Synthetic Biomimetic Liposomes Harness Efferocytosis Machinery for Highly Efficient Macrophages-Targeted Drug Delivery to Alleviate Inflammation.

Macrophages play pivotal roles in the regulation of inflammatory responses and tissue repair, making them a prime target for inflammation alleviation. However, the accurate and efficient macrophages targeting is still a challenging task. Motivated by the efficient and specific removal of apoptotic cells by macrophages efferocytosis, a novel biomimetic liposomal system called Effero-RLP (Efferocytosis-mediated Red blood cell hybrid Liposomes) is developed which incorporates the membrane of apoptotic red blood cells (RBCs) with liposomes for the purpose of highly efficient macrophages targeting. Rosiglitazone (ROSI), a PPARγ agonist known to attenuate macrophage inflammatory responses, is encapsulated into Effero-RLP as model drug to regulate macrophage functions in DSS-induced colitis mouse model. Intriguingly, the Effero-RLP exhibits selective and efficient uptake by macrophages, which is significantly inhibited by the efferocytosis blocker Annexin V. In animal models, the Effero-RLP demonstrates rapid recognition by macrophages, leading to enhanced accumulation at inflammatory sites. Furthermore, ROSI-loaded Effero-RLP effectively alleviates inflammation and protects colon tissue from injury in the colitis mouse model, which is abolished by deletion of macrophages from mice model. In conclusion, the study highlights the potential of macrophage targeting using efferocytosis biomimetic liposomes. The development of Effero-RLP presents novel and promising strategies for alleviating inflammation.

Recombinant expression and immune function analysis of C-reactive protein (CRP) from Hexagrammos otakii.

C-reactive protein (CRP) belongs to the short-chain pentraxin family and functions as a soluble pattern recognition molecule (PRM) aiding in host defense against pathogens. In the present study, a CRP gene, designated HoCRP, was cloned from Hexagrammos otakii for the first time. The full length of the HoCRP cDNA sequence is 821 bp, which contains an open reading frame (ORF) of 675 bp encoding a 224 amino acid protein. The deduced protein is predicted to have a theoretical isoelectric point (pI) of 5.30 and a molecular weight of 25.4 kDa. The recombinant HoCRP protein (rHoCRP) was expressed in E. coli to further characterize the functions of HoCRP. Saccharide binding experiments demonstrated that rHoCRP exhibited a high affinity for various pathogen-associated molecular patterns (PAMPs). Furthermore, bacterial binding and agglutination assays indicated that rHoCRP had the capability to recognize a wide spectrum of microorganisms. These findings suggest that HoCRP functions not only as a PRM for binding PAMPs but also as an immune effector molecule. Considering the role CRP plays in the classical complement pathway, the interaction between rHoCRP and rHoC1q was assessed and proven by a Pull-down and Elisa assay, which implied that rHoCRP may be able to activate complement. In addition, phagocytosis enhancement by rHoCRP in the presence or absence of complement components was analysed by flow cytometry. The results showed that rHoCRP could synergistically enhance the phagocytosis of RAW264.7 cells with complement, providing further evidence of complement activation by rHoCRP through the opsonization of specific complement components. In summary, our findings suggest that rHoCRP may play a crucial role in host antibacterial defense by recognizing pathogens, activating the complement system, and enhancing macrophage function.

Molecular characterization and functional analysis of peroxiredoxin 1 (Prx1) from roughskin sculpin (Trachidermus fasciatus).

Peroxiredoxin1(Prx1), also known as natural killer enhancing factor A (NKEF-A), is a crucial antioxidant involving in various cellular activities and immune response against bacterial and viral infection in fish. In the present study, a full-length Prx1 cDNA sequence (TfPrx1) was firstly cloned from roughskin sculpin (Trachidermus fasciatus), which was composed of 1044 bp nucleotides encoding a peptide of 199 amino acids with a molecular weight of 22.35 kDa and a theoretical pI of 6.42, respectively. The predicted peptide was a typical 2-cys Prx containing two conserved characteristic motifs 43FYPLDFTFVCPTEI56 and 170GEVCPA175 with the two conserved peroxidatic and resolving cysteine residuals forming disulfide bond. Quantitative real-time PCR analysis showed that TfPrx1 was ubiquitously expressed in all tested tissues with the highest expression in the intestine. It could be significantly induced following LPS injection and heavy metal exposure. Recombinant TfPrx1 (rTfPrx1) displayed insulin disulfide reduction and ROS-scavenging activity in a concentration-dependent manner, and further exhibited DNA and cytoprotective effects under oxidative stress. These results suggested that TfPrx1 protein may play an important role in fish immune protection from oxidative damage.

"Ice and Fire" Supramolecular Cell-Conjugation Drug Delivery Platform for Deep Tumor Ablation and Boosted Antitumor Immunity.

Cancer recurrence and metastasis are two major challenges in the current clinical therapy. In this work, a novel diketopyrrolopyrrole-based photothermal reagent (DCN) with unique J-aggregation-induced redshift is synthesized to achieve efficient tumor thermal ablation under safe power (0.33 W cm-2 ). Meanwhile, S-nitroso-N-acetylpenicillamine (SNAP) is co-loaded with near-infrared-absorbing DCN in amphiphilic polymers to realize heat-induced massive release of nitric oxide (NO), which can form oxidant peroxynitrite (ONOO- ) to active matrix metalloproteinases (MMPs), thereby degrading the compact tumor extracellular matrix to improve the ablation depth and infiltration of immune cells. Through a facile supramolecular assembly method, the DCN/SNAP nanoparticles are anchored to liquid-nitrogen-frozen cancer cells, achieving enhanced antitumor immune responses and effective inhibition of distant tumors and pulmonary metastases after only one treatment. The safety and effectiveness of this supramolecular cell-conjugation platform are verified by 2D/3D cellular experiments and bilateral tumor model, confirming the thermal-ablation-gas-permeation-antigen-presentation therapeutic mode has promising anticancer prospects.

Pathogenic bacteria defense and complement activation function analysis of Collectin-10 from Hexagrammos otakii.

With the tremendous success of the artificial breeding of Hexagrammos otakii, the yield has been substantially improved. However, intensive farming often results in bacterial diseases; hence it is imperative to find new antimicrobial molecules. In the present study, we identified a homologous cDNA fragment of collectin-10 from H. otakii, designated as HoCL-10. The cDNA length is 899 bp, which contains an open reading frame (ORF) of 816 bp encoding a secreted protein with 272 amino acid residues. The peptide of HoCL-10 contains an N-terminal collagen domain, a neck region, and a C-terminal carbohydrate recognition domain. The qRT-PCR results revealed that HoCL-10 mRNA was highest expressed in the liver and skin and was significantly induced post-LPS stimulation. The sugar and bacteria binding assay suggested that the recombinant HoCL-10 (rHoCL-10) could recognize various pathogen-associated molecular patterns (PAMPs) and bacteria. For effect on cells, rHoCL-10 enhanced the phagocytosis and migration ability of the macrophage indicated using pro-phagocytosis assay and trans-well assay. To determine the role of HoCL-10 in the complement system, the interaction between HoCL-10 and mannose-binding lectin associated serine protease 1, 2 (MASP-1, 2) were analyzed and demonstrated using ELISA and Far-western. And in vivo, the concentration of membrane-attack complex (MAC) in fish plasma was significantly down-regulated post-injection with HoCL-10 antibody. Finally, the bacteria challenge experiment was performed, implying that HoCL-10 may assist the host in defending against microbial invasion. The findings suggest that HoCL-10 may play crucial roles in host defense against microorganisms, possibly through opsonizing pathogens and activating the complement system.

Molecular characterization and antibacterial activities of a goose-type lysozyme gene from roughskin sculpin (Trachidermus fasciatus).

Lysozymes, acting as antimicrobial molecules, play a vital role in the host's innate immune response to pathogen infections. In the present study, a g-type lysozyme gene termed Tf-LyzG from roughskin sculpin, Trachidermus fasciatus was firstly reported. The deduced amino acid sequence of Tf-LyzG contained 188 residues and possessed conserved catalytic residues (Glu71, Asp84, and Asp95). Gene expression analysis revealed that Tf-LyzG was widely distributed in the tested eleven tissues with the highest expression in the gill and could be significantly induced post lipopolysaccharide (LPS) challenge. The lysozyme activity of the purified recombinant protein (rTf-LyzG) was found to be most active at pH 5.5 and 37 °C. rTf-LyzG exhibited a wide spectrum of potent bacteriolytic activity against four Gram-positive bacteria and six Gram-negative bacteria. It also displayed a high affinity to polysaccharides on bacteria surfaces including LPS, lipoteichoic acid (LTA), and peptidoglycan (PGN). rTf-LyzG was capable of binding and agglutinating all nine bacteria. Flow cytometry assay further revealed that rTf-LyzG could disrupt the membrane of Micrococcus lysodeikticus which is confirmed by scanning electron microscope (SEM) analysis that reveals blebs around the bacterial cell membrane. In summary, these data indicate that Tf-LyzG is of great importance in the fish immune response against pathogens invasion.

Cyclodextrin-Derived ROS-Generating Nanomedicine with pH-Modulated Degradability to Enhance Tumor Ferroptosis Therapy and Chemotherapy.

Nowadays, destruction of redox homeostasis to induce cancer cell death is an emerging anti-cancer strategy. Here, the authors utilized pH-sensitive acetalated ß-cyclodextrin (Ac-ß-CD) to efficiently deliver dihydroartemisinin (DHA) for tumor ferroptosis therapy and chemodynamic therapy in a synergistic manner. The Ac-ß-CD-DHA based nanoparticles are coated by an iron-containing polyphenol network. In response to the tumor microenvironment, Fe2+ /Fe3+ can consume glutathione (GSH) and trigger the Fenton reaction in the presence of hydrogen peroxide (H2 O2 ), leading to the generation of lethal reactive oxygen species (ROS). Meanwhile, the OO bridge bonds of DHA are also disintegrated to enable ferroptosis of cancer cells. Their results demonstrate that these nanoparticles acted as a ROS generator to break the redox balance of cancer cells, showing an effective anticancer efficacy, which is different from traditional approaches.

Molecular characterization and functional analysis of Trx and Trp14 in roughskin sculpin (Trachidermus fasciatus).

Thioredoxins (Trxs) are a family of small and highly conserved proteins which play crucial roles in the maintenance and regulation of the cellular redox homeostasis. In this study, the full-length cDNAs of thioredoxin 1 (TfTrx1) and thioredoxin-related protein of 14 kDa (TfTrp14) were isolated from roughskin sculpin (Trachidermus fasciatus). TfTrx1 is 662 bp in length with a 336-bp open reading frame (ORF) that encodes for a peptide with 111 amino acids, and TfTrp14 consists of 1066 bp with a 372-bp ORF that is translated to 123 amino acids. TfTrx1 and TfTrp14 contain highly conserved catalytic site motif CGPC and CPDC, respectively. Tissue distribution analysis indicated that both genes were broadly expressed in all examined tissues with the highest expression of TfTrx1 in the blood and TfTrp14 in the brain. In post-LPS and heavy metal challenge, the mRNA of both genes was significantly increased in the skin, liver, spleen, and brain at various times. The results of western blot detection displayed that the time of the induced maximum protein expression was 6-h post-LPS injection in the skin and liver, which were slightly delayed compared with that of 2 h at mRNA level. The recombinant TfTrp14 and TfTrx1 proteins were expressed in E. coli BL21 (DE3). The increase of the fluorescence intensity in rTfTrx1 and rTfTrp14 suggested the redox state changes in the microenvironment around tryptophan residues. Both of the recombinant proteins exhibited concentration-dependent disulfide reductase activity towards insulin, and the catalytic activity of rTfTrx1 was much higher than that of rTfTrp14.

Molecular characterization and functional analysis of the bactericidal permeability-increasing protein/LPS-binding protein (BPI/LBP) from roughskin sculpin (Trachidermus fasciatus).

Bactericidal permeability-increasing protein (BPI) and lipopolysaccharide (LPS) binding proteins (LBP) both play important roles in innate immunity against bacterial infection. Herein, we identified a novel full-length cDNA sequence of BPI/LBP from Trachidermus fasciatus (designated as TfBPI/LBP). The full-length cDNA sequence of TfBPI/LBP was 1594bp, which contains an open reading frame (ORF) of 1422bp encoding a secreted protein with 473 amino acid residues. Similar to BPI/LBPs from other teleost and mammals, the peptide of TfBPI/LBP contains an N-terminal BPI/LBP/CETP domain with an LPS-binding motif and a C-terminal BPI/LBP/CETP domain BPI2. Multiple alignments and phylogenetic analysis supported that TfBPI/LBP was a new member of the vertebrate BPI/LBP family. TfBPI/LBP gene was ubiquitously expressed in all detected tissues, with the most abundant in the liver, and could be significantly induced in the skin, blood, liver, spleen post LPS challenge. The recombinant N-terminal domain of TfBPI/LBP (designated as rTfBPI/LBPN) was successfully expressed in Escherichia coli. Sugar binding assay showed that rTfBPI/LBPN could bind to LPS, peptidoglycan (PGN), and lipoteichoic acid (LTA), with the highest affinity to LPS. The results of bacteria binding and agglutinating assay revealed that rTfBPI/LBPN could bind and agglutinate to all of the 9 kinds of bacteria we used. Moreover, membrane integrity analysis indicated that rTfBPI/LBPN could increase the membrane permeability of bacteria. These results suggested that BPI/LBP may play crucial roles in host defense against microorganisms, possibly through non-selective bacterial recognition and induction of membrane penetration.