Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line

The role of mechanosensitive (MS) Ca2+-permeable ion channels in platelets is unclear, despite the importance of shear stress in platelet function. We sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca2+ entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy. Cells were attached to microscope slides within flow chambers that allowed application of physiological and pathological shear stress. Arterial shear (1002.6s-1) induced a sustained increase in intracellular calcium ([Ca2+]i) in Meg-01 cells and enhanced the frequency of repetitive Ca2+ transients by 80% in platelets. These Ca2+ increases were abrogated by the MS channel inhibitor GsMTx-4 or by chelation of extracellular Ca2+. Thrombus formation was studied on collagen-coated surfaces using 3,3'-dihexyloxacarbocyanine iodide (DiOC6)-stained platelets. In addition, [Ca2+]i and functional responses of washed platelet suspensions were studied with Fura-2 and light transmission aggregometry, respectively. Thrombus size was reduced 50% by GsMTx-4 independently of P2X1 receptors. In contrast, GsMTx-4 had no effect on collagen-induced aggregation and on Ca2+ influx via TRPC6 or Orai1 channels, and caused only a minor inhibition of P2X1-dependent Ca2+ entry. The Piezo1 agonist, Yoda1, potentiated shear-dependent platelet Ca2+ transients by 170%. Piezo1 mRNA transcripts and protein were detected in both platelets and Meg-01 cells using qRT-PCR and Western blotting. We conclude that platelets and Meg-01 cells express the MS cation channel Piezo1, which may contribute to Ca2+ entry and thrombus formation under arterial shear stress

Upskilling and reskilling in the United Arab Emirates : Future-proofing careers with AI skills

The rapid evolution of technology, particularly in artificial intelligence (AI), is reshaping the global job market at an unprecedented pace. This transformative wave presents challenges and opportunities, especially for the United Arab Emirates (UAE), as it endeavours to future-proof its workforce. This article examines the imperative of upskilling and reskilling as a strategic response to the dynamic demands of the contemporary job landscape in the UAE. Focusing on the pivotal role of AI skills, the paper explores the current state of the UAE workforce, highlighting the vulnerabilities of existing job roles and industries to technological disruptions. The global context of upskilling initiatives provides a backdrop to understand the urgency of this issue and offers insights into successful implementation strategies

Increasing the expression of calcium-permeable TRPC3 and TRPC7 channels enhances constitutive secretion

The hTRPC [human TRPC (canonical transient receptor potential)] family of non-selective cation channels is proposed to mediate calcium influx across the plasma membrane via PLC (phospholipase C)-coupled receptors. Heterologously expressed hTRPC3 and hTRPC7 have been localized at the cell surface; however, a large intracellular component has also been noted but not characterized. In the present study, we have investigated the intracellular pool in COS-7 cells and have shown co-localization with markers for both the TGN (trans-Golgi network) and the cis-Golgi cisternae by immunofluorescence microscopy. Addition of BFA (Brefeldin A) to cells expressing hTRPC3 or hTRPC7 resulted in the redistribution of the Golgi component to the endoplasmic reticulum, indicating that this pool is present in both the Golgi stack and the TGN. Expression of either TRPC3 or TRPC7, but not TRPC1 or the cell surface marker CD8, resulted in a 2–4-fold increase in secreted alkaline phosphatase in the extracellular medium. Based on these results, we propose that an additional function of these members of the hTRPC family may be to enhance secretion either by affecting transport through the Golgi stack or by increasing fusion at the plasma membrane

The Serine 814 of TRPC6 Is Phosphorylated under Unstimulated Conditions

TRPC are nonselective cation channels involved in calcium entry. Their regulation by phosphorylation has been shown to modulate their routing and activity. TRPC6 activity increases following phosphorylation by Fyn, and is inhibited by protein kinase G and protein kinase C. A previous study by our group showed that TRPC6 is phosphorylated under unstimulated conditions in a human embryonic kidney cells line (HEK293). To investigate the mechanism responsible for this phosphorylation, we used a MS/MS approach combined with metabolic labeling and showed that the serine at position 814 is phosphorylated in unstimulated cells. The mutation of Ser814 into Ala decreased basal phosphorylation but did not modify TRPC6 activity. Even though Ser814 is within a consensus site for casein kinase II (CK2), we showed that CK2 is not involved in the phosphorylation of TRPC6 and does not modify its activity. In summary, we identified a new basal phosphorylation site (Ser814) on TRPC6 and showed that CK2 is not responsible for the phosphorylation of this site

Mapping and characterization of the X-linked dyskeratosis congenita (DKC) gene

We report the precise mapping and characterization of the genomic structure of the human homolog of the rat gene for the nucleolar protein NAP57, which has been reported to be responsible for X-linked dyskeratosis congenita (DKC). This single-copy gene, now called DKC, is transcribed from a CpG island 60 kb centromeric to the factor VIII gene in distal Xq28 and lies tail to tail with the palmitoylated erythrocyte membrane protein gene, MPP1. DKC comprises 15 exons spanning at least 16 kb and is transcribed into a widely expressed 2.6-kb message. Several functional motifs of DKC are assigned to coding sequences specified by individual exons. Analysis of normal female DNA revealed the presence of two polymorphisms in the DKC exons, while mutation analysis of a DKC patient identified a novel single amino acid missense mutation in exon 4. The latter together with exon 3 contain five of the six missense mutations reported so far in the DKC gene