In the early Drosophila embryo, epithelial cells begin to adopt a cell fate. At gastrulation, there is an asymmetric partitioning of the endoplasmic reticulum in a symmetrically dividing cell population before cell fate selection. These results highlight the changes in organelle distribution before asymmetric divisions.

Multi-organ fibrosis among end stage renal disease (ESRD) patients cannot be explained by uremia alone. Despite mitigation of thrombosis during hemodialysis (HD), subsequent platelet dysfunction and tissue dysregulation are less understood. We comprehensively profiled plasma and platelets from ESRD patients before and after HD to examine HD-modulation of platelets beyond thrombotic activation. Basal plasma levels of proteolytic regulators and fibrotic factors were elevated in ESRD patients compared to healthy controls, with isoform-specific changes during HD. Platelet lysate (PL) RNA transcripts for growth and coagulative factors were elevated post-HD, with upregulation correlated to HD vintage. Platelet secretome correlations to plasma factors reveal acutely induced pro-fibrotic platelet phenotypes in ESRD patients during HD characterized by preferentially enhanced proteolytic enzyme translation and secretion, platelet contribution to inflammatory response, and increasing platelet dysfunction with blood flow rate (BFR) and Vintage. Compensatory mechanisms of increased platelet growth factor synthesis with acute plasma matrix metalloproteinase (MMP) and tissue inhibitor of MMPs (TIMP) increases show short-term mode-switching between dialysis sessions leading to long-term pro-fibrotic bias. Chronic pro-fibrotic adaptation of platelet synthesis were observed through changes in differential secretory kinetics of heterogenous granule subtypes. We conclude that chronic and acute platelet responses to HD contribute to a pro-fibrotic milieu in ESRD.

Activation of circulating platelets by receptor binding and subsequent coagulation events are defined by a well characterized physiological response. However, the growing prevalence of chronic kidney disease (CKD) and implication of platelet-released factors in worsening cardiovascular outcomes with hemodialysis warrant further investigation into the mechanobiology of platelet degranulation. The significant drops in pressure caused by high friction across the hemodialysis flow circuit present an overlooked platelet stimulant not involving immobilization as a driver for cytoskeletal rearrangement. In this study, platelets from healthy and dialysis (pre- and post-treatment) donors were cyclically depressurized in static suspension to measure changes in physiology by integrin αIIbβ3 activation and surface P-selectin expression. The progressive increase in CD62P with no changes in PAC1 over pressure-cycling duration regardless of uremia signifies that hydrostatic depressurization involves a novel agonist-free mechanism leading to platelet degranulation as a unique case in which CD62P and PAC1 do not interchangeably indicate platelet activation. Subsequent stimulation using ADP further suggests that sustained depressurization regimens desensitize integrin αIIbβ3 activation. Variability in platelet response caused by uremia and CKD are observed by elevated baseline PAC1 in pre-dialysis samples, PAC1 retention after ADP exposure, and maximum CD62P with ADP independent of pressure. Theory for hydrostatic pressure-induced degranulation circumventing integrin-initiated signal transduction is here presented based on the Starling Equation.

Purpose: Gene therapy employing AAV vector-mediated gene delivery has undergone substantial growth in recent years with promising results in both preclinical and clinical studies, as well as emerging regulatory approval. However, the lack of methods for quantifying the efficacy of gene therapy from cellular delivery of gene editing technology to specific functional outcomes remains an obstacle for the efficient development of gene therapy treatments. Building upon prior works that utilized a genetically encoded Lysine Rich Protein as a chemical exchange saturation transfer (CEST) reporter, we hypothesized that AAV viral capsids may generate endogenous CEST contrast from the large number of surface lysine residues. Methods: Water-suppressed NMR and NMR-CEST experiments were performed on isolated solutions of AAV serotypes 1-9 on a Bruker 800MHz vertical scanner. A series of in vitro experiments were performed for thorough testing of NMR-CEST contrast of AAV2 capsids under varying pH, density, biological transduction stage, and later across multiple serotypes and mixed biological media. Reverse transcriptase (RT)-polymerase chain reaction (PCR) was used to quantify virus concentration. Subsequent experiments determined the pH-dependent exchange rate and optimized CEST saturation schemes for AAV contrast detection at 7 T. Results: NMR-CEST experiments revealed CEST contrast up to 52% for AAV2 viral capsids between 0.6-0.8 ppm. Evaluation of CEST contrast generated by AAV2 demonstrates high levels of CEST contrast across a variety of chemical environments, concentrations, and saturation schemes. AAV2 CEST contrast displayed significant positive correlations with capsid density (R2>0.99, P<0.001), pH (R2=0.97, P=0.01), and viral titer per cell count (R2=0.92, P<0.001). Transition to a preclinical field strength yielded up to 11.8% CEST contrast following optimization of saturation parameters. Conclusion: AAV2 viral capsids exhibit strong capacity as an endogenous CEST contrast agent and can potentially be used for monitoring and evaluation of AAV vector-mediated gene therapy protocols.

Multi-organ fibrosis among end stage renal disease (ESRD) patients cannot be explained by uremia alone. Despite mitigation of thrombosis during hemodialysis (HD), subsequent platelet dysfunction and tissue dysregulation are less understood. We comprehensively profiled plasma and platelets from ESRD patients before and after HD to examine HD-modulation of platelets beyond thrombotic activation. Basal plasma levels of proteolytic regulators and fibrotic factors were elevated in ESRD patients compared to healthy controls, with isoform-specific changes during HD. Platelet lysate (PL) RNA transcripts for growth and coagulative factors were elevated post-HD, with upregulation correlated to HD vintage. Platelet secretome correlations to plasma factors reveal acutely induced pro-fibrotic platelet phenotypes in ESRD patients during HD characterized by preferentially enhanced proteolytic enzyme translation and secretion, platelet contribution to inflammatory response, and increasing platelet dysfunction with blood flow rate (BFR) and Vintage. Compensatory mechanisms of increased platelet growth factor synthesis with acute plasma matrix metalloproteinase (MMP) and tissue inhibitor of MMPs (TIMP) increases show short-term mode-switching between dialysis sessions leading to long-term pro-fibrotic bias. Chronic pro-fibrotic adaptation of platelet synthesis were observed through changes in alpha-granule- and lysosome-filling behaviors. We conclude that chronic and acute platelet responses to HD contribute to a pro-fibrotic milieu in ESRD.