Polydopamine-armored zeolitic imidazolate framework-8-incorporated zwitterionic hydrogel with multifunctional properties for infected wound healing.

Diabetic skin wound healing is compromised by bacterial infections, oxidative stress, and vascular disruption, leading to delayed recovery and potential complications. This study developed an antibacterial, antioxidant, and adhesive hydrogel dressing that promotes rapid bacterial-infected diabetic wound healing using the biological macromolecule of polydopamine (PDA). This hydrogel comprised PDA-armored zeolitic imidazolate framework-8 nanoparticles (PDA@ZIF-8 NPs) combined with a second armor of zwitterionic polymer network (poly(acrylamide-co-sulfobetaine methacrylate); PAS), realizing low concentration Zn2+ release, good adhesion (14.7 kPa for porcine skin), and improved tensile strength (83.2 kPa). The hydrogel exhibited good antibacterial efficacy against both Staphylococcus aureus (S. aureus, 92.8 %), Escherichia coli (E. coli, 99.6 %) and methicillin-resistant S. aureus (MRSA, 99.2 %), which was attributed to the properties of the incorporated PDA@ZIF-8 NPs. Notably, in vitro, the PDA@ZIF-8 PAS hydrogel not only promoted fibroblast proliferation and migration but also facilitated endothelial cell angiogenesis. In vivo, the PDA@ZIF-8 PAS hydrogel retained its Zn2+-releasing function and effectively suppressed bacterial growth in infected wounds, thereby accelerating the regeneration of both normal and diabetic wounds. This multiarmored hydrogel is a promising sustained-release carrier for functional metal ions and drugs, making it applicable for not only skin healing, but potentially the regeneration of other complex tissues.

Yeast perilipin Pet10p/Pln1p interacts with Erg6p in ergosterol metabolism.

Lipid droplets (LD) are highly dynamic organelles specialized for the regulation of energy storage and cellular homeostasis. LD consist of a neutral lipid core surrounded by a phospholipid monolayer membrane with embedded proteins, most of which are involved in lipid homeostasis. In this study, we focused on one of the major LD proteins, sterol C24-methyltransferase, encoded by ERG6. We found that the absence of Erg6p resulted in an increased accumulation of yeast perilipin Pet10p in LD, while the disruption of PET10 was accompanied by Erg6p LD over-accumulation. An observed reciprocal enrichment of Erg6p and Pet10p in pet10Δ and erg6Δ mutants in LD, respectively, was related to specific functional changes in the LD and was not due to regulation on the expression level. The involvement of Pet10p in neutral lipid homeostasis was observed in experiments that focused on the dynamics of neutral lipid mobilization as time-dependent changes in the triacylglycerols (TAG) and steryl esters (SE) content. We found that the kinetics of SE hydrolysis was reduced in erg6Δ cells and the mobilization of SE was completely lost in mutants that lacked both Erg6p and Pet10p. In addition, we observed that decreased levels of SE in erg6Δpet10Δ was linked to an overexpression of steryl ester hydrolase Yeh1p. Lipid analysis of erg6Δpet10Δ showed that PET10 deletion altered the composition of ergosterol intermediates which had accumulated in erg6Δ. In conclusion, yeast perilipin Pet10p functionally interacts with Erg6p during the metabolism of ergosterol.

Co-immunoprecipitation for identifying protein-protein interaction on lipid droplets.

The lipid droplet (LD) is a conserved organelle that exists in almost all organisms, ranging from bacteria to mammals. Dysfunctions in LDs are linked to a range of human metabolic syndromes. The formation of protein complexes on LDs is crucial for maintaining their function. Investigating how proteins interact on LDs is essential for understanding the role of LDs. We have developed an effective method to uncover protein-protein interactions and protein complexes specifically on LDs. In this method, we conduct co-immunoprecipitation (co-IP) experiments using LD proteins extracted directly from isolated LDs, rather than utilizing proteins from cell lysates. To elaborate, we begin by purifying LDs with high-quality and extracting LD-associated proteins. Subsequently, the co-IP experiment is performed on these LD-associated proteins directly, which would enhance the co-IP experiment specificity of LD-associated proteins. This method enables researchers to directly unveil protein complexes on LDs and gain deeper insights into the functional roles of proteins associated with LDs.

LET-767 determines lipid droplet protein targeting and lipid homeostasis.

Lipid droplets (LDs) are composed of a core of neutral lipids wrapped by a phospholipid (PL) monolayer containing several hundred proteins that vary between different cells or organisms. How LD proteins target to LDs is still largely unknown. Here, we show that RNAi knockdown or gene mutation of let-767, encoding a member of hydroxysteroid dehydrogenase (HSD), displaced the LD localization of three well-known LD proteins: DHS-3 (dehydrogenase/reductase), PLIN-1 (perilipin), and DGAT-2 (diacylglycerol O-acyltransferase 2), and also prevented LD growth in Caenorhabditis elegans. LET-767 interacts with ARF-1 (ADP-ribosylation factor 1) to prevent ARF-1 LD translocation for appropriate LD protein targeting and lipid homeostasis. Deficiency of LET-767 leads to the release of ARF-1, which further recruits and promotes translocation of ATGL-1 (adipose triglyceride lipase) to LDs for lipolysis. The displacement of LD proteins caused by LET-767 deficiency could be reversed by inhibition of either ARF-1 or ATGL-1. Our work uncovers a unique LET-767 for determining LD protein targeting and maintaining lipid homeostasis.

Isolation and proteomic study of fish liver lipid droplets.

Lipid droplets (LDs) are a neutral lipid storage organelle that is conserved in almost all species. Excessive storage of neutral lipids in LDs is directly associated with many metabolic syndromes. Zebrafish is a better model animal for the study of LD biology due to its transparent embryonic stage compared to other organisms. However, the study of LDs in fish has been difficult due to the lack of specific LD marker proteins and the limitation of purification technology. In this paper, the purification and proteomic analysis of liver LDs of fish including zebrafish and Carassius auratus were performed for the first time. 259 and 267 proteins were identified respectively. Besides most of the identified proteins were reported in previous LD proteomes of mammals, indicating the similarity between mammal and fish LDs. We also identified many unique proteins of liver LDs in fish that are involved in the regulation of LD dynamics. Through morphological and biochemical analysis, we found that the marker protein Plin2 of zebrafish LD was located on LDs in Huh7 cells. These results will facilitate further study of LDs in fish and liver metabolic diseases using fish as a model animal.

Construction and application of artificial lipoproteins using adiposomes.

Lipoproteins are complex particles comprised of a neutral lipid core wrapped with a phospholipid monolayer membrane and apolipoproteins on the membrane, which is closely associated with metabolic diseases. To facilitate the elucidation of its formation and dynamics, as well as its applications, we developed an in vitro system in which adiposomes, consisting of a hydrophobic core encircled by a monolayer-phospholipid membrane, were engineered into artificial lipoproteins (ALPs) by recruiting one or more kinds of apolipoproteins, for example, apolipoprotein (Apo) A-I, ApoE, ApoA-IV, and ApoB. In vitro and in vivo studies demonstrated the stability and biological activity of ALPs derived from adiposomes, which resembles native lipoproteins. Of note, adiposomes bearing ApoE were internalized via clathrin-mediated endocytosis following LDLR binding and were delivered to lysosomes. On the other hand, adiposomes bearing ApoA-IV mimicked the existing form of endogenous ApoA-IV and exhibited significant improvement in glucose tolerance in mice. In addition, the construction process was simple, precise, reproducible, as well as easy to adjust for mass production. With this experimental system, different apolipoproteins can be recruited to build ALPs for some biological goals and potential applications in biomedicine.

Measurement of ATGL activity using adiposomes.

Adipose triacylglycerol lipase (ATGL) is a dynamic lipid droplet-associated protein involved in cellular lipolysis, which is conserved from bacteria to humans. Recent methods that measure the enzymatic activity of ATGL in vitro are established using lipid emulsions. However, the lipid emulsion platforms contain various membranous structures which reduce the accuracy of enzymatic activity determination. Therefore, a new platform and corresponding method are required for accurate measurement of ATGL enzymatic activity that represents cellular lipid and energy homeostasis. Adiposomes are artificial lipid nanostructures mimicking lipid droplets. Employing adiposome as a platform, we have developed an assay to measure the enzymatic activity of ATGL in vitro. Here, a detailed protocol is described to explain how to measure the activity of ATGL using adiposomes. This method successfully proves the concept of lipid droplet-mimetic lipase activity determining platform and provides a tool to identify the active sites of lipases.

ROS conversion promotes the bactericidal efficiency of Eosin Y based photodynamic therapy.

Photodynamic therapy (PDT) is becoming an efficient antibacterial strategy without drug-resistance. Here, we report a promising reactive oxygen species (ROS) conversion strategy to increase the antibacterial efficiency of an Eosin Y (EOS)-based PDT system. Based on visible-light illumination, EOS generates a high concentration of singlet oxygen (1O2) in the solution. With the introduction of HEPES in the EOS system, it can almost completely convert 1O2 to hydrogen peroxide (H2O2). The orders-of-magnitude increases in the half-lives of the ROS (H2O2vs.1O2) present in the solution can enable more persistent oxidation ability. Thus, it is able to increase the bactericidal efficiency (against S. aureus) from 37.9% to 99.9%, promote the inactivation efficiency of methicillin-resistant S. aureus (MRSA) from 26.9% to 99.4%, and enhance the eradication rate of MRSA biofilm from 69% to 90%. Further in vivo investigation showed that the increased oxidation ability of the EOS/HEPES PDT system can enable quicker healing and maturing (even better than that for vancomycin administration) of MRSA-infected skin wounds on rats. This strategy may find many creative applications for the efficient eradication of bacteria and other pathogenic microorganisms.

An Adhesive/Anti-Adhesive Janus Tissue Patch for Efficient Closure of Bleeding Tissue with Inhibited Postoperative Adhesion.

Most of the current bioadhesives cannot perform well on bleeding tissues while postoperative adhesion is a general but serious clinical issue. Here, a three-layer biodegradable Janus tissue patch (J-TP) that is able to simultaneously enable efficient closure of bleeding wounds with significantly promoted clotting ability and suppressed postoperative adhesion of tissues is reported. A dry adhesive hydrogel bottom layer of the J-TP can form rapid (within 15 s) and strong (tensile strength up to 98 kPa) adhesion to bleeding/wet tissues with high bursting pressure (about 312.5 mmHg on a sealed porcine skin) through hydrogen binding and covalent conjugation between the carboxyl & N-hydroxy succinimide (NHS) groups of hydrogel and the primary amine groups of tissues, while the phosphonic motifs can significantly reduce blood loss (by 81% on a rat bleeding liver model) of bleeding wounds. A thin polylactic acid (PLA) middle layer can improve the tensile strength (by 132%) of the J-TP in wet conditions while the grafted zwitterionic polymers can effectively prevent postoperative tissue adhesion and inflammatory reaction. This J-TP may be a promising tissue patch to assist the clinical treatment of injured bleeding tissues with inhibited postoperative adhesion.

One amino acid drives the lipid droplet targeting sequence of a new noncoding RNA-encoded protein to mitochondrion.

Over the past decade, the majority of the mammalian genome considered to be noncoding has been revealed to be able to produce proteins. Many RNA molecules, mis-annotated as noncoding, actually are predicted to code for proteins. Some of those proteins have been identified and verified to play critical roles in multiple biological processes. The lipid droplet (LD) is a unique cellular organelle bound with a phospholipid monolayer membrane, and is closely associated with cellular lipid metabolism and metabolic disorders. However, it is still unclear how a protein targets to LDs. Here we identified a new protein on LDs, LDANP2, which is encoded by noncoding RNA, through a proteomics-based strategy. The key sequence for its localization on LDs, Truncation 3, is predicted to form an amphipathic helix. Surprisingly, the deletion of the first amino acid in Truncation 3 resulted in mitochondrial localization. How the types of amino acids would determine the LD or mitochondrial localizations of the protein was studied. The findings introduce a useful strategy to mine for new proteins and would provide clues to the understanding of how a protein would find its right organelle, with phospholipid monolayer or bilayer membrane.

Long-term antibacterial, antioxidative, and bioadhesive hydrogel wound dressing for infected wound healing applications.

Bacterial infection and oxidative stress hinder clinical wound healing. Therefore, wound dressings with antibacterial and antioxidative properties are urgently needed. In this study, a type of quaternized lignin (QL) functionalized poly(hexamethylene biguanide) hydrochloride (PHMB) complex incorporated polyacrylamide (QL-PHMB-PAM) hydrogel was developed as a multifunctional dressing material for the promotion of infected wound repair. Owing to the abundant catechol groups of quaternized lignin, the QL-PHMB-PAM hydrogel exhibited robust repeatable adhesiveness to various substrates with antioxidative properties. Additionally, the antibacterial components of PHMB in the QL-PHMB-PAM composite hydrogel showed high efficiency and long-term antibacterial activity against Staphylococcus aureus (S.aureus), Escherichia coli (E.coli), and methicillin-resistant S. aureus (MRSA; up to 100%). Furthermore, in vivo experiments indicated that this multifunctional hydrogel accelerated the healing of S. aureus-infected wounds by promoting the reconstruction of blood vessels and hair follicles. These results demonstrate that this antioxidative, antibacterial, and bioadhesive hydrogel is a promising alternative wound dressing material for the prevention of bacterial infections and the acceleration of infected wound regeneration.

Zwitterionic betaines over HEPES as the new generation biocompatible pH buffers for cell culture.

Good's buffers have been widely applied in cell/organ culture over the past half a century as biocompatible pH stabilizers. However, the emergence of severe adverse effects, such as cellular uptake, lysosomal autophagic activation, and visible light-induced cytotoxicity, raises serious questions over its biocompatibility while underlying mechanism was unclear. Here we report that riboflavin (RF, component of cell culture medium) generates 1O2, ·OH, and O2 •- under visible light exposure during regular cell manipulation. These short half-life reactive oxygen species (ROS) react with tertiary amine groups of HEPES, producing 106.6 µM of H2O2. Orders of magnitude elevated half-life of ROS in the medium caused severe cytotoxicity and systematic disorder of normal cell functions. We have further designed and validated zwitterionic betaines as the new generation biocompatible organic pH buffers, which is able to completely avoid the adverse effects that found on HEPES and derivate Good's buffers. These findings may also open a new avenue for zwitterionic betaine based materials for biomedical applications.

Inulin reduces liver triacylglycerol by increasing lipid droplet lipolysis in fat-loaded mice.

Increased consumption of high-fat low-fiber foods has been shown to contribute to the development of metabolic syndromes, such as fatty liver, obesity, diabetes, et al. Fermentable dietary fiber, such as inulin, is broadly used to mitigate host metabolic abnormalities. In this work, we studied systematically the effect of inulin on mice with metabolic disorders, induced by either short- or long-term high-fat feeding. As expected, inulin reduced the body weight of mice in both groups. However, it was found that inulin feeding could only increase energy expenditure, alleviate adiposity, and improve glucose intolerance in mice fed with high-fat diet (HFD) for 1 month but not for 4 months. Surprisingly, inulin supplementation could alleviate HFD-induced hepatic steatosis, mediated through increasing adipose triglyceride lipase (ATGL) on liver lipid droplets, in both groups. Gut microbiota in the short- and long-term fat-loaded mice were shown to be modulated differently, which may mediate the differential effects of inulin. These results may help in understanding the role and mechanism of fermentable fiber regulating host metabolism.

Protein sample preparation for tissue distribution study.

PURPOSE: The distribution and expression level of a protein among animal tissues is indicative of its possible roles. It is important to establish a generally applicable method to prepare protein samples with high-quality and achieve near 100% recovery of proteins from animal tissues. EXPERIMENTAL DESIGN: During preparation, to sufficiently dissolve and maintain stability of almost all proteins from tissues, as well as to avoid most contaminations affecting protein detection, 2×SDS Sample Buffer, sonication and trichloroacetic acid precipitation are applied. RESULTS: Here we provide a relatively simple, reproducible, and broadly applicable method for studying protein distribution in most tissues, in which the issues resulting from protein degradation and modification during sample preparation and assay interference by other cellular components like neutral lipids and glycogen could be overcome. Furthermore, this method represents the protein content by equal wet tissue mass, which is a better means to present the expression level of a protein in various tissues. High-quality protein samples from almost all tissues could be prepared. CONCLUSIONS AND CLINICAL RELEVANCE: The samples produced are amenable to tissue distribution analysis by Western blotting and for silver/Coomassie staining, proteomics, and other protein analyses, which would contribute to potential biomarkers or treatments for a disease.

Layer-by-layer construction of zwitterionic/biguanide polymers on silicone rubber as an antifouling and bactericidal coating.

Biofilm formation on biomedical devices is a major cause of device-associated infection. Traditional antibiotic treatment for biofilm-associated infection increases the risk of multidrug resistance. Thus, there is an urgent need to develop antibiotic-free strategies to prevent biofilm formation on biomedical devices. Herein, we report a layer-by-layer strategy to construct an antifouling and bactericidal dual-functional coating for silicone rubber (SR)-based substrates. Five zwitterionic active ester copolymers, p(SBMA-co-NHSMA), with varied zwitterionic pSBMA components that ranged from 50 to 90% (molar ratio) were precisely prepared. Based on -NH2/NHS chemistry, a zwitterionic pSBMA antifouling coating was successfully constructed on an -NH2-activated SR surface, while a biguanide polymer (PHMB) bactericidal coating was consequently tethered. The relationship between the composition of the polymeric coating and the overall antibacterial property (antifouling and bactericidal) that was endowed to the SR surface was established. The in vitro and in vivo results consistently showed that the optimal p(SBMA-co-NHSMA) copolymer (SBMA/NHSMA with molar percentage of 70/30) synergistically utilized antifouling and bactericidal abilities to endow a highly efficient overall antibacterial property (near 100% antibacterial ratios) to SR70-PHMB substrates without compromising cellular viability. This strategy may be applied to the many SR-based biomedical implants and devices where an antibacterial surface is required.

Zwitterionic/phosphonate copolymer coatings endow excellent antifouling properties and robust re-mineralization ability of dentine substrates.

Inhibition of biofilm formation and induction of the re-mineralization of damaged dental tissues are two major strategies to combat dental hypersensitivity (DH). However, single component synthetic materials normally cannot fulfil these two functions during the repairing of damaged dental tissues. Here, we report zwitterionic phosphorylcholine based polymers to be a new type of dual functional coating for the repairing of DH. Zwitterionic/phosphonate copolymers, p(DEMMP-co-MPC), bearing varied zwitterionic contents (95 and 75 mol%) were prepared through conventional radical copolymerization. 1H NMR spectroscopy clearly indicated the precise preparation of the copolymers. The copolymers can be easily coated on dentine substrates based on the high affinity between the phosphonate group and the calcium phosphate minerals of the dentine substrates, as evidenced by XPS and water contact angle measurements. Antifouling evaluations indicated that zwitterionic coating can efficiently inhibit protein adsorption (BSA, egg white, and milk, by 85%) and bacterial adhesion (by 97.1%) on dentine substrates. Furthermore, in vitro and in vivo experiments consistently indicated that the zwitterionic coating could not only induce the robust re-mineralization of dentine surfaces, but also template the extensive re-mineralization of dentine tubules to a similar level of pristine dentine. Both the antifouling properties and the re-mineralization potency are positively correlated with the content of zwitterionic pMPC in the coating copolymer. These findings may provide the zwitterionic phosphorylcholine based materials to be a promising candidate to treat dental hypersensitivity and other related dental diseases.

Tertiary amines convert 1O2 to H2O2 with enhanced photodynamic antibacterial efficiency.

Photodynamic inactivation (PDI) is a promising approach to combat the increasing global multi-drug resistance crisis. However, the very short half-life of 1O2 and the inevitable photobleaching of photosensitizer (PS) are the inherent drawbacks that largely compromise its therapeutic efficiency. Here, we report a ROS conversion strategy that simultaneously addresses these issues. Based on a photodynamic model system where riboflavin (RF) served as the PS, we have clearly shown that about 93.2% of 1O2 could be converted to hydrogen peroxide (H2O2) in the presence of tertiary amine. The less reactivity of H2O2 (v.s.1O2) could retard the photobleaching of riboflavin by 88.9%. Orders of magnitude extended half-life of ROS (H2O2v.s.1O2) and retarded photobleaching of RF synergistically provide a more persistent oxidization that increased the oxidation capacity of the photodynamic model system by 56.6%. Consequently, it is able to improve the therapeutic efficiencies from 89.6% to 99.1% in combating methicillinresistant S. aureus (MRSA) and from 64.0% to 92.0% in eradicating S. aureus biofilm on biomaterials within a 5-min simulated sunlight illumination. The reinforced photodynamic model system could also significantly accelerate the healing & maturing of MRSA infected skin wound as compared to that of clinically used vancomycin. The generality of "ROS conversion" among different amines and different photosensitizers have been verified. These findings may inspire many creative approaches to increase the antibacterial efficiency of current photodynamic treatments for diverse applications.

Zwitterionic/active ester block polymers as multifunctional coatings for polyurethane-based substrates.

Bacterial-associated infection, blood coagulation, and tissue adhesion are severe issues associated with biomedical implants and devices in clinic applications. Here, we report a general strategy to simultaneously tackle these issues on polyurethane (PU)-based substrates. Taking advantage of reversible addition-fragmentation chain transfer (RAFT) polymerization, well-defined zwitterionic/active ester block polymers (pSBMA-b-pNHSMA) with an identical pNHSMA segment (polymerization degree of 15) but varied zwitterionic pSBMA segments (polymerization degrees of 40 and 100) were precisely prepared. The pSBMA-b-pNHSMA block polymers could be easily covalently constructed on PU substrates that were pretreated with a polydopamine coating based on highly efficient anime-active ester chemistry, as evidenced by the water contact angle and XPS tests. The relationship between the length of pSBMA segments in the coating and the antifouling ability of PU substrates was established. The results indicated that block polymers with a pSBMA segment of 40 repeat units could significantly prevent protein adsorption, bacterial/platelet adhesion, and cell attachment on PU substrates within 24 h, while a longer pSBMA segment (repeat units of 100) could endow long-term antibacterial (14 days without biofilm formation) and anti-cell attachment (5 days without cell attachment) properties to the PU substrates. Furthermore, the coating significantly improved the surface lubricating property of PU substrates without compromising on the mechanical property. This strategy may find many applications in PU-based implants and devices.

Protocol for using artificial lipid droplets to study the binding affinity of lipid droplet-associated proteins.

Here, we present a protocol to construct artificial lipid droplets to study the binding affinity of lipid droplet-associated proteins. We provide procedures to construct adiposomes and prepare recombinant lipid droplet-associated proteins. Then we describe approaches to measure the number density of perilipin 2 on natural lipid droplets, construct artificial lipid droplets, and determine the binding affinity of perilipin 2 on artificial lipid droplets. This protocol can be adapted to determine the binding properties of various lipid droplet-associated proteins. For complete details on the use and execution of this protocol, please refer to Ma et al. (2021).