Use of All-Male cyp17a1-Deficient Zebrafish (Danio rerio) for Evaluation of Environmental Estrogens.

Natural and synthetic environmental estrogens (EEs) are widespread and have received extensive attention. Our previous studies demonstrated that depletion of the cytochrome P450 17a1 gene (cyp17a1) leads to all-testis differentiation phenotype in zebrafish and common carp. In the present study, cyp17a1-deficient zebrafish with defective estrogen biosynthesis were used for the evaluation of EEs, as assessed by monitoring vitellogenin (vtg) expression. A rapid and sensitive assessment procedure was established with the 3-day administration of estradiol (E2), followed by examination of the transcriptional expression of vtgs in our cyp17a1-deficient fish. Compared with the control fish, a higher E2-mediated vtg upregulation observed in cyp17a1-deficient zebrafish exposed to 0.1 µg/L E2 is known to be estrogen receptor-dependent and likely due to impaired in vivo estrogen biosynthesis. The more responsive vtg expression in cyp17a1-deficient zebrafish was observed when exposed to 200 and 2000 µg/L bisphenol A (BPA) and perfluoro-1-octanesulfonate (PFOS). The estrogenic potentials of E2, BPA, and PFOS were compared and assessed by the feminization effect on ovarian differentiation in cyp17a1-deficient zebrafish from 18 to 50 days postfertilization, based on which a higher sensitivity of E2 in ovarian differentiation than BPA and PFOS was concluded. Collectively, through the higher sensitivity to EEs and the capacity to distinguish chemicals with different estrogenic potentials exhibited by the all-male cyp17a1-deficient zebrafish with impaired estrogen biosynthesis, we demonstrated that they can be used as an excellent in vivo model for the evaluation of EEs. Environ Toxicol Chem 2024;43:1062-1074. © 2024 SETAC.

Enhanced insulin activity achieved in VDRa/b ablation zebrafish.

Introduction: 1α,25-dihydroxyvitamin D3 (1α,25[OH]2VD3) is a hormone known for its key roles in calcium absorption and nutrient metabolism. In teleost fishes, 1α,25(OH)2VD3 insufficiency causes impaired glucose metabolism and lipid oxidation. However, the cascade and mechanisms of 1α,25(OH)2VD3 and the vitamin d receptor (VDR) signaling are unclear. Results: In this study, two genes (vdra and vdrb) encoding paralogs of VDRs were genetically knocked out in zebrafish. Growth retardation and accumulated visceral adipose tissue have been observed in vdra -/-;vdrb -/- deficient line. In the liver elevated accumulation of triglycerides and suppressed lipid oxidation were detected. Morover significantly elevated 1α,25(OH)2VD3 levels were detected in vdra-/-;vdrb-/- zebrafish due to cyp24a1 transcription repression. Furthermore VDRs ablation Enhanced insulin signaling including elevated insulin/insra trancriptional levels, glycolysis, lipogenesis and promoted AKT/mTOR activity. Discussion: In conclusion, our present studies provides a zebrafish model with an elevated 1α,25(OH)2VD3 levels in vivo. The 1α,25(OH)2VD3/VDRs signaling promote lipid oxidation activity. However 1α,25(OH)2VD3 activity of regulation of glucose homeostasis through Insulin/Insr was independent of nuclear VDRs in teleosts.

Effects of Different Doses of Dexmedetomidine Combined with Thoracic Paravertebral Nerve Block Anesthesia on Agitation and Hemodynamics in Patients Undergoing Thoracotomy during Recovery.

Objective: To investigate the effect of different doses of dexmedetomidine combined with thoracic paravertebral nerve block anesthesia on agitation and hemodynamics in patients undergoing thoracotomy during recovery. Methods: One hundred patients who underwent thoracotomy in our hospital from August 2018 to April 2021 were enrolled and assigned (1 : 1 : 1 : 1) into 4 groups via the random number table method. The patients in the control group were treated with double-lumen tube general anesthesia + ropivacaine for thoracic paravertebral nerve block anesthesia; patients in experimental group A received double-lumen general anesthesia +0.5 µg·kg-1 dexmedetomidine + ropivacaine for thoracic paravertebral nerve block anesthesia; patients in experimental group B received thoracic paravertebral nerve block anesthesia with double-lumen general anesthesia +1.0 µg·kg-1 dexmedetomidine + ropivacaine; patients in experimental group C received thoracic paravertebral nerve block anesthesia with double-lumen general anesthesia +1.5 µg·kg-1 dexmedetomidine + ropivacaine. The postoperative recovery time and visual analog scale (VAS), level of hemodynamics (heart rate (HR), mean arterial pressure (MAP)), agitation during the recovery period, and complications were compared amongst the 4 groups of patients at different time points. Results: The postoperative VAS scores of patients in groups B2 and B3 were slightly lower than those of patients in groups A and B1, but a one-way analysis of variance revealed no statistical difference in the postoperative recovery time and VAS pain scores of the four groups (P > 0.05), and the recovery time of patients in experimental group C was slightly higher than that of patients in group B2. At T0 and T1, there was no significant difference in the levels of HR and MAP among the four groups (P > 0.05). The levels of HR and MAP of the patients in groups B2 and B3 were significantly different from the patients in the control group and experimental group A at T2 and T3 (P < 0.05). The patients in experimental group B and experimental group C showed better outcomes than those in the control group and experimental group A in the assessment of agitation during the recovery period (P < 0.05). There was no significant difference in the incidence of complications among the four groups (P > 0.05). Conclusion: In line with the principle of preference for a small anesthesia dose, 1.0 µg·kg-1 dose of dexmedetomidine combined with ropivacaine produces a pronounced efficacy in patients undergoing thoracotomy. It effectively controls the occurrence of agitation during the recovery period and maintains the stability of the patient's hemodynamics, with a high clinical safety profile.

Characterization of the Interrenal Gland and Sexual Traits Development in cyp17a2-Deficient Zebrafish.

Unlike the Cytochrome P450, family 17, subfamily A, member 1 (Cyp17a1), which possesses both 17α-hydroxylase and 17,20-lyase activities involved in the steroidogenic pathway that produces androgens and estrogens, Cytochrome P450, family 17, subfamily A, polypeptide 2 (Cyp17a2) possesses only 17α-hydroxylase activity and is known essential for the synthesis of cortisol. Besides with expressed in testes and ovaries, where the cyp17a1 is mainly expressed, cyp17a2 is also expressed in the interrenal gland in fish. Until now, the roles of cyp17a2 in fish, especially in sexual traits development and hypothalamic-pituitary-interrenal (HPI) axis, are poorly studied. To investigate the roles of Cyp17a2 in teleosts, the cyp17a2-null zebrafish was generated and analyzed by us. The significantly decreased cortisol concentration was observed both in the cyp17a2-deficient males and females at adult stage. The interrenal gland enlargement, increased pituitary proopiomelanocortin a (pomca) expression, decreased locomotion activity and response to light-stimulated stress were observed in cyp17a2-deficient fish. Intriguingly, the cyp17a2-deficient males were fertile and with normal breeding tubercles on the pectoral fin, but females were infertile, deficient in genital papilla and with decreased gonadosomatic index (GSI). The increased progesterone (P4), 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) and 11-ketotestosterone (11-KT) in the cyp17a2-deficient males and females were observed. The increased concentration of testosterone (T) and estradiol (E2) was observed in cyp17a2-/- females and cyp17a2-/- males, respectively. By examining the ovaries development of cyp17a2-deficient fish at 3 months postfertilization (mpf), we observed that the oocytes were over-activated. Taken together, our findings demonstrate that Cyp17a2 is indispensable for production and physiology of cortisol, and cyp17a2-deficiency resulted in diminished cortisol but accumulated P4 and DHP, which may result in the over-activated oocytes in cyp17a2-deficient females.

Characterization of a novel broad-spectrum endolysin PlyD4 encoded by a highly conserved prophage found in Aeromonas hydrophila ST251 strains.

Aeromonas hydrophila is a zoonotic pathogen that exhibits high level resistance to classic antibiotics and is a heavy burden for aquaculture industry. Lytic enzymes encoded by phages or prophages have shown potential for use against pathogenic bacteria. In this study, an intact prophage (named phAhD4) was identified from A. hydrophila D4. phAhD4 is highly conserved in all 10 published A. hydrophila sequence type (ST) 251 strains and is unique to the ST251 strains. The unique endolysin PlyD4, encoded by phAhD4, was obtained by prokaryotic expression. PlyD4 showed bactericidal activity against a broad range of bacterial species in vitro, including A. hydrophila, Aeromonas veronii, Vibrio parahemolyticus, Pseudomonas aeruginosa, and so on. Synergistically with 5 mmol/L ethylene diamine tetraacetic acid (EDTA), the ratio of the optical density at 600 nm (OD600) of PlyD4 treatment versus the OD600 with no PlyD4 treatment for most tested strains decreased from 1 to 0.1-0.8 within 2 h. PlyD4 exhibited optimal activity at 28 °C and maintained high activity over a wide pH range (pH 6-10). Divalent metal ions conferred significant enhancement to PlyD4 lytic activity at low concentrations (0.1 mmol/L). In vivo, a 4.5 µg dose of PlyD4 protected 75.0% (15/20) of zebrafish in a bacteremia model of A. hydrophila D4 infection. These results indicated that PlyD4 was an effective therapeutic agent against multiple aquaculture-related pathogens. To the best of our knowledge, this study is the first to report on an A. hydrophila prophage endolysin that exerts antibacterial activity against a broad range of pathogens. KEY POINTS: • The prophage phAhD4 is highly conserved in 10 published A. hydrophila ST251 strains. • PlyD4 exerts antibacterial activity against multiple aquaculture-related pathogens. • PlyD4 conferred protection against A. hydrophila infection in a zebrafish model.