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Mutations in the calreticulin gene (CALR) were recently identified in approximately 70–80% of patients with JAK2-V617F-negative essential thrombocytosis and primary myelofibrosis. All frameshift mutations generate a recurring novel C-terminus. Here we provide evidence that mutant calreticulin does not accumulate efficiently in cells and is abnormally enriched in the nucleus and extracellular space compared to wildtype calreticulin. The main determinant of these findings is the loss of the calcium-binding and KDEL domains. Expression of type I mutant CALR in Ba/F3 cells confers minimal IL-3-independent growth. Interestingly, expression of type I and type II mutant CALR in a non-hematopoietic cell line does not directly activate JAK/STAT signaling compared to JAK2-V617F expression. These results led us to investigate paracrine mechanisms of JAK/STAT activation. Here we show that conditioned media from cells expressing type I mutant CALR exaggerate cytokine production from normal monocytes with or without treatment with a toll-like receptor agonist. These effects are not dependent on the novel C-terminus. These studies offer novel insights into the mechanism of JAK/STAT activation in patients with JAK2-V617F-negative essential thrombocytosis and primary myelofibrosis.

Nile tilapia (Oreochromis niloticus) is the world’s most widely cultured fish species. Therefore, its nutritional physiology is of great interest from an aquaculture perspective. Studies conducted on several fish species, including tilapia, demonstrated the beneficial effects of dietary salt supplementation on growth; however, the mechanism behind these beneficial effects is still not fully understood. The fish intestine is a complex system, with functions, such as nutrient absorption, ion equilibrium and acid-base balance that are tightly linked and dependent on each other's activities and products. Ions are the driving force in the absorption of feed components through pumps, transporters and protein channels. In this study, we examined the impact of 5% increase in dietary NaCl on protein, lipid, ash and dry matter digestibility, as well as on the expression of intestinal peptide transporters (PepTs) and ion pumps (Na+/K+-ATPase, V-H+-ATPase, N+/H+-Exchanger) in Nile tilapia. In addition, effects on the gut microbiome were evaluated. Our results show that dietary salt supplementation significantly increased digestibility of all measured nutritional components, peptide transporters expression and ion pumps activity. Moreover, changes in the gut microbial diversity were observed, and were associated with lipid digestibility and Na+/K+-ATPase expression.

Estradiol-17beta (E2) maintains high cAMP levels and meiotic arrest in zebrafish oocytes through activation of G protein-coupled estrogen receptor (GPER). The catecholestrogen 2-hydroxyestradiol-17beta (2-OHE2) has an opposite effect to that of E2 on oocyte maturation (OM) and cAMP levels in Indian catfish oocytes. We tested the hypothesis that 2-OHE2 is produced in zebrafish ovaries and promotes the resumption of oocyte meiosis through its action as a GPER antagonist. Ovarian 2-OHE2 production by estrogen-2-hydroxylase (EH) was up-regulated by gonadotropin treatment at the onset of OM, consistent with a physiological role for 2-OHE2 in regulating OM. The increases in EH activity and OM were blocked by treatment with CYP1A1 and CYP1B1 inhibitors. Expression of cyp1a, cyp1b1, and cyp1c mRNAs was increased by gonadotropin treatment, further implicating these Cyp1s in 2-OHE2 synthesis prior to OM. Conversely, aromatase activity and cyp19a1 mRNA expression declined during gonadotropin induction of OM. 2-OHE2 treatment significantly increased spontaneous OM in defolliculated zebrafish oocytes and reversed the inhibition of OM by E2 and the GPER agonist G-1. 2-OHE2 was an effective competitor of [(3)H]-E2 binding to recombinant zebrafish GPER expressed in HEK-293 cells. 2-OHE2 also antagonized estrogen actions through GPER on cAMP production in zebrafish oocytes, resulting in a reduction in cAMP levels. Stimulation of OM by 2-OHE2 was blocked by pretreatment of defolliculated oocytes with the GPER antibody. Collectively, the results suggest that 2-OHE2 functions as a GPER antagonist and promotes OM in zebrafish through blocking GPER-dependent E2 inhibition of the resumption of OM.

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