyright © 2021 Elsevier B.V. All rights reserved.)

E. coli histone-like protein HU has been shown to interact with different topological forms of DNA. Using radiolabeled HU, we examine the effects of DNA supercoiling on HU-DNA interactions. We show that HU binds preferentially to negatively supercoiled DNA and that the affinity of HU for DNA increases with increases in the negative superhelical density of DNA. Binding of HU to DNA is most sensitively influenced by DNA supercoiling within a narrow but physiologically relevant range of superhelicity ( σ = -0.06-0). Under stoichiometric binding conditions, the affinity of HU for negatively supercoiled DNA ( σ = -0.06) is more than 10 times higher than that for relaxed DNA at physiologically relevant HU/DNA mass ratios (e.g., 1:10). This binding preference, however, becomes negligible at HU/DNA mass ratios higher than 1:2. At saturation, HU binds both negatively supercoiled and relaxed DNA with similar stoichiometries, i.e., 5-6 base pairs per HU dimer. In our chemical crosslinking studies, we demonstrate that HU molecules bound to negatively supercoiled DNA are more readily crosslinked than those bound to linear DNA. At in vivo HU/DNA ratios, HU appears to exist predominantly in a tetrameric form on negatively supercoiled DNA and in a dimeric form on linear DNA. Using a DNA ligase-mediated nick closure assay, we show that approximately 20 HU dimers are required to constrain one negative supercoil on relaxed DNA. Although fewer HU dimers may be needed to constrain one negative supercoil on negatively supercoiled DNA, our results and estimates of the cellular level of HU argue against a major role for HU in constraining supercoils in vivo. We discuss our data within the context of the dynamic distribution of the HU protein in cells, where temporal and local changes of DNA supercoiling are known to take place.

Well dispersibility of 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO)-oxidized nanochitins under alkaline conditions supports the effective chemical crosslinking between nanochitin and epichlorohydrin. The storage modulus of nanochitin hydrogels can be promoted by approximately 10 times as the nanochitin-to-epichlorohydrin mass ratio changes from 4:1 (120 Pa) to 1:4 (1200 Pa). Besides the enhanced mechanical property of hydrogels, the inter-structure of resulting cryogels is found controllable. With increasing epichlorohydrin dosage, the inter-structure of cryogels transforms from a typical fiber-like to honeycomb-like texture. The balance between chemical crosslinking effect and electrostatic repulsion between nanochitins is believed to result this controllable inter-structure. Further immersing into acetic acid solution can greatly enhance the mechanical strength of nanochitin hydrogels due to the introduction of physical crosslinking domains by shielding the electrostatic repulsion, the storage modulus becomes two times higher after immersing in 50% (w/w) acetic acid solution, while the surface area of nanochitin cryogels decreases due to the denser structure., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The rheological changes that occur during the chemical gelation of semidilute solutions of chitosan in the presence of the low-toxicity agent glyceraldehyde (GCA) are presented and discussed in detail. The entanglement concentration for chitosan solutions was found to be approximately 0.2 wt.% and the rheological experiments were carried out on 1 wt.% chitosan solutions with various amounts of GCA at different temperatures (25 °C and 40 °C) and pH values (4.8 and 5.8). High crosslinker concentration, as well as elevated temperature and pH close to the pK a value (pH ≈ 6.3-7) of chitosan are three parameters that all accelerate the gelation process. These conditions also promote a faster solid-like response of the gel-network in the post-gel region after long curing times. The mesh size of the gel-network after a very long (18 h) curing time was found to contract with increasing level of crosslinker addition and elevated temperature. The gelation of chitosan in the presence of other chemical crosslinker agents (glutaraldehyde and genipin) is discussed and a comparison with GCA is made. Small angle neutron scattering (SANS) results reveal structural changes between chitosan solutions, incipient gels, and mature gels.

The interfacial compatibility of the natural filler and synthetic polymer is the key performance characteristic of biocomposites. The fillers are chemically modified, or coupling agents and compatibilisers are used to ensure optimal filler-polymer compatibility. Hence, we have investigated the effect of compatibilisation strategies of olive pits (OP) flour content (10, 20, 30, and 40%wt.) filled with recycled low-density polyethylene (rLDPE) on the chemical, physical, mechanical, and thermal behaviour of the developed biocomposites. In this study, we aim to investigate the filler-polymer compatibility in biocomposites by employing novel strategies for the functionalisation of OP filler and/or rLDPE matrix. Specifically, four cases are considered: untreated OP filled rLDPE (Case 1), treated OP filled rLDPE (Case 2), treated OP filled functionalised rLDPE (Case 3), and treated and functionalised OP filled functionalised rLDPE (Case 4). In general, the evaluation of the performance of biocomposites facilitated the application of OP industrial waste as an eco-friendly reinforcing agent for rLDPE-based biocomposites. Furthermore, surface treatment and compatibilisation improved the properties of the developed biocomposites over untreated filler or uncoupled biocomposites. Besides that, the compatibilisers used aided in reducing water uptake and improving thermal behaviour, which contributed to the stability of the manufactured biocomposites.

Hydrogels are key components in bioink formulations to ensure printability and stability in biofabrication. In this study, a well-known Diels-Alder two-step post-polymerization modification approach is introduced into thermogelling diblock copolymers, comprising poly(2-methyl-2-oxazoline) and thermoresponsive poly(2-n-propyl-2-oxazine). The diblock copolymers are partially hydrolyzed and subsequently modified by acid/amine coupling with furan and maleimide moieties. While the thermogelling and shear-thinning properties allow excellent printability, trigger-less cell-friendly Diels-Alder click-chemistry yields long-term shape-fidelity. The introduced platform enables easy incorporation of cell-binding moieties (RGD-peptide) for cellular interaction. The hydrogel is functionalized with RGD-peptides using thiol-maleimide chemistry and cell proliferation as well as morphology of fibroblasts seeded on top of the hydrogels confirm the cell adhesion facilitated by the peptides. Finally, bioink formulations are tested for biocompatibility by incorporating fibroblasts homogenously inside the polymer solution pre-printing. After the printing and crosslinking process good cytocompatibility is confirmed. The established bioink system combines a two-step approach by physical precursor gelation followed by an additional chemical stabilization, offering a broad versatility for further biomechanical adaptation or bioresponsive peptide modification., (© 2021 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.)

Background: Proteins are highly dynamic and their biological function is controlled by not only temporal abundance changes but also via regulated protein-protein interaction networks, which respond to internal and external perturbations. A wealth of novel analytical reagents and workflows allow studying spatiotemporal protein environments with great granularity while maintaining high throughput and ease of analysis., Areas Covered: We review technology advances for measuring protein-protein proximity interactions with an emphasis on proximity labeling, and briefly summarize other spatiotemporal approaches including protein localization, and their dynamic changes over time, specifically in human cells and mammalian tissues. We focus especially on novel technologies and workflows emerging within the past 5 years. This includes enrichment-based techniques (proximity labeling and crosslinking), separation-based techniques (organelle fractionation and size exclusion chromatography), and finally sorting-based techniques (laser capture microdissection and mass spectrometry imaging)., Expert Opinion: Spatiotemporal proteomics is a key step in assessing biological complexity, understanding refined regulatory mechanisms, and forming protein complexes and networks. Studying protein dynamics across space and time holds promise for gaining deep insights into how protein networks may be perturbed during disease and aging processes, and offer potential avenues for therapeutic interventions, drug discovery, and biomarker development.

Theory predicts that the net charge (Z) of a protein can be altered by the net charge of a neighboring protein as the two approach one another below the Debye length. This type of charge regulation suggests that a protein's charge and perhaps function might be affected by neighboring proteins without direct binding. Charge regulation during protein crowding has never been directly measured due to analytical challenges. Here, we show that lysine specific protein crosslinkers (NHS ester-Staudinger pairs) can be used to mimic crowding by linking two non-interacting proteins at a maximal distance of ~7.9 Å. The net charge of the regioisomeric dimers and preceding monomers can then be determined with lysine-acyl "protein charge ladders" and capillary electrophoresis. As a proof of concept, we covalently linked myoglobin (Z monomer  = -0.43 ± 0.01) and α-lactalbumin (Z monomer  = -4.63 ± 0.05). Amide hydrogen/deuterium exchange and circular dichroism spectroscopy demonstrated that crosslinking did not significantly alter the structure of either protein or result in direct binding (thus mimicking crowding). Ultimately, capillary electrophoretic analysis of the dimeric charge ladder detected a change in charge of ΔZ = -0.04 ± 0.09 upon crowding by this pair (Z dimer  = -5.10 ± 0.07). These small values of ΔZ are not necessarily general to protein crowding (qualitatively or quantitatively) but will vary per protein size, charge, and solvent conditions., (© 2021 The Protein Society.)

Physically crosslinked hydrogels with thixotropic properties attract considerable attention in the biomedical research field because their self-healing nature is useful in cell encapsulation, as injectable gels, and as bioinks for three-dimensional (3D) bioprinting. Here, we report the formation of thixotropic hydrogels containing nanofibers of double-hydrophobic elastin-like polypeptides (ELPs). The hydrogels are obtained with the double-hydrophobic ELPs at 0.5 wt%, the concentration of which is an order of magnitude lower than those for previously reported ELP hydrogels. Although the kinetics of hydrogel formation is slower for the double-hydrophobic ELP with a cell-binding sequence, the storage moduli G' of mature hydrogels are similar regardless of the presence of a cell-binding sequence. Reversible gel-sol transitions are demonstrated in step-strain rheological measurements. The degree of recovery of the storage modulus G' after the removal of high shear stress is improved by chemical crosslinking of nanofibers when intermolecular crosslinking is successful. This work would provide deeper insight into the structure-property relationships of the self-assembling polypeptides and a better design strategy for hydrogels with desired viscoelastic properties.

Nanogels represent an innovative platform for tunable drug release and targeted therapy in several biomedical applications, ranging from cancer to neurological disorders. The design of these nanocarriers is a pivotal topic investigated by the researchers over the years, with the aim to optimize the procedures and provide advanced nanomaterials. Chemical reactions, physical interactions and the developments of engineered devices are the three main areas explored to overcome the shortcomings of the traditional nanofabrication approaches. This review proposes a focus on the current techniques used in nanogel design, highlighting the upgrades in physico-chemical methodologies, microfluidics and 3D printing. Polymers and biomolecules can be combined to produce ad hoc nanonetworks according to the final curative aims, preserving the criteria of biocompatibility and biodegradability. Controlled polymerization, interfacial reactions, sol-gel transition, manipulation of the fluids at the nanoscale, lab-on-a-chip technology and 3D printing are the leading strategies to lean on in the next future and offer new solutions to the critical healthcare scenarios.

The twin-arginine translocase (Tat) mediates the transport of already-folded proteins across membranes in bacteria, plants and archaea. TatA is a small, dynamic subunit of the Tat-system that is believed to be the active component during target protein translocation. TatA is foremost characterized as a bitopic membrane protein, but has also been found to partition into a soluble, oligomeric structure of yet unknown function. To elucidate the interplay between the membrane-bound and soluble forms we have investigated the oligomers formed by Arabidopsis thaliana TatA. We used several biophysical techniques to study the oligomeric structure in solution, the conversion that takes place upon interaction with membrane models of different compositions, and the effect on bilayer integrity upon insertion. Our results demonstrate that in solution TatA oligomerizes into large objects with a high degree of ordered structure. Upon interaction with lipids, conformational changes take place and TatA disintegrates into lower order oligomers. The insertion of TatA into lipid bilayers causes a temporary leakage of small molecules across the bilayer. The disruptive effect on the membrane is dependent on the liposome's negative surface charge density, with more leakage observed for purely zwitterionic bilayers. Overall, our findings indicate that A. thaliana TatA forms oligomers in solution that insert into bilayers, a process that involves reorganization of the protein oligomer., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Acellular nerve allografts (ANGs) represent a promising alternative in nerve repair. Our aim is to improve the structural and biomechanical properties of biocompatible Sondell (SD) and Roosens (RS) based ANGs using genipin (GP) as a crosslinker agent ex vivo. The impact of two concentrations of GP (0.10% and 0.25%) on Wistar rat sciatic nerve-derived ANGs was assessed at the histological, biomechanical, and biocompatibility levels. Histology confirmed the differences between SD and RS procedures, but not remarkable changes were induced by GP, which helped to preserve the nerve histological pattern. Tensile test revealed that GP enhanced the biomechanical properties of SD and RS ANGs, being the crosslinked RS ANGs more comparable to the native nerves used as control. The evaluation of the ANGs biocompatibility conducted with adipose-derived mesenchymal stem cells cultured within the ANGs confirmed a high degree of biocompatibility in all ANGs, especially in RS and RS-GP 0.10% ANGs. Finally, this study demonstrates that the use of GP could be an efficient alternative to improve the biomechanical properties of ANGs with a slight impact on the biocompatibility and histological pattern. For these reasons, we hypothesize that our novel crosslinked ANGs could be a suitable alternative for future in vivo preclinical studies.

Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast Saccharomyces cerevisiae as a model system for understanding peptide-GPCR interactions. It explored the structure and function of synthetic α-factor analogs and biosynthetic receptor domains, as well as designed mutations of Ste2p. The results and conclusions are described using the nuclear magnetic resonance interrogation of synthetic Ste2p transmembrane domains (TMs), the fluorescence interrogation of agonist and antagonist binding, the biochemical crosslinking of peptide analogs to Ste2p, and the phenotypes of receptor mutants. We identified the ligand-binding domain in Ste2p, the functional assemblies of TMs, unexpected and interesting ligand analogs; gained insights into the bound α-factor structure; and unraveled the function and structures of various Ste2p domains, including the N-terminus, TMs, loops connecting the TMs, and the C-terminus. Our studies showed interactions between specific residues of Ste2p in an active state, but not resting state, and the effect of ligand activation on the dimerization of Ste2p. We show that, using a battery of different biochemical and genetic approaches, deep insight can be gained into the structure and conformational dynamics of GPCR-peptide interactions in the absence of a crystal structure.

Chemical crosslinking-mass spectrometry (XL-MS) is a valuable technique for gaining insights into protein structure and the organization of macromolecular complexes. XL-MS data yield inter-residue restraints that can be compared with high-resolution structural data. Distances greater than the crosslinker spacer-arm can reveal lowly populated "excited" states of proteins/protein assemblies, or crosslinks can be used as restraints to generate structural models in the absence of structural data. Despite increasing uptake of XL-MS, there are few tools to enable rapid and facile mapping of XL-MS data onto high-resolution structures or structural models. PyXlinkViewer is a user-friendly plugin for PyMOL v2 that maps intra-protein, inter-protein, and dead-end crosslinks onto protein structures/models and automates the calculation of inter-residue distances for the detected crosslinks. This enables rapid visualization of XL-MS data, assessment of whether a set of detected crosslinks is congruent with structural data, and easy production of high-quality images for publication., (© 2020 The Authors. Protein Science published by Wiley Periodicals LLC. on behalf of The Protein Society.)

Detailed molecular information on G-actin assembly into filaments (F-actin), and their structure, dynamics, and interactions, is essential for understanding their cellular functions. Previous studies indicate that a flexible DNase I binding loop (D-loop, residues 40-50) plays a major role in actin's conformational dynamics. Phalloidin, a "gold standard" for actin filament staining, stabilizes them and affects the D-loop. Using disulfide crosslinking in yeast actin D-loop mutant Q41C/V45C, light-scattering measurements, and cryoelectron microscopy reconstructions, we probed the constraints of D-loop dynamics and its contribution to F-actin formation/stability. Our data support a model of residues 41-45 distances that facilitate G- to F-actin transition. We report also a 3.3-Å resolution structure of phalloidin-bound F-actin in the ADP-Pi-like (ADP-BeFx) state. This shows the phalloidin-binding site on F-actin and how the relative movement between its two protofilaments is restricted by it. Together, our results provide molecular details of F-actin structure and D-loop dynamics., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The fabrication of polymer micelles, both with ample intracorporeal circulation stability and fast release within cancerous cells, is still facing challenges. Herein, we fabricated a strategy to improve the stability of polymer micelles using pseudo- and chemical crosslinking jointly. To be specific, a star-shaped polymer (TMP-PGMA- g -PEG) with trimethylolpropane (TMP) as the inner core was synthesized with glycidyl methacrylate (GMA), followed by the graft reaction with amine-terminated poly(ethylene glycol) (mPEG-NH 2 ). Star polymer micelle-based nanomedicines (TPP/paclitaxel (PTX)) were obtained using paclitaxel (PTX) as a model drug and polymer micelles (TPP) as carriers, which were constructed by TMP-PGMA- g -PEG. The star core and arms behaved as pseudo crosslinkers, which reduced their critical micelle concentration (CMC) values and improved their stability; profoundly, cystamine was used as a chemical crosslinker to react with the rest of the epoxy groups of TPP or TPP/PTX and further improve their stability. Finally, dual pseudo and chemical crosslinked star polymer micelles (CTPP) and micelle-based nanomedicines (CTPP/PTX) were obtained. The results demonstrated that CTPP/PTX with combined stability design presented excellent stability both in vitro and in vivo physiological conditions. Notably, cystamine not only served as a crosslinker but also had a reduction-responsive disulfide bond to achieve fast release inside cancer cells with a high level of glutathione (GSH). This smart design effectively resolved the antinomy that the polymer micelle delivery system could not cause rapid release in tumor sites when it possesses extreme stability resulted from chemical crosslinking. Both in vitro and in vivo experiments clearly stated the advantages of CTPP/PTX, including excellent stability, fast reduction-responsive release, and remarkable antitumor efficacy.

Pure gelatin hydrogel (PG) has limited practical applications due to their thermal instability and unfavorable mechanical properties. To overcome these limitations, dually crosslinked hydrogels were developed by imparting chemical crosslinking to existing physically crosslinked gelatin hydrogel networks using succinoglycan dialdehyde (SGDA) as a macromolecular crosslinker. SGDA-reinforced gelatin hydrogels (SGDA/Gels) displayed an 11 times higher compressive stress under identical deformation strain and a 1040% improvement in storage modulus (G') than PG. In addition, chemical crosslinking induced by SGDA increased the thermal stability of SGDA/Gels, such that they did not decompose at 60 °C, as confirmed by oscillatory temperature ramp experiments. The newly synthesized SGDA/Gels with reinforced networks and thermal stability exhibit potential for long-term use as controlled drug delivery carriers and 3D cell culture scaffolds for tissue engineering., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Fibrin glue has been widely used as a surgical sealing and hemostatic agent. Its application is restricted due to poor tissue adhesion and low mechanical strength. To develop better tissue sealant and hemostatic agent, this study prepared the injectable hydrogels by chemically cross-linking gelatin (G) with or without hyaluronic acid (HA) in situ at a mild condition. The rheological analysis, Fourier transform infrared spectroscopy, swelling, proteolytic degradation, biocompatibility, tissue sealing, and hemostatic ability of the hydrogels were investigated. It was found that the chemical cross-linking rapidly formed in both self-crosslinking gelatin (sc-G) and gelatin/hyaluronate acid (G/HA) hydrogels. The hydrogels could be degraded by trypsin and had a desirable biocompatibility. The tissue sealing ability of the hydrogels was superior to fibrin glue. Furthermore, the G/HA hydrogel had similar hemostatic performance as fibrin glue, and was better than that of gelatin hydrogel. The results in the study indicated that the G/HA hydrogel could be used in clinic as a tissue sealant or surgical hemostat., (© 2019 Wiley Periodicals, Inc.)

Stretchable and tough hydrogels have drawn a lot of attention recently. Due to their unique properties, they have great potential in the application in areas such as mechanical sensing, wound healing, and drug delivery. In this review, we will summarize recent developments of stretchable and tough hydrogels in these areas.

Herein the construction of a strong gelatin hydrogel is presented by using pullulan dialdehyde (PDA) as a macromolecular crosslinker. The resultant PDA crosslinked gelatin hydrogels (G-PDA) exhibit extremely high mechanical strength, manifested in the achieved optimal compressive stress of 5.80 MPa at 80% strain, which is up to 152 times higher than pure gelatin hydrogel. The G-PDA were characterized by Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). The extent of crosslinking was determined by ninhydrin assay. The results suggested that the synergistic effect of dual-crosslinking, which is composed of short- and long-range covalent crosslinking and thermoreversible physical crosslinking, may played a key role in enhancing the load-bearing capacity of ensuing hydrogels. The swelling and enzymatic degradation of G-PDA are gradually limited with increasing PDA concentration. The result from MTT assay demonstrated that G-PDA is non-cytotoxic against MC3T3 cells, regardless of the concentrations of PDA., (Copyright © 2019 Elsevier Ltd. All rights reserved.)