Exosomes from bacteria (Streptococcus parauberis) challenged olive flounder (Paralichthys olivaceus): Isolation, molecular characterization, wound healing, and regeneration activities.

Exosomes are a group of extracellular vesicles carrying membrane proteins, lipids, RNAs, and, cytosolic proteins, which play key role in intercellular communication and homeostasis. This study describes the isolation, physicochemical, morphological and molecular characterization, toxicity, wound healing, and regeneration properties of plasma derived exosomes from naive (phosphate-buffered saline [PBS]-injected; PBS-Exo) and Streptococcus parauberis-challenged (Sp-Exo) olive flounder (Paralichthys olivaceus). The average diameters of PBS-Exo and Sp-Exo were 120.5 ± 6.1 and 113.1 ± 9.3 nm, respectively, and they presented unique cup shape morphologies. Both exosomes exhibited classical tetraspanin surface markers (CD81, CD9, and CD63) and were enriched with acetylcholinesterase. High-throughput miRNA profiling revealed differentially expressed miRNAs (log2 fold change ≥1; P < 0.05), including 14 known and 22 novel miRNAs, in Sp-Exo. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that the target genes of the miRNAs contribute towards various physiological and immunological functions, including wound healing and fin regeneration. Sp-Exo exhibited a rapid wound healing (cell migration) capacity in human fibroblast cells, and its mRNA and protein expression patterns corroborated its activity. Higher larval fin regeneration was more prevalent in Sp-Exo than in PBS-Exo, which further confirmed its functional significance. Our study provides the first basic physiochemical, morphometric, molecular (miRNA profiling), and wound healing evidences of Sp-Exo in olive flounder and highlights important miRNA cargoes in exosomes that may be potential therapeutic candidates in wound healing.

Octopromycin: Antibacterial and antibiofilm functions of a novel peptide derived from Octopus minor against multidrug-resistant Acinetobacter baumannii.

The emergence of carbapenem-resistant Acinetobacter baumannii has increased the risk of nosocomial infections, which pose a huge health threat. There is an urgent need to develop alternative therapies, including broad-spectrum antimicrobial peptides. In this study, we designed, characterized, and studied the antibacterial, antibiofilm effects and possible mode of actions of a novel synthetic peptide Octopromycin, derived from the proline-rich protein 5 of Octopus minor. Octopromycin consists of 38 amino acids, (+5) net positive charge, high hydrophobic residue ratio (36%), and two α-helix secondary structures. The minimum inhibitory concentration and minimum bactericidal concentration against A. baumannii were 50 and 200 µg/mL, respectively. Time-kill kinetics and bacterial viability assays confirmed the concentration-dependent antibacterial activity of Octopromycin. Field emission scanning electron microscopy images clearly showed ultrastructural alterations in Octopromycin-treated A. baumannii cells. Propidium iodide penetrated into Octopromycin-treated A. baumannii cells, demonstrating the loss of cell membrane integrity. Octopromycin treatment increased the production of reactive oxygen species in a concentration-dependent manner, and it inhibited the biofilm formation and showed biofilm eradication activity against A. baumannii. In vitro and in vivo safety evaluation revealed that Octopromycin was nontoxic to HEK293T and Raw 264.7 cells (<400 µg/mL), as well as mice red blood cells (<300 µg/mL), and zebrafish embryos (<4 µg/mL). An in vivo study results revealed that the A. baumannii-infected fish treated with Octopromycin exhibited a significantly higher relative percent survival (37.5%) than the infected mock-treated fish with PBS (16.6%). Furthermore, a decreased bacterial load and fewer alterations in histological analysis confirmed the successful control of A. baumannii by Octopromycin in vivo. Collectively, the results indicate that the antibacterial peptide Octopromycin may achieve rapid control of A. baumannii through multi-target interactions; it presents a desirable therapeutic option for the prevention and control of the infections.

Spirulina maxima derived marine pectin promotes the in vitro and in vivo regeneration and wound healing in zebrafish.

Purified bioactive components of marine algae have shown great pharmaceutical and biomedical potential, including wound healing activity. However, the activity of Spirulina maxima is the least documented with regard to wound healing potential. In the present study, we investigated the regenerative and wound healing activities of a Spirulina (Arthrospira) maxima based pectin (SmP) using in vitro human dermal fibroblasts (HDFs) and in vivo zebrafish model. SmP treated (12.5-50 µg/mL) HDFs showed increased cell proliferation by 20-40% compared to the untreated HDFs. Moreover, in vitro wound healing results in HDFs demonstrated that SmP decreased the open wound area % in concentration-dependent manner at 12.5 (32%) and 25 µg/mL (12%) compared to the control (44%). Further, zebrafish larvae displayed a greater fin regenerated area in the SmP exposed group at 25 (0.48 mm2) and 50 µg/mL (0.51 mm2), whereas the untreated group had the lowest regenerated area (0.40 mm2) at 3 days post amputation. However, fin regeneration was significantly (P < 0.001) higher only in the SmP treated group at 50 µg/mL. Furthermore, the open skin wound healing % in adult zebrafish was significantly higher (P < 0.05) after topical application (600 µg/fish) of SmP (46%) compared to the control (38%). Upregulation of genes such as tgfß1, timp2b, mmp9, tnf-α, and il-1ß, and chemokines such as cxcl18b, ccl34a.4, and ccl34b.4, in the muscle and kidney tissues of SmP treated fish compared to the respective control group was demonstrated using qRT-PCR. Histological analysis results further supported the rapid epidermal growth and tissue remodeling in SmP treated fish, suggesting that SmP exerts positive effects associated with wound healing. Therefore, SmP can be considered a potential regenerative and wound healing agent.

Succinyl chitosan gold nanocomposite: Preparation, characterization, in vitro and in vivo anticandidal activity.

Herein, we have successfully synthesized a novel N-Succinyl chitosan/gold nanocomposite (N-SuC/Au NC) using N-SuC and gold(III) chloride, and investigated the biocompatibility and antifungal activity. The synthesized N-SuC/Au NC was characterized by UV-visible spectroscopy, X-ray diffraction, field emission scanning electron microscope, and inductively coupled plasma atomic emission spectroscopy. The N-SuC/Au NC exhibited a strong inhibition effect towards pathogenic Candida albicans. Morphological analysis revealed the destruction of C. albicans cell membrane due to N-SuC/Au NC treatment. The in vitro and in vivo toxicity of N-SuC/Au NC was analyzed with HEK293T mammalian cells and zebrafish larvae, respectively. The synthesized N-SuC/Au NC demonstrated no cytotoxicity towards HEK293T cells up to 1200 µg/mL concentration. The survival rate of the zebrafish larvae at 120 hpf, was found as 100% up to 1200 µg/mL of N-SuC/Au NC exposure. The in vivo studies further confirmed the inhibitory effects of N-SuC/Au NC on the formation of C. albicans hyphae in infected zebrafish muscle tissue.

Marine Microalgae, Spirulina maxima-Derived Modified Pectin and Modified Pectin Nanoparticles Modulate the Gut Microbiota and Trigger Immune Responses in Mice.

This study evaluated the modulation of gut microbiota, immune responses, and gut morphometry in C57BL/6 mice, upon oral administration of S. maxima-derived modified pectin (SmP, 7.5 mg/mL) and pectin nanoparticles (SmPNPs; 7.5 mg/mL). Metagenomics analysis was conducted using fecal samples, and mice duodenum and jejunum were used for analyzing the immune response and gut morphometry, respectively. The results of metagenomics analysis revealed that the abundance of Bacteroidetes in the gut increased in response to both modified SmP and SmPNPs (75%) as compared with that in the control group (66%), while that of Firmicutes decreased in (20%) as compared with that in the control group (30%). The mRNA levels of mucin, antimicrobial peptide, and antiviral and gut permeability-related genes in the duodenum were significantly (p < 0.05) upregulated (> 2-fold) upon modified SmP and SmPNPs feeding. Protein level of intestinal alkaline phosphatase was increased (1.9-fold) in the duodenum of modified SmPNPs feeding, evidenced by significantly increased goblet cell density (0.5 ± 0.03 cells/1000 µm2) and villi height (352 ± 10 µm). Our results suggest that both modified SmP and SmPNPs have the potential to modulate gut microbial community, enhance the expression of immune related genes, and improve gut morphology.

Novel pectin isolated from Spirulina maxima enhances the disease resistance and immune responses in zebrafish against Edwardsiella piscicida and Aeromonas hydrophila.

In this study, we demonstrate the enhanced disease resistance and positive immunomodulation of novel pectin isolated from Spirulina maxima (SmP) in zebrafish model. Zebrafish larvae exposed to SmP had significantly (p < 0.05) higher cumulative percent survival (CPS) at 25 (44.0%) and 50 µg/mL (67.0%) against Edwardsiella piscicida compared to the control. However, upon Aeromonas hydrophila challenge, SmP exposed larvae at 50 µg/mL had slightly higher CPS (33.3%) compared to control group (26.7%). SmP supplemented zebrafish exhibited the higher CPS against E. piscicida (93.3%) and A. hydrophila (60.0%) during the early stage of post-infection (<18 hpi). qRT-PCR results demonstrated that exposing (larvae) and feeding (adults) of SmP, drive the modulation of a wide array of immune response genes. In SmP exposed larvae, up-regulation of the antimicrobial enzyme (lyz: 3.5-fold), mucin (muc5.1: 2.84, muc5.2: 2.11 and muc5.3: 2.40-fold), pro-inflammatory cytokines (il1ß: 1.79-fold) and anti-oxidants (cat: 2.87 and sod1: 1.82-fold) were identified. In SmP fed adult zebrafish (gut) showed >2-fold induced pro-inflammatory cytokine (il1ß) and chemokines (cxcl18b, ccl34a.4 and ccl34b.4). Overall results confirmed the positive modulation of innate immune responses in larval stage and it could be the main reason for developing disease resistance against E. piscicida and A. hydrophila. Thus, non-toxic, natural and biodegradable SmP could be considered as the potential immunomodulatory agent for sustainable aquaculture.