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The large yellow croaker (Larimichthys crocea) is an economically important marine species with the highest annual production among the farmed marine fishes in China. However, the aquaculture industry of this species is suffering from severe problems that include weakened disease resistance, decreased growth rate, and reduced meat quality due to frequent inbreeding. Genome editing, which has a huge potential for solving those problems by introducing favorable genetic changes, is not yet available for the large yellow croaker. Here, we pioneered the techniques of embryo microinjection and genome editing using the CRISPR/Cas9 system in this species. Recombinant plasmids encoding green fluorescent protein (GFP) were introduced into the fertilized eggs of L. crocea by microinjection before the chorion had hardened. A high survival rate (40%) and GFP-positive larvae rate (81.8%) were achieved, indicating that the microinjection technique in L. crocea was successfully established. On this basis, Cas9 mRNA and sgRNA targeting the tyrosinase a gene in L. crocea (Lc-tyra) were coinjected into fertilized eggs of L. crocea. Mutant individuals with insertion and deletion mutations of Lc-tyra were detected. These results indicated that the CRISPR/Cas9-based genome editing technology established herein could efficiently introduce mutations at a specific site in the L. crocea genome. This method provides the potential for genetic improvement and functional genomic study in this species. This is the first report on effective CRISPR/Cas9-based genome editing in L. crocea. [ABSTRACT FROM AUTHOR]

The glucagon receptor (GCGR) is a potential target for diabetes therapy. Several emerging GCGR antagonism-based therapies are under preclinical and clinical development. However, GCGR antagonism, as well as genetically engineered GCGR deficiency in animal models, are accompanied by α-cell hyperplasia and hyperglucagonemia, which may limit the application of GCGR antagonism. To better understand the physiological changes in α cells following GCGR disruption, we performed single cell sequencing of α cells isolated from control and gcgr-/- (glucagon receptor deficient) zebrafish. Interestingly, beyond the α-cell hyperplasia, we also found that the expression of gcga, gcgb, pnoca, and several glucagon-regulatory transcription factors were dramatically increased in one cluster of gcgr-/- α cells. We further confirmed that glucagon mRNA was upregulated in gcgr-/- animals by in situ hybridization and that glucagon promoter activity was increased in gcgr-/-;Tg(gcga:GFP) reporter zebrafish. We also demonstrated that gcgr-/- α cells had increased glucagon protein levels and increased granules after GCGR disruption. Intriguingly, the increased mRNA and protein levels could be suppressed by treatment with high-level glucose or knockdown of the pnoca gene. In conclusion, these data demonstrated that GCGR deficiency not only induced α-cell hyperplasia but also increased glucagon expression in α cells, findings which provide more information about physiological changes in α-cells when the GCGR is disrupted. [ABSTRACT FROM AUTHOR]

Background: The overlap guiding tube (OGT) method, which was designed by our team to assist in overlap esophagojejunostomy, could potentially provide new perspectives for esophagojejunostomy. The application of the OGT-assisted method was first explored by our team and has not yet been reported., Methods: This cohort study analyzed the 3 month outcomes of 38 gastric/gastroesophageal junction (G/GEJ) tumor patients who underwent OGT-assisted overlap esophagojejunostomy., Results: There were 27 males and 11 females, aged 40-82 years. All patients underwent surgery successfully. The success rate of inserting anvil fork into esophageal lumen at first attempt was 97.4%. The total operation time, esophagojejunostomy time, volume of intraoperative blood loss, and length of surgical incision were 317.6 ± 51.5 min, 20.8 ± 3.8 min, 50.0 (range 15.0-200.0) ml, and 5.0 (range 4.0-8.0) cm, respectively. No procedures were converted to other laparoscopic anastomosis techniques or open approaches. The time to postoperative initial flatus, liquid diet intake, soft diet intake, and length of postoperative hospital stay were 3.0 (range 1.0-6.0) days, 4.0 (range 2.0-9.0)days, 6.0 (range 3.0-11.0) days, and 8.5 (range 6.0-16.0) days, respectively. Overall, postoperative complications were observed in 8 (21.1%) patients. Among them, one patients developed esophagojejunal anastomotic leakage. After 3 months of follow-up, none of the patients developed anastomotic stenosis or experienced unplanned secondary surgery or perioperative death., Conclusions: OGT-assisted overlap esophagojejunostomy for patients with G/GEJ tumors is safe and feasible, with good short-term effects. OGT method has a satisfactory success rate of inserting anvil fork into esophageal lumen at first attempt and could prevent from developing esophageal submucosa pseudocanals., (© 2022. The Author(s).)