RESUMO
Several of our internal organs, including heart, lungs, stomach, and spleen, develop asymmetrically along the left-right (LR) body axis. Errors in establishing LR asymmetry, or laterality, of internal organs during early embryonic development can result in birth defects. In several vertebrates-including humans, mice, frogs, and fish-cilia play a central role in establishing organ laterality. Motile cilia in a transient embryonic structure called the "left-right organizer" (LRO) generate a directional fluid flow that has been proposed to be detected by mechanosensory cilia to trigger asymmetric signaling pathways that orient the LR axis. However, the mechanisms that control the form and function of the ciliated LRO remain poorly understood. In the zebrafish embryo, precursor cells called dorsal forerunner cells (DFCs) develop into a transient ciliated structure called Kupffer's vesicle (KV) that functions as the LRO. DFCs can be visualized and tracked in the embryo, thereby providing an opportunity to investigate mechanisms that control LRO development. Previous work revealed that proliferation of DFCs via mitosis is a critical step for developing a functional KV. Here, we conducted a targeted pharmacological screen to identify mechanisms that control DFC proliferation. Small molecule inhibitors of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) were found to reduce DFC mitosis. The SERCA pump is involved in regulating intracellular calcium ion (Ca2+) concentration. To visualize Ca2+ in living embryos, we generated transgenic zebrafish using the fluorescent Ca2+ biosensor GCaMP6f. Live imaging identified dynamic cytoplasmic Ca2+ transients ("flux") that occur unambiguously in DFCs. In addition, we report Ca2+ flux events that occur in the nucleus of DFCs. Nuclear Ca2+ flux occurred in DFCs that were about to undergo mitosis. We find that SERCA inhibitor treatments during DFC proliferation stages alters Ca2+ dynamics, reduces the number of ciliated cells in KV, and alters embryo laterality. Mechanistically, SERCA inhibitor treatments eliminated both cytoplasmic and nuclear Ca2+ flux events, and reduced progression of DFCs through the S/G2 phases of the cell cycle. These results identify SERCA-mediated Ca2+ signaling as a mitotic regulator of the precursor cells that give rise to the ciliated LRO.
RESUMO
BACKGROUND: Cow's milk protein allergy (CMA) is the second most common food allergy in Singapore. However, there is limited data on local paediatric CMA. OBJECTIVE: We aimed to describe the demographics, clinical characteristics, natural history and diagnostic performance of skin prick test (SPT) and cow's milk-specific immunoglobulin E (CM-IgE) in Singaporean children diagnosed with IgE-mediated CMA. METHODS: A retrospective review of medical records was conducted for children with an SPT performed to cow's milk between 2011 and 2016. RESULTS: There were 355 patients included, 313 cow's milk allergic and 42 cow's milk tolerant. The median age of reaction was 6 months (IQR 4-8). The most common allergic presentation was cutaneous reactions, followed by gastrointestinal reactions. Six patients (1.9%) reported anaphylaxis at initial presentation and 16 children (5.1%) experienced anaphylaxis to cow's milk at least once in their lifetime. Most of the CMA patients (81.8%) acquired natural tolerance by 6 years old. SPT to cow's milk of ≥ 7 mm and CM-IgE of ≥ 13 kU/L showed good discriminative abilities in predicting a failed oral food challenge (OFC) outcome. CONCLUSIONS: CMA is a food allergy which commonly presents during infancy, and parents need to be aware of the likelihood of severe allergic reactions, including anaphylaxis. Prognosis for CMA is generally favourable. Future prospective cohort studies are required to better understand the natural history and better define the diagnostic cut-off values for allergy testing in our population.
