Oxidative Cross-linking Research Articles

Noncanonical Amino Acid Incorporation Modulates Condensate States of Intrinsically Disordered Proteins in Escherichia coli Cells.

Biomolecular condensates are distinct subcellular structures with on-demand material states and dynamics in living cells. However, strategies for modulating their material states and physicochemical properties are lacking. Here, we report a chemical strategy for modulating the condensate states of intrinsically disordered proteins in bacterial Escherichia coli cells. This is achieved by noncanonical amino acid (DOPA) incorporation into model resilin-like proteins (RLPs) to endow autonomous oxidative and coordinative cross-linking mechanisms. Biogenesis of spherical gel-like condensates is achieved upon heterologous expression of the DOPA-incorporated RLP in the cells at 30 °C. We reveal that liquid-liquid phase separation underlies the formation of liquid condensates, and this liquid-like state is metastable and its dynamics is compromised by the oxidative and coordinative cross-linkings that ultimately drive the liquid-to-gel transition. Therefore, this study has deepened our understanding of biomolecular condensation and offers a new chemical strategy to expand the landscape of condensation phenotypes of living cells.

Synergistic effect of Hypoxic Conditioning and Cell-Tethering Colloidal Gels enhanced Productivity of MSC Paracrine Factors and Accelerated Vessel Regeneration.

Microporous hydrogels have been widely used for delivering therapeutic cells. However, several critical issues, such as the lack of control over the harsh environment they are subjected to under pathological conditions and rapid egression of cells from the hydrogels, have produced limited therapeutic outcomes. To address these critical challenges, cell-tethering and hypoxic conditioning colloidal hydrogels containing mesenchymal stem cells (MSCs) are introduced to increase the productivity of paracrine factors locally and in a long-term manner. Cell-tethering colloidal hydrogels that are composed of tyramine-conjugated gelatin prevent cells from egressing through on-cell oxidative phenolic crosslinks while providing mechanical stimulation and interconnected microporous networks to allow for host-implant interactions. Oxygenating microparticlesencapsulated in tyramine-conjugated colloidal microgels continuously generated oxygen for 2 weeks with rapid diffusion, resulting in maintaining a mild hypoxic condition while MSCs consumed oxygen under severe hypoxia. Synergistically, local retention of MSCs within the mild hypoxic-conditioned and mechanically robust colloidal hydrogels significantly increased the secretion of various angiogenic cytokines and chemokines. The oxygenating colloidal hydrogels induced anti-inflammatory responses, reduced cellular apoptosis, and promoted numerous large blood vessels in vivo. Finally, mice injected with the MSC-tethered oxygenating colloidal hydrogels significantly improved blood flow restoration and muscle regeneration in a hindlimb ischemia (HLI) model.

Synergistic tuning of microstructure and morphology in carbon molecular sieve hollow fibers for propylene/propane separation

Asymmetric carbon molecular sieve (CMS) hollow fiber membranes with tunable micro‐ and macro‐structural morphologies for energy efficient propylene‐propane separation are reported here. A sub‐glass transition temperature (sub‐Tg) thermal oxidative crosslinking strategy enables simultaneous optimization of the intrinsic molecular sieving properties while also reducing the thickness of the CMS “skin” derived from the 6FDA:BPDA/DAM polyimide precursors. Such synergistic tuning of CMS microstructure and macroscopic morphology of CMS hollow fibers enables significantly increased propylene permeance (reaching 186.5 GPU) while maintaining an appealing propylene/propane selectivity of 13.3 for 50/50 propylene/propane mixed gas feeds. Our findings reveal a more refined and versatile tool than available with previous O2‐doping pretreatments. The advanced approach here should be broadly useful to other polyimide precursors and diverse gas pairs.

Synergistic Tuning of Microstructure and Morphology in Carbon Molecular Sieve Hollow Fibers for Propylene/Propane Separation.

Asymmetric carbon molecular sieve (CMS) hollow fiber membranes with tunable micro- and macro-structural morphologies for energy efficient propylene-propane separation are reported here. A sub-glass transition temperature (sub-Tg) thermal oxidative crosslinking strategy enables simultaneous optimization of the intrinsic molecular sieving properties while also reducing the thickness of the CMS "skin" derived from the 6FDA : BPDA/DAM polyimide precursors. Such synergistic tuning of CMS microstructure and macroscopic morphology of CMS hollow fibers enables significantly increased propylene permeance (reaching 186.5 GPU) while maintaining an appealing propylene/propane selectivity of 13.3 for 50/50 propylene/propane mixed gas feeds. Our findings reveal a more refined and versatile tool than available with previous O2-doping pretreatments. The advanced approach here should be broadly useful to other polyimide precursors and diverse gas pairs.

A tough, strong, and fast-curing phenolic resin enabled by dopamine-grafted chitosan and polyethyleneimine-functionalized graphene

Phenolic resins are widely used for outdoor and structural wood-based panels; however, they are challenged by high curing temperatures, low curing rates, and high brittleness. Inspired by lobster epidermis hardening, a tough, strong, and fast-curing phenolic resin (named DCS/PG/PF) was proposed herein. In this approach, dopamine-grafted chitosan (DCS) and polyethyleneimine-functionalized graphene (PEI@G) were incorporated into neat phenol formaldehyde (PF) resin. The gel time and maximum curing temperature of DCS/PG/PF resin were considerably reduced from 445 s and 147.8 °C for the neat PF resin to 317 s and 127.8 °C, respectively. This was attributed to the oxidative crosslinking of catechol moieties in DCS and amino groups in PEI@G within the naturally alkaline environment of phenolic resins in addition to the high reactivity between catechol moieties and PF chains as well as between amino and PF chains. The prepared resin demonstrated a dry bonding strength of 2.56 MPa, wet bonding strength of 1.81 MPa, and debonding work of 0.714 J, exhibiting a considerable increase of 16.9 %, 52.1 %, and 95.1 %, respectively, compared with those of the PF resin. These improvements were attributed to the dense organic–inorganic hybrid crosslinking network formed in the DCS/PG/PF. Furthermore, the DCS/PG/PF resin exhibited enhanced thermal stability.

Oxidative regulation and cytoprotective effects of γ-polyglutamic acid on surimi sol subjected to freeze-thaw process

Frozen surimi sol incline to protein oxidation, but the quality control strategies based on oxidation remain limited. Hence, the antioxidant and cryoprotective effects of γ-polyglutamic acid (γ-PGA) on freeze-thawed salt-dissolved myofibrillar protein (MP) sol were investigated. Results showed that γ-PGA could effectively regulate protein oxidation of MP sol during freeze-thawing with lower carbonyl contents and less oxidative cross-linking. Meanwhile, γ-PGA primely maintained sol protein structures, showing reduction of 15.28% of salt soluble protein contents at γ-PGA addition of 0.04% under unoxidized condition. Additionally, compared to the control group without oxidation treatment, cooking loss of heat-induced gel with 0.04% γ-PGA decreased by 47.19%, while gel strength obviously increased by 57.22% respectively. Overall, moderate γ-PGA addition (0.04%) could inhibit protein oxidation of sol, further improving frozen stability of sol through hydrogen bonds and hydrophobic interaction, but excessive γ-PGA was adverse to sol quality due to severe cross-linking between γ-PGA and MP.

Upscaling Osteoclast Generation by Enhancing Macrophage Aggregation Using Hollow Microgels.

Osteoclasts, the bone resorbing cells of hematopoietic origin formed by macrophage fusion, are essential in bone health and disease. However, in vitro research on osteoclasts remains challenging due to heterogeneous cultures that only contain a few multinucleated osteoclasts. Indeed, a strategy to generate homogeneous populations of multinucleated osteoclasts in a scalable manner has remained elusive. Here, the investigation focuses on whether microencapsulation of human macrophages in microfluidically generated hollow, sacrificial tyramine-conjugated dextran (Dex-TA) microgels could facilitate macrophage precursor aggregation and formation of multinucleated osteoclasts. Therefore, human mononuclear cells are isolated from buffy coats and differentiated toward macrophages. Macrophages are encapsulated in microgels using flow focus microfluidics and outside-in enzymatic oxidative phenolic crosslinking, and differentiated toward osteoclasts. Morphology, viability, and osteoclast fusion of microencapsulated cells are assessed. Furthermore, microgels are degraded to allow cell sorting of released cells based on osteoclastic marker expression. The successful encapsulation and osteoclast formation of human macrophages in Dex-TA microgels are reported for the first time using high-throughput droplet microfluidics. Intriguingly, osteoclast formation within these 3D microenvironments occurs at a significantly higher level compared to the conventional 2D culture system. Furthermore, the feasibility of establishing a pure osteoclast culture from cell transfer and release from degradable microgels is demonstrated.

Accelerated multistep thermal stabilization of polyacrylonitrile fibers using an ethylenediamine pretreatment

The polyacrylonitrile multifilament yarn underwent a multistep heat treatment process including stabilization times ranging from 5 to 75 min following impregnation with a 30% ethylenediamine (EDA) aqueous solution. A series of measurements were employed to determine the structure and properties of thermally stabilized PAN samples. These included fiber thickness, fiber density, flame testing, tensile testing, X-ray diffraction, thermal analysis (differential scanning calorimetry and thermogravimetric analysis), and infrared spectroscopy. The results from XRD and IR spectroscopy indicated that the rate of aromatization reactions increased with longer stabilization times. A detailed examination of the XRD curves obtained through curve-fitting procedures suggested a rapid transformation of the original structure into a disordered amorphous phase containing pre-graphitic domains, evidenced by a gradual reduction in the degree of apparent crystallinity of the original PAN sample. The integration of EDA before the thermal stabilization stage significantly reduced the cyclization time of nitrile groups in the PAN polymer structure, thereby accelerating the stabilization reactions. This chemical pretreatment also improved the thermal stability of the samples by promoting oxidative cross-linking of the PAN polymer chains. After a 75 min multistep stabilization, the carbon yield at 1000 °C was 70.5%. Conversion index values, calculated using IR, XRD, and DSC methods, were 98.3%, 94.8%, and 89.5% respectively for the 75 min sample. These findings highlight the importance of EDA in accelerating the formation of an aromatic structure, which is critical for withstanding the high temperatures of subsequent carbonization stages.Graphical abstract

Enzymatically cross-linked arabinoxylan hydrogel: Effect of glucose oxidase on the gel characteristics and oral colon-targeted delivery for Naja atra neurotoxin

Water-extracted arabinoxylan (WEAX) can form covalent hydrogels and has potential applications in colon-targeted drug delivery. However, it is not clear which structural characteristics of WEAX gels can better facilitate drug release in the colon as a microbial triggered system. In this study, different rheological properties of WEAX gels were prepared through oxidative cross-linking using glucose oxidase (GOX) with varying amounts for oral colon-targeted released Naja atra neurotoxin (NANT). It was found that the WEAX hydrogel with an adequate strong cross-linking degree minimized NANT loss in the gastrointestinal tract and improved its release in the colon in vitro. Moreover, NANT was fixed to the wall of the gel network by noncovalent binding and could maintain its secondary structure in the WEAX–NANT hydrogel. The WEAX–NANT hydrogel had an obvious analgesic effect in the in vivo mice test. The results revealed that the WEAX–NANT hydrogel with a strong cross-linking degree was better at releasing NANT.

Detection of a Kinetically Competent Compound-I Intermediate in the Vancomycin Biosynthetic Enzyme OxyB.

Cytochrome P450 enzymes are abundantly encoded in microbial genomes. Their reactions have two general outcomes, one involving oxygen insertion via a canonical "oxygen rebound" mechanism and a second that diverts from this pathway and leads to a wide array of products, notably intramolecular oxidative cross-links. The antibiotic of-last-resort, vancomycin, contains three such cross-links, which are crucial for biological activity and are installed by the P450 enzymes OxyB, OxyA, and OxyC. The mechanisms of these enzymes have remained elusive in part because of the difficulty in spectroscopically capturing transient intermediates. Using stopped-flow UV/visible absorption and rapid freeze-quench electron paramagnetic resonance spectroscopies, we show that OxyB generates the highly reactive compound-I intermediate, which can react with a model vancomycin peptide substrate in a kinetically competent fashion to generate product. Our results have implications for the mechanism of OxyB and are in line with the notion that oxygen rebound and oxidative cross-links share early steps in their catalytic cycles.

Effects of Laccase and Transglutaminase on the Physicochemical and Functional Properties of Hybrid Lupin and Whey Protein Powder.

Plant-based protein is considered a sustainable protein source and has increased in demand recently. However, products containing plant-based proteins require further modification to achieve the desired functionalities akin to those present in animal protein products. This study aimed to investigate the effects of enzymes as cross-linking reagents on the physicochemical and functional properties of hybrid plant- and animal-based proteins in which lupin and whey proteins were chosen as representatives, respectively. They were hybridised through enzymatic cross-linking using two laccases (laccase R, derived from Rhus vernicifera and laccase T, derived from Trametes versicolor) and transglutaminase (TG). The cross-linking experiments were conducted by mixing aqueous solutions of lupin flour and whey protein concentrate powder in a ratio of 1:1 of protein content under the conditions of pH 7, 40 °C for 20 h and in the presence of laccase T, laccase R, or TG. The cross-linked mixtures were freeze-dried, and the powders obtained were assessed for their cross-linking pattern, colour, charge distribution (ζ-potential), particle size, thermal stability, morphology, solubility, foaming and emulsifying properties, and total amino acid content. The findings showed that cross-linking with laccase R significantly improved the protein solubility, emulsion stability and foaming ability of the mixture, whereas these functionalities were lower in the TG-treated mixture due to extensive cross-linking. Furthermore, the mixture treated with laccase T turned brownish in colour and showed a decrease in total amino acid content which could be due to the enzyme's oxidative cross-linking mechanism. Also, the occurrence of cross-linking in the lupin and whey mixture was indicated by changes in other investigated parameters such as particle size, ζ-potential, etc., as compared to the control samples. The obtained results suggested that enzymatic cross-linking, depending on the type of enzyme used, could impact the physicochemical and functional properties of hybrid plant- and animal-based proteins, potentially influencing their applications in food.

Determining sulfur speciation in oxidatively crosslinked degradable polymers using sulfur K‐edge X‐ray absorption spectroscopy

AbstractA new family of water‐degradable elastic polymers prepared by oxidative crosslinking of the parent polythionolactones shows promise in a broad range of applications, but the compositions of these materials elude conventional analytical methods. Here we use sulfur K‐edge X‐ray absorption spectroscopy to quantify the amounts of thioether, disulfide, sulfone, sulfate ester, and thionoester in each polymer and to rule out the presence of several other functional groups, including sulfonate, thiosulfonate, sulfate, and sulfoxide. We rationalize this speciation as a function of linker flexibility in the context of sulfinyl cycloaddition reactions and propose a mechanism of aggregation for the oxidized polymers. Our results correlate with swelling ratios but not with porosity nor crosslink density measurements, demonstrating the importance of pairing mechanical and chemical techniques when characterizing heterogeneous organic polymers. Finally, we take advantage of the proximity of the gold M4,5‐edges to the sulfur K‐edge to analyze the binding and reactivity of Au(III) with the crosslinked polymers.

Additive-free Absorbable Keratin Sponge With Procoagulant Activity for Noncompressible Hemostasis.

The development of a natural, additive-free, absorbable sponge with procoagulant activity for noncompressible hemostasis remains a challenging task. In this study, we extracted high molecular weight keratin (HK) from human hair and transformed it into a hemostatic sponge with a well-interconnected pore structure using a foaming technique, freeze-drying, and oxidation cross-linking. By controlling the cross-linking degree, the resulting sponge demonstrated excellent liquid absorption ability, shape recovery characteristics, and robust mechanical properties. The HK10 sponge exhibited rapid liquid absorption, expanding up to 600% within 5 s. Moreover, the HK sponge showed superior platelet activation and blood cell adhesion capabilities. In SD rat liver defect models, the sponges demonstrated excellent hemostatic performance by sealing the wound and expediting coagulation, reducing the hemostatic time from 825 to 297 s. Furthermore, HK sponges have excellent biosafety, positioning them as a promising absorbable sponge with the potential for the treatment of noncompressible hemostasis.

Multiscale damage evolution and fracture for thermal aging energetic material based on mesoscopic observation and molecular dynamics simulation

As a composite energetic material composed of filler and matrix, the mechanical properties of HTPB propellants change constantly due to the aging of each component and component interfaces during long-term storage. However, the study of a single component or performance parameter limits disclosure of the complex aging mechanism and mechanical property evolution. Therefore, based on accelerated thermal aging experiments, this study integrated gas detection, contact angle testing, in situ loading experiments, digital image correlation numerical analysis, and all-atom molecular dynamic simulations to explore the underlying mechanism of structural morphology and mechanical property changes during propellant aging. The results showed that the aging reaction was dominated by degradation chain breaking in the early stage and oxidative crosslinking in the later stage. Degradation chain breaking enhanced interfacial bonding between the two phases, while oxidative crosslinking deteriorated interfacial bonding. Compared with lossless specimens, short-term thermal aging weakened the strength of the matrix and enhanced bonding interfaces. During in situ loading, interfacial pore expansion and matrix damage and fracture occurred simultaneously and the matrix became the key damage source. However, with the extension of thermal aging time, the strength of the matrix increased, bonding interface weakened, and bonding interfaces became a weak point of loading. This made the high-strain area change from the matrix at the initial loading stage to the bonding interface and the damage of the bonding interface caused whole fracture of the specimen. The external loading resulted in the rearrangement of interfacial molecular chains, change of molecular spacing, and initiation and expansion of micropores between molecular chains. The tractor-separation curve conformed to the exponential cohesion model and the damage mode of the interface layer changed between adhesion and cohesion due to thermal aging.

Experimental measurement and molecular dynamics simulation analysis of thermal aging performance in composite solid propellants

As a complex mixed system, the aging mechanism of high-energy composite materials involves the aging of each component itself, interactions between components, and transformation of microstructural and thermodynamic properties. The study of a single component or performance parameter has great limitations for elaborating the cognitive complexity of aging processes, making it difficult to reveal the underlying principles of physicochemical property transformation. Thus, it can only be applied as an empirical summary. In view of this, this study comprehensively utilized numerical analysis and experimental detection to explore the evolution of micromolecular conformation and changes in macro aging performance from four aspects of composite systems, including binder aging, plasticizer migration, bond interface debonding, and mechanical property transformation. There was good consistency observed between molecular simulation and thermal aging experiments, indicating that there was basically no post curing process for HTPB propellants using TDI as the curing agent. In the early stage of aging, scission chain was the main process and, in the later stage of aging, oxidation crosslinking the main process. Oxidative crosslinking increased the mean square radius of gyration and glass transition temperature of the binder system, reduced the peak value of radial distribution function, and revealed the reason for the change of propellant hardness and CC and CO double bond content. However, chain degradation increased the number of polar groups, enhanced intermolecular polarity, reduced the solubility parameter, and increased the free volume fraction of the mixed system and the dioctyl sebacate diffusion coefficient. This resulted in an increase in the tangent value of the HTPB propellant loss angle from 0.43 to 0.44° and a decrease in the glass transition temperature from 203.3 to 202.0 K. The binding energy at the interfaces of AP/HTPB and Al/HTPB increased first and then decreased, which was consistent with SEM images of bonding interfaces. The joint effect of binding energy and mechanical properties of the binder system promoted the transformation of the stress-strain curve of aging propellant.

Evaluation of the abilities of atomic oxygen/ultraviolet resistance for the filled silicone rubber coating: Structures, properties, and aging mechanism

A series of experimental techniques were used to study the erosion-damage effect of a silica-filled silicone rubber coating exposed to simulated space environment (atomic oxygen and ultraviolet irradiation). The elastomer is known to be chemically and thermally stable, but there are insufficient data on its durability in the space environment. In the present work, atomic oxygen doses up to 3.8 × 1021 atoms·cm−2 and ultraviolet irradiation doses up to 360 equivalent sun-hours (ESH) were applied from Space Environment Simulation Measurement System, respectively. Since the aging and failure processes of the filled silicone rubber relate to different aspects of chemical structure and physics form, therefore, this study explored the use of chemical-mechanics methods to comprehensively evaluate the erosion-damage effect of materials. Morphology and chemical changes due to radiation were investigated by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS), and nanoindentation test probed surface mechanical properties changed with exposure dose. Results demonstrated that, with increased exposure time and irradiation-induced free radicals，the aggregation state and molecular chain structure of the silica-filled silicone rubber have changed, which caused an increase in surface hardness and modulus. Taken together, the results indicated that oxidative cross-linking was the dominant degradation mechanism under the irradiation dose in this paper.

Bioinspired strategy for the fabrication of fast‐curing, tough, and high‐strength phenolic resins

AbstractPhenolic resins (PRs) find extensive use in adhesives and constructions, but they suffer from low curing rate and high brittleness. Herein, we drew inspiration from the lobster sclerotization process and proposed a simple approach to fabricate PRs by integrating pyrogallol‐functionalized graphene nanoplatelets (PG@GnPs), amine‐terminated hyperbranched phosphonitrilic chloride trimer (HPNC), and phenol formaldehyde (PF) resin. The resulting PRs exhibited significant improvements in comprehensive performance. The onset curing temperature decreased from 115.6 to 101.7°C, while the adhesion work increased by 126.5% and the toughness increased by 494%. Additionally, the wet bonding strength increased by 68.7% and the flexural strength increased by 208%. The accelerated curing rate was attributable to the oxidative crosslinking between catechol moieties in pyrogallol and amino groups in HPNC under alkaline conditions, as well as the high reactivity between pyrogallol or amino groups and PF chains. The outstanding mechanical properties were resulted from the synergistic enhancement effects of PG@GnPs and HPNC, which effectively transferred and absorbed energy upon loading. Overall, this work presented an attractive design strategy for developing high‐performance and multifunctional polymer composites.Highlights A bioinspired strategy was proposed for preparing advanced phenolic resins. PG@GnPs were produced via a green and low‐cost ball milling method. Adhesion work and toughness increased by 126.5% and 494%, respectively. Bonding and flexural strength have improved by 68.7% and 208%, respectively. Significantly accelerated curing rate was achieved.

Effect of carbonyl-amino condensation, non-covalent cross-linking and conformational changes induced by ultrasound-assisted Maillard reaction on lysinoalanine formation in silkworm pupa protein

The inhibition of cross-linked lysinoalanine (LAL) formation in silkworm pupa protein isolates (SPPI) by Maillard reaction (using varying xylose concentration) and ultrasound treatment was studied. Results showed that sonicated SPPI was effectively grafted with high concentration of xylose (5 %), resulting in the lowest LAL content, which was 48.75 % and 30.64 % lower than the control and ultrasound-treated samples, respectively. Chemical bond analysis showed that the combined treatment destroyed the ionic bonds, intrachain (g-g-t), and interchain (g-g-g) disulfide bonds, but stimulated the polymerization of hydrogen and hydrophobic bonds between SPPI and xylose, and as well enhanced the net negative charge between SPPI/Xylose complexes. The particles of the complexes were more loose, dispersed and rough, and had a stronger hydrophilic microenvironment, accompanied by alterations in microscopic, secondary and tertiary structures. Ultrasound treatment induced the breakdown of the oxidative cross-linking in SPPI, and promoted the sulfhydryl group-dehydroalanine binding and the carbonyl-amino condensation of the protein and xylose, and thus inhibited the formation of cross-linked LAL. Furthermore, the physicochemical and structural parameters were highly interrelated with cross-linked LAL content (|r| > 0.9). The outcomes provided a novel avenue and theoretical basis for minimizing LAL formation in SPPI and improving the nutrition and safety of SPPI.

Identification of tyrosine brominated extracellular matrix proteins in normal and fibrotic lung tissues

Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within the extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation of bromide into tyrosine residues. In this work, we sought to identify the major target proteins for tyrosine bromination by HOBr or by PXDN-mediated oxidation in ECM from mouse teratocarcinoma PFHR9 cells. We detected 61 bromotyrosine (BrY)-containing peptides representing 23 proteins in HOBr-modified ECM from PFHR9 cells, among which laminins displayed the most prominent bromotyrosine incorporation. Moreover, we also found that laminin α1, laminin β1, and tubulointerstitial nephritis antigen-like (TINAGL1) contained BrY in untreated PFHR9 cells, which depended on PXDN. We extended these analyses to lung tissues from both healthy mice and mice with experimental lung fibrosis, and in lung tissues obtained from human subjects. Analysis of ECM-enriched mouse lung tissue extracts showed that 83 ECM proteins were elevated in bleomycin-induced fibrosis, which included various collagens and laminins, and PXDN. Similarly, mRNA and protein expression of PXDN and laminin α/β1 were enhanced in fibrotic mouse lung tissues, and also in mouse bone-marrow-derived macrophages or human fibroblasts stimulated with transforming growth factor β1, a profibrotic growth factor. We identified 11 BrY-containing ECM proteins, including collagen IV α2, collagen VI α1, TINAGL1, and various laminins, in both healthy and mouse fibrotic lung tissues, although the relative extent of tyrosine bromination of laminins was not significantly increased during fibrosis. Finally, we also identified 7 BrY-containing ECM proteins in human lung tissues, again including collagen IV α2, collagen VI α1, and TINAGL1. Altogether, this work demonstrates the presence of several bromotyrosine-modified ECM proteins, likely involving PXDN, even in normal lung tissues, suggesting a potential biological function for these modifications.

Chemically engineered exogenous organic reactions in living cells for in situ fluorescence imaging and biomedical applications.

The unique microenvironment within living cells, characterized by high glutathione levels, reactive oxygen species concentrations, and active enzymes, facilitates the execution of chemical reactions. Recent advances in organic chemistry and chemical biology have leveraged living cells as reactors for chemical synthesis. This review summarizes recent reports on key intracellular in situ synthesis processes, including the synthesis of near-infrared fluorescent dyes, intracellular oxidative cross-linking, bioorthogonal reactions, and intracellular polymerization reactions. These methods have been applied to fluorescence imaging, tumor treatment, and the enhancement of biological functions. Finally, we discuss the challenges and opportunities in the field of in situ intracellular synthesis. We aim to guide the design of chemical molecules for in situ synthesis, improving the efficiency and control of artificial reactions in living cells, and ultimately achieving cell factory-like exogenous biological synthesis, biological function enhancement, and biomedical applications.