Knock-out of vasotocin reduces reproductive success in female zebrafish, Danio rerio.

The vertebrate nonapeptide vasotocin/vasopressin is evolutionarily highly conserved and acts as neuromodulator and endocrine/paracrine signaling molecule. Circumstantial and mechanistic evidence from pharmacological manipulations of the vasotocin system in several teleost fishes suggest sex- and species-specific reproductive roles of vasotocin. While effects of vasotocin on teleost reproductive physiology involve both courtship behaviors and the regulation of the hypothalamic-pituitary-gonadal (HPG) axes, comprehensive studies investigating behavioral and physiological reproductive consequences of genetic ablation of vasotocin in a genetically tractable fish model, such as the zebrafish, are currently lacking. Here, we report the generation of homozygous CRISPR/Cas9-based vasotocin gene knock-out zebrafish. Breeding pairs of vasotocin knock-out fish produce significantly fewer fertilized eggs per clutch compared to wildtype fish, an effect coincident with reduced female quivering courtship behavior. Crossbreeding experiments reveal that this reproductive phenotype is entirely female-dependent, as vasotocin-deficient males reproduce normally when paired with female wild-type fish. Histological analyses of vasotocin knock-out ovaries revealed an overall reduction in oocytes and differential distribution of oocyte maturation stages, demonstrating that the reproductive phenotype is linked to oocyte maturation and release. Ovarian hormone quantification and gene expression analysis in mutant fish indicated reduced synthesis of Prostaglandin F2α, a hormone involved in ovarian maturation, egg release and regulation of female courtship behavior in some cyprinids. However, acute injection of vasotocin did not rescue the female mutant reproductive phenotype, suggesting a contribution of organizational effects of vasotocin. Together, this study provides further support for emerging roles of vasotocin in female teleost reproduction in an important teleost model species.

Novel Antimicrobial Peptide "Octoprohibitin" against Multidrug Resistant Acinetobacter baumannii.

Octoprohibitin is a synthetic antimicrobial peptide (AMP), derived from the prohibitin-2 gene of Octopus minor. It showed substantial activity against multidrug resistant (MDR) Acinetobacter baumannii with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 200 and 400 µg/mL, respectively. Time-kill kinetics and bacterial viability assays confirmed the concentration-dependent antibacterial activity of octoprohibitin against A. baumannii. The morphology and ultrastructure of A. baumannii were altered by treatment with octoprohibitin at the MIC and MBC levels. Furthermore, propidium iodide-fluorescein diacetate (PI-FDA) staining and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining of octoprohibitin-treated A. baumannii revealed membrane permeability alterations and reactive oxygen species (ROS) generation, respectively. Agarose gel retardation results confirmed the DNA-binding ability of octoprohibitin to the genomic DNA of A. baumannii. Furthermore, octoprohibitin showed concentration-dependent inhibition of biofilm formation and eradication. The minimum biofilm inhibition concentration (MBIC) and minimum biofilm eradication concentration (MBEC) of octoprohibitin were 1000 and 1460 µg/mL, respectively. Octoprohibitin produced no significant cytotoxicity up to 800 µg/mL, and no hemolysis was observed up to 400 µg/mL. Furthermore, in vivo analysis in an A. baumannii-infected zebrafish model confirmed the effective bactericidal activity of octoprohibitin with higher cumulative survival percent (46.6%) and fewer pathological signs. Histological analysis showed reduced alterations in the gut, kidney, and gill tissues in the octoprohibitin-treated group compared with those in the phosphate-buffered saline (PBS)-treated group. In conclusion, our results suggest that octoprohibitin is a potential antibacterial and antibiofilm agent against MDR A. baumannii.

Antimicrobial and Anti-Biofilm Peptide Octominin for Controlling Multidrug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a serious nosocomial pathogen with multiple drug resistance (MDR), the control of which has become challenging due to the currently used antibiotics. Our main objective in this study is to determine the antibacterial and antibiofilm activities of the antimicrobial peptide, Octominin, against MDR A. baumannii and derive its possible modes of actions. Octominin showed significant bactericidal effects at a low minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of 5 and 10 µg/mL, respectively. Time-kill kinetic analysis and bacterial viability tests revealed that Octominin showed a concentration-dependent antibacterial activity. Field-emission scanning electron microscopy (FE-SEM) analysis revealed that Octominin treatment altered the morphology and membrane structure of A. baumannii. Propidium iodide (PI) and reactive oxygen species (ROS) generation assays showed that Octominin increased the membrane permeability and ROS generation in A. baumannii, thereby causing bacterial cell death. Further, a lipopolysaccharides (LPS) binding assay showed an Octominin concentration-dependent LPS neutralization ability. Biofilm formation inhibition and eradication assays further revealed that Octominin inhibited biofilm formation and showed a high biofilm eradication activity against A. baumannii. Furthermore, up to a concentration of 100 µg/mL, Octominin caused no hemolysis and cell viability changes in mammalian cells. An in vivo study in zebrafish showed that the Octominin-treated group had a significantly higher relative percentage survival (54.1%) than the untreated group (16.6%). Additionally, a reduced bacterial load and fewer alterations in histological analysis confirmed the successful control of A. baumannii by Octominin in vivo. Collectively, these data suggest that Octominin exhibits significant antibacterial and antibiofilm activities against the multidrug-resistant A. baumannii, and this AMP can be developed further as a potent AMP for the control of antibiotic resistance.

Spirulina maxima Derived Pectin Nanoparticles Enhance the Immunomodulation, Stress Tolerance, and Wound Healing in Zebrafish.

In this study, Spirulina maxima derived pectin nanoparticles (SmPNPs) were synthesized and multiple biological effects were investigated using in vitro and in vivo models. SmPNPs were not toxic to Raw 264.7 cells and zebrafish embryos up to 1 mg/mL and 200 µg/mL, respectively. SmPNPs upregulated Il 10, Cat, Sod 2, Def 1, Def 2, and Muc 1 in Raw 264.7 cells and tlr2, tlr4b, tlr5b, il1ß, tnfα, cxcl8a, cxcl18b, ccl34a.4, ccl34b.4, muc5.1, muc5.2, muc5.3, hamp, cstd, hsp70, cat, and sod1 in the larvae and adult zebrafish, suggesting immunomodulatory activity. Exposure of larvae to SmPNPs followed by challenge with pathogenic bacterium Aeromonas hydrophila resulted a two-fold reduction of reactive oxygen species, indicating reduced oxidative stress compared to that in the control group. The cumulative percent survival of larvae exposed to SmPNPs (50 µg/mL) and adults fed diet supplemented with SmPNPs (4%) was 53.3% and 76.7%, respectively. Topical application of SmPNPs on adult zebrafish showed a higher wound healing percentage (48.9%) compared to that in the vehicle treated group (38.8%). Upregulated wound healing markers (tgfß1, timp2b, mmp9, tnfα, il1ß,ccl34a.4, and ccl34b.4), enhanced wound closure, and restored pigmentation indicated wound healing properties of SmPNPs. Overall, results uncover the multiple bioactivities of SmPNPs, which could be a promising biocompatible candidate for broad range of aquatic and human therapies.