Establishment and characterization of a new cell line from the muscle of humpback grouper (Cromileptes altivelis).

The humpback grouper (Cromileptes altivelis) is a commercially important species of the family Epinephelidae. With the development in aquaculture industry, C. altivelis breeding has gradually increased in volumetric production, leading to the occurrence of various diseases. In this study, we established a new cell line (CAM) derived from the muscle tissue of C. altivelis. Our results showed that the optimal growth temperature and working concentration of fetal bovine serum (FBS) of CAM cells were 28 °C and 15%, respectively. DNA sequencing and comparative analysis of 18S rRNA gene sequence showed that CAM cell line was originated from C. altivelis. Chromosome analysis showed that the modal chromosome number of CAM cells was 48. After transfection using pEGFP-N3 plasmid, CAM cells exhibited high transfection efficiency, indicating that CAM cells could be used in foreign gene expression studies. Further, cytotoxicity analysis revealed that CAM cells were sensitive to Vibrio harveyi and Edwardsiella tarda. Moreover, the cytotoxicity of heavy metals (Hg, Cd, and Cu) to CAM cells was dose-dependent. This CAM cell line might be used as an ideal tool in vitro for analyzing and understanding the mechanisms of pathogenesis, host-pathogen interactions, and toxicity assay of heavy metals.

Evaluation of Lactococcus lactis HNL12 combined with Schizochytrium limacinum algal meal in diets for humpback grouper (Cromileptes altivelis).

The humpback grouper (Cromileptes altivelis) is a commercially valuable species of the family Epinephelidae; however, its marketization suffers from slow growth speed, low survival rate, and various pathogenic diseases. Lactococcus lactis and Schizochytrium limacinum are commonly used as immunostimulants due to their health benefits for the aquatic organisms. In the present study, we assessed the effects of dietary supplementation with L. lactis HNL12 combined with S. limacinum algal meal on the growth performances, innate immune response, and disease resistance of C. altivelis against Vibrio harveyi. The results showed that fish fed with a combination diet of L. lactis and S. limacinum exhibited significantly higher final weight, percent weight gain, and specific growth rate compared with groups fed with them alone. A bacterial challenge experiment indicated that the group fed with the L. lactis combined with S. limacinum diet achieved the highest relative percent of survival value (68.63%), suggesting that L. lactis and S. limacinum significantly improved the disease resistance against V. harveyi after a 4-week feeding trial. Moreover, the respiratory burst activity of macrophages of fish fed with a L. lactis combined with S. limacinum diet was significantly higher than that of fish fed the control diet after 1, 2, and 3 weeks of feeding. The serum superoxide dismutase of fish fed with a L. lactis combined with S. limacinum diet significantly increased compared to those fed the control diet after 1 and 2 weeks of feeding, while the serum alkaline phosphatase of fish fed with a L. lactis combined with S. limacinum diet after 2 and 4 weeks was significantly increased, compared to the control group. The serum lysozyme activities of fish fed with a L. lactis combined with S. limacinum diet significantly increased compared to the control group after 2 weeks of feeding. Furthermore, transcriptome sequencing of the C. altivelis head kidney was conducted to explore the immune-regulating effects of the L. lactis combined with S. limacinum diet on C. altivelis. A total of 86,919 unigenes, annotated by at least one of the reference databases (Nr, Swiss-Prot, GO, COG, and KEGG), were assembly yielded by de novo transcriptome. In addition, 157 putative differentially expressed genes (DEGs) were identified between the L. lactis combined with S. limacinum group and the control group. For pathway enrichment, the DEGs were categorized into nine KEGG pathways, which were mainly related to infective diseases, antigen processing and presentation, digestive system, and other immune system responses. The findings of this study suggest that the L. lactis combined with S. limacinum diet can induce positive effects on the growth, immunity, and disease resistance of C. altivelis against V. harveyi. This study expands our understanding of the synergistic combinations of probiotics and prebiotics in aquaculture.

Comparative analysis of the expression patterns of IL-1ß, IL-11, and IL-34 in golden pompano (Trachinotus ovatus) following different pathogens challenge.

Interleukins (ILs) are a subgroup of cytokines, which are molecules involved in the intercellular regulation of the immune system. These cytokines have been extensively studied in mammalian models, but systematic analyses of fish are limited. In the current study, 3 IL genes from golden pompano (Trachinotus ovatus) were characterized. The IL-1ß protein contains IL-1 family signature motif, and four long helices (αA - αD) in IL-11 and IL-34, which were well conserved. All 3 ILs clustered phylogenetically with their respective IL relatives in mammalian and other teleost species. Under normal physiological conditions, the expression of IL-1ß, IL-11, and IL-34 were detected at varied levels in the 11 tissues examined. Most of the 3 ILs examined were highly expressed in liver, spleen, kidney, gill, or skin. Following pathogenic bacterial, viral, or parasitic challenge, IL-1ß, IL-11, and IL-34 exhibited distinctly different expression profiles in a time-, tissue-, and pathogen-dependent manner. In general, IL-1ß was expressed at higher levels following challenge with all pathogens examined than was observed for IL-11 and IL-34. Furthermore, Streptococcus agalactiae and Cryptocaryon irritans caused higher levels of IL-1ß and IL-11 expression than Vibrio harveyi and viral nervous necrosis virus (VNNV). The increased expression of IL-34 caused by VNNV and C. irritans were higher than that caused by V. harveyi and S. agalactiae. These results suggest that these 3 ILs in T. ovatus may play different effect pathogen type specific responses.

MHC class IIα polymorphism and its association with resistance/susceptibility to Vibrio harveyi in golden pompano (Trachinotus ovatus).

The major histocompatibility complex (MHC) plays an important role in the vertebrate immune response to antigenic peptides, and it is essential for recognizing foreign pathogens in organisms. In this study, MHC class IIα (Trov-MHC IIα) from the golden pompano (Trachinotus ovatus) was first cloned and identified. The gene structure of Trov-MHC IIα was contained four exons and three introns. High levels of polymorphism were found in the exon 2 of Trov-MHC IIα. A total of 29 different MHC class IIα alleles with high polymorphism were identified from 80 individuals. The ratio of non-synonymous substitutions (dN) to synonymous substitutions (dS) was 3.157 (>1) in the peptide binding regions (PBRs) of Trov-MHC IIα, suggesting positive balancing selection. Six alleles were selected to analyze the association between alleles and resistance/susceptibility to Vibrio harveyi in golden pompano. The results showed that Trov-DAA*6401 and Trov-DAA*6702 alleles were associated with the resistance to V. harveyi in golden pompano, while alleles Trov-DAA*6304 and Trov-DAA*7301 were associated with the susceptibility to V. harveyi in golden pompano. This study confirmed the association between alleles of MHC class IIα and disease resistance, and also detected some alleles which might be correlated with high V. harveyi-resistance. These disease resistance-related MHC alleles could be used as potential genetic markers for molecular marker-assisted selective breeding in the golden pompano.

A cuttlefish ink nanoparticle-reinforced biopolymer hydrogel with robust adhesive and immunomodulatory features for treating oral ulcers in diabetes.

Oral ulcers can be managed using a variety of biomaterials that deliver drugs or cytokines. However, many patients experience minimal benefits from certain medical treatments because of poor compliance, short retention times in the oral cavity, and inadequate drug efficacy. Herein, we present a novel hydrogel patch (SCE2) composed of a biopolymer matrix (featuring ultraviolet-triggered adhesion properties) loaded with cuttlefish ink nanoparticles (possessing pro-healing functions). Applying a straightforward local method initiates the formation of a hydrogel barrier that adheres to mucosal injuries under the influence of ultraviolet light. SCE2 then demonstrates exceptional capabilities for near-infrared photothermal sterilization and neutralization of reactive oxygen species. These properties contribute to the elimination of bacteria and the management of the oxidation process, thus accelerating the healing phase's progression from inflammation to proliferation. In studies involving diabetic rats with oral ulcers, the SCE2 adhesive patch significantly quickens recovery by altering the inflamed state of the injured area, facilitating rapid re-epithelialization, and fostering angiogenesis. In conclusion, this light-sensitive hydrogel patch offers a promising path to expedited wound healing, potentially transforming treatment strategies for clinical oral ulcers.

An Immunomodulatory Hydrogel by Hyperthermia-Assisted Self-Cascade Glucose Depletion and ROS Scavenging for Diabetic Foot Ulcer Wound Therapeutics.

Current therapeutic protocols for diabetic foot ulcers (DFUs), a severe and rapidly growing chronic complication in diabetic patients, remain nonspecific. Hyperglycemia-caused inflammation and excessive reactive oxygen species (ROS) are common obstacles encountered in DFU wound healing, often leading to impaired recovery. These two effects reinforce each other, forming an endless loop. However, adequate and inclusive methods are still lacking to target these two aspects and break the vicious cycle. This study proposes a novel approach for treating DFU wounds, utilizing an immunomodulatory hydrogel to achieve self-cascade glucose depletion and ROS scavenging to regulate the diabetic microenvironment. Specifically, AuPt@melanin-incorporated (GHM3) hydrogel dressing is developed to facilitate efficient hyperthermia-enhanced local glucose depletion and ROS scavenging. Mechanistically, in vitro/vivo experiments and RNA sequencing analysis demonstrate that GHM3 disrupts the ROS-inflammation cascade cycle and downregulates the ratio of M1/M2 macrophages, consequently improving the therapeutic outcomes for dorsal skin and DFU wounds in diabetic rats. In conclusion, this proposed approach offers a facile, safe, and highly efficient treatment modality for DFUs.

Architecting polyelectrolyte hydrogels with Cu-assisted polydopamine nanoparticles for photothermal antibacterial therapy.

Polydopamine nanoparticles (PDA NPs) are an appealing biomimetic photothermal agent for photothermal antibacterial treatment because of their long-term safety, excellent photostability, accessible manufacturing, and good biodegradability. However, the low photothermal conversion efficiency (PCE) of PDA NPs requires high-power and long-term near-infrared light irradiation, which severely restricts their practical application. In this work, PDA@Cu NPs were fabricated by growing Cu NPs in situ on the surface of PDA and then introduced into a polyelectrolyte hydrogel precursor (cationic polyethyleneimine/anionic pectin, named as CPAP). The formulated photothermal platform possessed a high PCE (55.4%), almost twice as much as pure PDA NPs (30.8%). Moreover, the designed CPAP/PDA@Cu captured and killed some bacteria by electrostatic adsorption, which helped enhance the antibacterial performance. As expected, the formed CPAP/PDA@Cu that combined the advantageous features of PDA@Cu NPs (high PCE) and CPAP matrix (inherent antibacterial activity and preventing NPs aggregation) can efficiently kill bacteria both in vitro and in vivo under the help of near-infrared laser irradiation. Taken together, this study offers a promising strategy for constructing a facile and safe PDA-based photothermal agent for photothermal antibacterial therapy.

Engineering Robust Ag-Decorated Polydopamine Nano-Photothermal Platforms to Combat Bacterial Infection and Prompt Wound Healing.

Polydopamine (PDA) nanoparticles have emerged as an attractive biomimetic photothermal agent in photothermal antibacterial therapy due to their ease of synthesis, good biodegradability, long-term safety, and excellent photostability. However, the therapeutic effects of PDA nanoparticles are generally limited by the low photothermal conversion efficiency (PCE). Herein, PDA@Ag nanoparticles are synthesized via growing Ag on the surface of PDA nanoparticles and then encapsulated into a cationic guar gum (CG) hydrogel network. The optimized CG/PDA@Ag platform exhibits a high PCE (38.2%), which is more than two times higher than that of pure PDA (16.6%). More importantly, the formulated CG/PDA@Ag hydrogel with many active groups can capture and kill bacteria through effective interactions between hydrogel and bacteria, thereby benefiting the antibacterial effect. As anticipated, the designed CG/PDA@Ag system combined the advantages of PDA@Ag nanoparticles (high PCE) and hydrogel (preventing aggregation of PDA@Ag nanoparticles and possessing inherent antibacterial ability) is demonstrated to have superior antibacterial efficacy both in vitro and in vivo. This study develops a facile approach to boost the PCE of PDA for photothermal antibacterial therapy, providing a significant step forward in advancing the application of PDA nano-photothermal agents.

Facile formation of injectable quaternized chitosan/tannic acid hydrogels with antibacterial and ROS scavenging capabilities for diabetic wound healing.

The wound healing process of the diabetic wound is often hindered by excessive oxygen free radicals and infection. An ideal wound dressing should possess great reactive oxygen species (ROS) scavenging property and considerable antibacterial ability. In this study, we facilely constructed a novel hydrogel dressing with excellent ROS scavenging property and outstanding antibacterial performance by introducing tannic acid (TA) into quaternized chitosan (QCS) matrix. Attributing to the suitable physical crosslinking between TA and QCS, this QCS/TA hydrogel was endowed with injectable and self-healing properties, which could avoid the various external squeezing on the irregular shape by wound dressing. The results showed that it could promote coagulation, suppress inflammation and expedite collagen deposition in the skin defect model of diabetic rats. This study provides a facile and convenient method for constructing injectable hydrogel dressing, which has application potentials in the clinical management of diabetic wounds.

ε­Polylysine-stabilized agarose/polydopamine hydrogel dressings with robust photothermal property for wound healing.

Polydopamine (PDA) is emerging as an attractive photothermal agent due to its good photothermal performance and excellent biocompatibility. However, without chemical modification, PDA is normally unstable and usually leached out from the constructed biomaterials, realistically limiting its application space. Here, we constructed a new hydrogel dressing with robust and stable photothermal performance by introduction of ε-Polylysine (ε-PL) into agarose/PDA matrix to efficiently lock PDA. By optimizing PDA/ε-PL rational dose in agarose network structure, a hybrid agarose/PDA/ε-PL hydrogel (ADPH) with stable photothermal functionality and desirable physicochemical properties could be achieved. ADPH possessed satisfactory microbicidal efficacy in vivo, which enabled the bacteria-infected skin wound to be cured quickly by successful suppressing inflammation, accelerating collagen deposition and promoting angiogenesis in a bacterial-infected wound model. Collectively, this study illustrates a simple, convenient but powerful strategy to design functionally stable ADPH dressing for treating dermal wounds, which could open vistas in clinical wound management.