Cross-linking Mode Research Articles

Control of bacterial cell wall autolysins by peptidoglycan crosslinking mode

To withstand their internal turgor pressure and external threats, most bacteria have a protective peptidoglycan (PG) cell wall. The growth of this PG polymer relies on autolysins, enzymes that create space within the structure. Despite extensive research, the regulatory mechanisms governing these PG-degrading enzymes remain poorly understood. Here, we unveil a novel and widespread control mechanism of lytic transglycosylases (LTs), a type of autolysin responsible for breaking down PG glycan chains. Specifically, we show that LD-crosslinks within the PG sacculus act as an inhibitor of LT activity. Moreover, we demonstrate that this regulation controls the release of immunogenic PG fragments and provides resistance against predatory LTs of both bacterial and viral origin. Our findings address a critical gap in understanding the physiological role of the LD-crosslinking mode in PG homeostasis, highlighting how bacteria can enhance their resilience against environmental threats, including phage attacks, through a single structural PG modification.

Identification of a family of peptidoglycan transpeptidases reveals that Clostridioides difficile requires noncanonical cross-links for viability

Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen Clostridioides difficile, about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in C. difficile. We also show that C. difficile has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficile VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in C. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively.

Lightweight, compressible, elastic resorcinol-formaldehyde aerogels with cobalt-ligand-enhanced cross-linked skeletons

Resorcinol-formaldehyde (RF) aerogel materials exhibit remarkable properties such as low density, ablation resistance (high residual carbon rate), easily regulated pore structure and low thermal conductivity, making them promising for applications in adsorption, medicine and aerospace, among others. However, since their inception, RF aerogels have suffered from disadvantages such as brittleness and long preparation cycles, which have severely limited their widespread use. In order to improve the mechanical properties of RF aerogels, this paper presents a new synthetic route for obtaining strong elastic properties without introducing additional fiber reinforcement. Here, a lightweight, compressible, elastic resorcinol-formaldehyde aerogel with cobalt-ligand-enhanced cross-linked skeletons was prepared by sol-gel and freeze-drying methods using an alkali metal salt (cobalt (II) acetate) as a cross-linking agent and sodium acetate as a catalyst. The coordination of metal ions (Co2+) with resorcinol resulted in the formation of a structure analogous to a metal-phenolic network (MPN), which altered the crosslinking mode and degree of crosslinking of the RF aerogel. This process led to the thickening of the skeletal structure of the sample. Concurrently, a multilayered pore structure was generated within the interior under the appropriate pH conditions, which contributed to the strengthening of the skeleton of the samples and a reduction in thermal conductivity. The obtained RF-Co aerogel can withstand 68.6 % compression change without fracture and shows good elastic deformation at 35 % compression rate, while the aerogel exhibits low thermal conductivity (0.039 W (m K)−1) and low density (0.09 g cm−3).

Effect of Fe(III) on soybean protein-gallic acid hydrogel: Structure, functional properties, and digestive properties

A soy protein isolate-gallic acid (SPI-GA) complex was synthesized, mediated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide. Different amounts of Fe3+ were then added to the SPI-GA complex to produce hydrogels (SPA@Fe) containing metal-phenolic network without heating the protein. Spectral analysis revealed that these hydrogels contained multiple modes of cross-linking, such as Schiff bases, hydrogen bonds, and coordination bonds. Coordination bonds formed between GA and Fe3+ mainly had a coordination number of three, and they changed the structure of the protein. Furthermore, these coordination bonds also endowed SPA@Fe with a more compact crosslinked network, thus improving the hydrogel's textural characteristics, thermal stability, storage modulus, and glucose adsorption capacity. The SPA@Fe hydrogel exhibited the best performance when the molar ratio of GA to Fe3+ was 1, with the thermal stability, hardness, springiness, and glucose adsorption capacity being 3.9, 1.84, 1.22, and 1.93 times higher than those of the SPI-GA hydrogel, respectively. Moreover, in-vitro digestion tests showed that SPA@Fe could control the release of loaded curcumin, had good capacity for adsorbing bile salt, and showed delayed degradation. This hydrogel system may offer a safe and feasible way to deliver bioactive substances such as curcumin.

Investigating the Effect of Ionic and Covalent Crosslinkers on the Properties of Marine-based Macroalgae Biofilm Composite

Macroalgae with nontoxicity, biodegradability and biocompatibility has attracted more attentions as a sustainable alternative towards petroleum-derived plastics. However, brittleness and high affinity towards water has limited marine-based macroalgae films to be widely used. Thus, this paper aims to compare the roles of calcium chloride as ionic cross-linker and acrylic resin as covalent cross-linker in agar-PEG plasticised biofilm composite. Agar biofilm composite with PEG 1000 40% were cross-linked with i) calcium chloride 1, 2, 3 and 4 (CaCl2/agar) wt/wt % at 2 or 4 min immersion time, and ii) acrylic resin at 10, 20, 30, 40 and 50 (acrylic resin/agar) wt/wt %. FTIR and TGA proved that CaCl2 and acrylic resin managed to cross-link agar-PEG films ionically and covalently respectively. In general, films cross-linked with CaCl2 produced highest affinity towards water than films cross-linked with acrylic resin. Highest tensile strength, highest tensile modulus and lowest elongation at break was achieved when films cross-linked with CaCl2. Films cross-linked with CaCl2 has higher degradation rate than films cross-linked with acrylic resin. In conclusion, different mode of cross-linking (ionic or covalent) imparted different properties on the agar-PEG film.

Applications and Recent Developments of Hydrogels in Ophthalmology.

Hydrogels are a type of functional polymer material with a three-dimensional network structure composed of physically or chemically cross-linked polymers. All hydrogels have two common features: first, their structure contains a large number of hydrophilic groups; therefore, they have a high water content and can swell in water. Second, they have good regulation, and the physical and chemical properties of their cross-linked network can be changed by environmental factors and deliberate modification methods. In recent years, the application of hydrogels in ophthalmology has gradually attracted attention. By selecting an appropriate composition and cross-linking mode, hydrogels can be used in different fields for various applications, such as gel eye drops, in situ gel preparation, intravitreal injection, and corneal contact lenses. This Review provides a detailed introduction to the classification of hydrogels and their applications in glaucoma, vitreous substitutes, fundus diseases, corneal contact lenses, corneal diseases, and cataract surgery.

Engineering biodegradable controlled gelatin-zein bilayer film with improved mechanical strength and flexibility

Compiling hydrophobic and hydrophilic proteins to form mechanically reliable bilayer film with improved water barrier properties and hydrophobicity to replace petroleum-based standards is challenging because fabricating compatible surfaces and flexible film with interlinked interfaces is complex. The mechanical properties of gelatin and zein layers exhibited unique variations, and synergistic enhancement of bilayer properties reflected robust layer-by-layer (LBL) interactions. To overcome the problem of impaired integrated surface, viscously cross-linked zein film forming solution (FFS) by glyoxal (GX) introduced compact and closely entangled network formation with cross-linking of gelatin film. Zein surface compatibility and flexibility by GX and polyethylene glycol (PEG 400) synergized with the gelatin layer closed to compare flexible plastic materials such as PE and PP besides modulated color and transparency. Enhancing flexible formation was mechanized by acetic acid as solvent of zein solution after long-range repulsion during casting on well-manageable plasticized and cross-linked gelatin film with glutaraldehyde (GA) and PEG 400, respectively. Elongation (E%) was achieved by nearly 192% with modified tensile strength (TS) and Young's modulus (YM). Cross-linkers, especially GX, resulted in lower water solubility (WS), and joint layers resulted in controlled water vapor permeability (WVP) of bilayer films. Decreased surface roughness and WS increased water contact angle (WCA). FTIR spectroscopy, XRD analysis, DMTA, and SDS-PAGE analyzed cross-linking mode and intensity. Hence, gelatin-zein bilayer films showed significant potential for improving value-added functional properties.

Narrow-Pore Engineering of Vinylene-Linked Covalent Organic Frameworks with Weak Interaction-Triggered Multiple Responses.

Vinylene-linked covalent organic frameworks (COFs) are emerging as promising crystalline materials, but their narrow pore engineering is severely impeded by the weak reversibility of the carbon-carbon double bond formation reaction, which has led to less exploration of their ultramicroporous structures and properties. Herein, we developed a single aromatic ring-based tetratopic monomer, tetramethylpyrazine, which undergoes a smooth Knoevenegal condensation at its four arylmethly carbon atoms with linear aromatic dialdehyde monomers upon the self-catalyzed activation of pyridine nitrogen-containing monomers in the presence of an organic anhydride. This has resulted in the formation of two vinylene-linked COFs, which both crystallized in orthorhombic lattices, and layered in AA stacking fashions along the vertical directions. They exhibit high surface areas and well-tailored ultramicropore sizes up to 0.5 nm. The unique cross-linking mode at two pairs of para-positions of each pyrazine unit through carbon-carbon double bonds afford them with π-extended conjugation over the in-plane backbones and substantial semiconducting characters. The resultant COFs can be well-dispersed in water to form stable sub-microparticles with negative charges (zeta potentials: ca. -30 mV), and exhibiting tunable aggregation behaviors through protonation/deprotonation. As a consequence, they exhibit pore-size-dependent colorimetric responses to various anions with different pKa values in high selectivity.

High uptake of citrus essential oil-loaded zein particles into E. coli tuned by the wet-crosslinked folic acid

Zein particles are increasingly used for encapsulation and delivery of hydrophobic bioactive. Conjugation of zein with multi-carboxylic acids by the alkali-catalysed and wet amidation process causes zein crosslinking and enables tuning the characteristics of zein particles. However, little is known about how the conjugation is associated with the crosslinking mode, i.e., intra and/or intermolecular crosslinks. In this study, folic acid (FoA) was conjugated with zein via the wet method, and citrus essential oil (CEO) was encapsulated within the constructed zein-FoA particles. Infra-red spectroscopy and nuclear magnetic resonance analysis indicated amide bond formation between zein and FoA showing that the content of primary amino groups in zein particles decreased due to conjugation. The mole fraction of FoA in the particles (χFoA) was measured as an index of conjugation extent and even though it did not exceed ∼0.1, conjugation influenced several characteristics of the particles. The contents of α-helix and β-turn structures decreased, and that of β-sheets increased with increasing χFoA. We hypothesized based on ζ-potential and hydrodynamic diameter results, as well as gel electrophoresis that conjugation with FoA caused formation of only intramolecular crosslinks at χFoA ≤ 0.035, but it additionally resulted in intermolecular crosslinks at χFoA ≥ 0.037. Conjugation with FoA at χFoA 0.091 resulted in an improvement of the zein particles biological performance. It caused an increase (∼30%) in the particle uptake by E. coli cells, as well as the magnitude of CEO delivery into the bacteria cells compared with non-conjugated particles.

Orthogonally crosslinked alginate conjugate thermogels with potential for cell encapsulation

Hydrogels with more than one mode of crosslinking have gained interest due to improved control over hydrogel properties such as mechanical strength using multiple stimuli. In this work, sodium alginate was covalently conjugated onto thermoresponsive polyurethanes to prepare hybrid polymers (EPC-Alg) that are responsive to both temperature and Ca2+, forming orthogonally crosslinked hydrogels which are non-toxic to cells. Notably, the crosslinks are fully reversible, allowing for gel strength to be modulated via selective removal of either stimulus, or complete deconstruction of the hydrogel network by removing both stimuli. Higher alginate fractions increased the hydrophilicity and Ca2+ response of the EPC-Alg hydrogel, enabling tunable modulation of the thermal stability, stiffness and gelation temperatures. The EPC-Alg hydrogel could sustain protein release for a month and encapsulate neural spheroids with high cell viability after 7-day culture, demonstrating feasibility towards 3D cell encapsulation in cell-based biomedical applications such as cell encapsulation and cell therapy.

Study on the 3D BioPrinting Technology of Hydrogel Tissue Engineering scaffolds

The precision molding capability of bio-3D printing is the basis for its application in tissue engineering, and SA-Gel two-component hydrogel has been widely used in bio-3D printing manufacturing of tissue engineering scaffolds. The extrusionability of hydrogel is the key point for the printing precision. In this paper, starting from the concentration, ratio, rheology and cross-linking mode of hydrogel, the research ideas of “single factor independent analysis” and multi-factor comprehensive application are adopted to solve the printing accuracy. Through the research and screening of printing parameters and printing compensation technology, the accurate printing of the 1D line segment and 2D grid structure at 45°C for SA-Gel hydrogel with 2% and 8% mass respectively have been achieved.

Insights into the mechanism on the high-temperature activity of transglutaminase from Bacillus clausii and its crosslinked mode at protein level

Finding new crosslinking enzymes and elucidating internal cross-linking mode are beneficial to meeting the requirement of various protein substrates and processing conditions in food industry. Transglutaminase (TG) can lead to the covalent crosslink of ε-(γ-glutamine)-lysine isopeptidic bonds in proteins through acyl-transfer reaction. This work reported the characterization of transglutaminases (TGs) from four Bacillus strains with obvious differences in amino acid sequences and enzymatic properties. Among them, TG from B. clausii (BCLTG) displayed the highest optimal temperature (70 °C) and specific activity (282 U/mg). The molecular dynamics simulation indicated that enlargement of substrate accessible tunnel in BCLTG with the increased temperature might result in the easy approach of substrate molecule to the active center. Moreover, BCLTG could cause the crosslinking of bovine serum albumin and almost all protein constituents in whey protein and soy protein isolate, and the crosslinking path of BCLTG towards three proteins was revealed. This study provides an ideal candidate for proteins crosslinking and theoretical foundation and preliminary information for applying TG in food processing.

Ab-Initio Investigations on Hydrogen Dissociation and Cross-Linking of Hydrocarbon Chains of Self-Assembled Monolayers of Alkanes

First-principles calculations were carried out to study the structural and electronic properties of hydrocarbon chains of self-assembled monolayers with hydrogen dissociation. It was found that the incoming hydrogen could lead to the formation of H2 molecules by stripping the nearby hydrogen atoms in the chains and thereby leave the neighboring carbon atoms to be free radicals. Two parallel hydrocarbon chains with dangling bonds can form a direct C-C bond, i.e., cross-linking happens between the two chains, which is ascribed to a charge accumulation in the cross-linking region. The polymerization of short molecules into long hydrocarbon chains through a different cross-linking mode is also discussed.

Sodium alginate-derived porous carbon: Self-template carbonization mechanism and application in capacitive energy storage

Sodium alginate (SA) is an environment-friendly and low-cost polysaccharide carbohydrate extracted from seaweed. As a carbon precursor, sodium alginate has the advantages of clear molecular structure, small molecular weight, and easy controls of the structure and composition of the product, but there have been few studies for the mechanism for SA carbonization. In this work, the carbon skeleton cross-linking mode, heteroatom doping and defect generation mechanism in the process of SA pyrolysis are clarified. Subsequently, based on the understanding of the carbonization mechanism of SA-derived carbon, we have prepared a stable SA-derived interconnected porous carbon by self-template method. The materials prepared by this method possess high oxygen content (17.6 at%) and high specific surface area (384.4 m2 g−1). Zn-ion hybrid capacitors (ZICs) device assembled with SA-derived porous carbon performs superior energy densities (based on cathode mass) of 78.35 and 35.56 Wh kg−1 at the power densities of 160 and 5120 W kg−1, respectively. This work deeply explained the carbonization mechanism of sodium alginate and evaluated the application prospects of SA-based carbon in ZICs comprehensively.

Fe3O4@PDA@PEI Core-Shell Microspheres as a Novel Magnetic Sorbent for the Rapid and Broad-Spectrum Separation of Bacteria in Liquid Phase.

Bacterial infection is a significant cause of morbidity and mortality to humans worldwide. Thus, a method for nonspecific, sensitive, and rapid enrichment of such bacteria is essential for bacteria detection and treatment. This study demonstrates a self-made core-shell Fe3O4@Polydopamine@Polyethyleneimine magnetic beads (Fe3O4@PDA@PEI MBs) with a high density positive charge-based magnetic separation scheme for the broad-spectrum rapid enrichment of microorganisms in the liquid phase. MBs with a high-density positive charge have a strong electrostatic attraction to most microorganisms in nature. Our scheme is as follows: (1) wrapping dopamine (DA) on the iron oxide through self-polymerization and wrapping PEI on the outermost shell layer in a mode of crosslinking with the PDA; (2) subsequently, the Fe3O4@PDA@PEI MBs were used to concentrate microorganisms from the sample solution; (3) performing magnetic separation and calculating the adsorption efficiency. The as-prepared Fe3O4@PDA@PEI MBs composite was carefully characterized by zeta potential analysis, Value stream-mapping (VSM), transmission electron microscopy (TEM), and Fourier transforms infrared spectrometry (FT-IR). In this study, both gram-positive and gram-negative bacteria could be captured in three minutes through electrostatic interaction. Furthermore, the adsorption efficiency on gram-negative (>98%) is higher than that on gram-positive (>95%), allowing for a simple, rapid assay to enrich organisms in resource-limited settings.

The effect of modifying the nanostructure of gelatin fiber scaffolds on early angiogenesis in vitro and in vivo

Early angiogenesis is one of the key challenges in tissue regeneration. Crosslinking mode and fiber diameter are critical factors to affect the adhesion and proliferation of cells. However, whether and how these two factors affect early angiogenesis remain largely unknown. To address the issue, the optimal crosslinking mode and fiber diameter of gelatin fiber membrane for early angiogenesis in vivo and in vitro were explored in this work. Compared with the post crosslinked gelatin fiber membrane with the same fiber diameter, the 700 nm diameter in situ crosslinked gelatin fiber membrane was found to have smaller roughness (230.67 ± 19 nm) and stronger hydrophilicity (54.77° ± 1.2°), which were suitable for cell growth and adhesion. Moreover, the in situ crosslinked gelatin fiber membrane with a fiber diameter of 1000 nm had significant advantages in early angiogenesis over the two with fiber diameters of 500 and 700 nm by up-regulating the expression of Ang1, VEGF, and integrin-β1. Our findings indicated that the in situ crosslinked gelatin fiber membrane with a diameter of 1000 nm might solve the problem of insufficient blood supply in the early stage of soft tissue regeneration and has broad clinical application prospects in promoting tissue regeneration.

Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli.

Bacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of β-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3→3 cross-links compared to 4→3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify among eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4→3 (MepM) and 3→3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4→3 and 3→3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4→3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of β-lactams.

Pectin hydrogels, aerogels, cryogels and xerogels: Influence of drying on structural and release properties

Pectin is an appealing material for biomedical applications because of its biocompatibility, bioactivity, biodegradability as well as its gelling and stabilizing properties. In this work, porous pectin cryogels, aerogels and xerogels were prepared via freeze-drying, sc drying and evaporative drying at 60 °C under low vacuum, respectively, starting from the same pectin precursor, a hydrogel. The physico-chemical and structural properties of the porous pectin matrices were investigated and related to the preparation conditions. The release of the model hydrophilic respiratory drug theophylline from all pectin networks, including the hydrogel, was studied and related to the network morphology and erosion profiles. The Korsmeyer-Peppas and Peppas-Sahlin models were used to determine the main physical mechanisms controlling the release. The release of theophylline can be tuned via the mode of pectin crosslinking in the initial hydrogel as well as via the drying method, demonstrating the potential of porous pectin matrices for drug delivery purposes.

Layer-by-layer self-assembly films for buccal drug delivery: The effect of polymer cross-linking

The goal of this study was the preparation of multilayer polyectrolyte films intended for buccal delivery of benzydamine (BZ). Another important issue was to evaluate the effect of polymer cross-linking as a means to enhance drug loading and ensure sustained drug release.LbL deposition technique was applied for the multilayer build-up. The deposition was performed by alternative dip-coating of corona pretreated polylactic acid substrates into chitosan and caseine solutions. Chitosan and casein deposition was followed by cross-linking with glutaraldehyde/sodium tripolyphosphate, and calcium chloride, respectively. For drug loading, the multilayers were soaked into benzydamine solution after chitosan cross-linking. The procedure was carried out until eight layers were laid. The formation of polyelectrolyte complex and the cross-linking occurrence were proven by ATR-FTIR. Further investigations on the morphology and topography of the samples were carried out by AFM. Moreover, swelling behavior, content uniformity, drug release and mucoadhesion were investigated.The experimental data displayed differences in the structure and surface properties of the films as per the cross-linking agent used. All the investigated parameters were influenced by the cross-linking mode. Double cross-linking of chitosan with glutaraldehyde and sodium tripolyphosphate proved to be the most appropriate method for the achievement of the desired goals.

The Tail of XCTK2 Contains Two Distinct Microtubule Binding Domains

Molecular motors organize the microtubules (MTs) that make up the mitotic spindle, and defects in motor function can cause improper spindle assembly that leads to chromosome mis-segregation. The Kinesin-14 family of molecular motors facilitates spindle pole focusing by cross-linking and sliding anti-parallel and parallel MTs through their motor and tail domains. How the tail contributes to these functions is unclear. Kinesin-14 tail MT binding is inhibited by importin α/β association through a nuclear localization signal (NLS). To understand how importin α/β association regulates the cross-linking and sliding activity of Kinesin-14s, we visualized Xenopus XCTK2 cross-linking and sliding in vitro. Surprisingly, importin α/β preferentially inhibited anti-parallel cross-linking and sliding. These results suggest that the tail may contain two different MT binding domains (MBDs) that recognize the anti-parallel and parallel MT orientations. To test this hypothesis, we created a deletion series of proteins from the N-terminus and C-terminus of the tail and tested them for MT binding. We identified two MBDs, which we call MBD1 and MBD2, that had MT binding abilities similar to the full-length tail. YPet-tagged MBD1, which contains the NLS, displayed robust FRET with importin α-CyPet and importin β, indicating tight association; whereas, MBD2 did not. In the presence of importin α/β, MBD1 was also completely inhibited from binding to MTs. These results are consistent with the model that MBD1 recognizes the anti-parallel conformation, and MBD2 recognizes the parallel conformation. We are currently generating site-specific mutations within MBD1 and MBD2 to inhibit MT binding so that we can demonstrate separation of cross-linking activities in the full-length protein. Once we identify the mutations that disrupt anti-parallel or parallel MT cross-linking, we will test how these modes of cross-linking and sliding contribute to spindle assembly.