A novel sorting signal for RNA packaging into small extracellular vesicles.

Extracellular vesicles (EVs) play a critical role in the transport of functional RNAs to target recipient cells in numerous physiological processes. The RNA profiles present in EVs differed significantly from those in the originating cells, suggesting selective and active loading of specific RNAs into EVs. Small EVs (sEVs) obtained by stepwise ultracentrifugation have been reported to contain non-sEV components. Analysis of sEVs separated from non-sEVs components revealed that microRNAs may not be released by sEVs. This has raised interest in other RNA types, such as mRNA, which may be functional molecules released by sEVs. However, the molecular mechanisms underlying selective loading of mRNA into sEVs remain unclear. Here, we show that the part of 3' untranslated region (UTR) sequence of RAB13 selectively enriches RNA in sEVs and serves as an RNA signal for loading into sEVs. Our results demonstrate that RAB13 is the most enriched RNA in sEVs, and this enrichment is primarily driven by its 3'UTR sequence. These findings highlight the potential of the RAB13 3'UTR sequence as an RNA signal that enables the loading of target RNA into sEVs. This technology has the potential to improve EV-based drug delivery and other applications.

Association Between Dietary Potassium Intake Estimated From Multiple 24-Hour Urine Collections and Serum Potassium in Patients With CKD.

Introduction: Limited and inconclusive evidence for the association of dietary potassium intake with serum potassium in chronic kidney disease (CKD) patients have been shown, though restricting dietary potassium has been recommended for CKD patients to prevent hyperkalemia. Multiple 24-hour urine collections are necessary to adequately assess potassium intake. We investigated associations of 24-hour urinary potassium excretion (UKV) with serum potassium in CKD outpatients based on multiple 24-hour urine collections. Methods: This retrospective cohort study was based on outpatients with CKD stages G3 to G5, median age of 72.0 years; and median follow-up of 3.9 months and 8.9 months, respectively, for analyses using 3-time measurement (N = 290 and 870 observations) and 7-time measurements (N = 220 and 1540 observations). The outcome was serum potassium. Results: Multivariable-adjusted mean difference in serum potassium (mEq/l) and odds ratio of hyperkalemia per 10 mEq/d increase in UKV were, respectively, 0.12 (95% confidence interval [CI]: 0.09-0.15) and 2.15 (1.70-2.73) in generalized estimating equations (GEEs) with 3-time measurements. The mean difference became more pronounced as CKD stages progressed: 0.08 (0.05-0.12), 0.12 (0.08-0.16), and 0.16 (0.12-0.20) for CKD G3, G4, and G5. Similar results were obtained from analyses using 7-time measurements and hierarchical Bayesian measurement error models treating measurement error of UKV adequately. Conclusion: We suggest significant but weak associations (R2: 0.08, 0.14, and 0.18 for CKD G3, G4, and G5) between serum potassium and dietary potassium intake estimated by multiple 24-hour urine collections in CKD patients. Further studies are needed to validate nutritional and clinical aspects of the associations.

Highly sensitive and non-disruptive detection of residual undifferentiated cells by measuring miRNAs in culture supernatant.

The clinical usage of induced pluripotent stem cell (iPSC)-derived regenerative medicine products is limited by the possibility of residual undifferentiated cells forming tumours after transplantation. Most of the existing quality control tests involve crushing of cells. As a result, the cells to be transplanted cannot be directly tested, thereby increasing the cost of transplantation. Therefore, we tested a highly sensitive and non-disruptive quality-testing method that involves measuring microRNAs (miRNAs) in culture supernatants released by cells. By measuring miR-302b in the culture supernatant, residual iPSCs were detected with higher sensitivity than by measuring LIN28 (Lin-28 Homolog A) in the cells. To use this method, we also monitored the progression of differentiation. Our novel highly sensitive and non-disruptive method for detecting residual undifferentiated cells will contribute to reducing the manufacturing cost of iPSC-derived products and improving the safety of transplantation.

Hybrid double-spiral microfluidic chip for RBC-lysis-free enrichment of rare cells from whole blood.

Drug selection and treatment monitoring via minimally invasive liquid biopsy using circulating tumor cells (CTCs) are expected to be realized in the near future. For clinical applications of CTCs, simple, high-throughput, single-step CTC isolation from whole blood without red blood cell (RBC) lysis and centrifugation remains a crucial challenge. In this study, we developed a novel cancer cell separation chip, "hybrid double-spiral chip", that involves the serial combination of two different Dean flow fractionation (DFF) separation modes of half and full Dean cycles, which is the hybrid DFF separation mode for ultra-high-throughput blood processing at high precision and size-resolution separation. The chip allows fast processing of 5 mL whole blood within 30 min without RBC lysis and centrifugation. RBC and white blood cell (WBC) depletion rates of over 99.9% and 99%, respectively, were achieved. The average recovery rate of spiked A549 cancer cells was 87% with as low as 200 cells in 5 mL blood. The device can achieve serial reduction in the number of cells from approximately 1010 cells of whole blood to 108 cells, and subsequently to an order of 106 cells. The developed method can be combined with measurements of all recovered cells using imaging flow cytometry. As proof of concept, CTCs were successfully enriched and enumerated from the blood of metastatic breast cancer patients (N = 10, 1-69 CTCs per 5 mL) and metastatic prostate cancer patients (N = 10, 1-39 CTCs per 5 mL). We believe that the developed method will be beneficial for automated clinical analysis of rare CTCs from whole blood.

Whole-mount single molecule FISH method for zebrafish embryo.

Noise in gene expression renders cells more adaptable to changing environment by imposing phenotypic and functional heterogeneity on genetically identical individual cells. Hence, quantitative measurement of noise in gene expression is essential for the study of biological processes in cells. Currently, there are two complementary methods for quantitatively measuring noise in gene expression at the single cell level: single molecule FISH (smFISH) and single cell qRT-PCR (or single cell RNA-seq). While smFISH has been developed for culture cells, tissue sections and whole-mount invertebrate organisms, the method has not been reported for whole-mount vertebrate organisms. Here, we report an smFISH method that is suitable for whole-mount zebrafish embryo, a popular vertebrate model organism for the studies of development, physiology and disease. We show the detection of individual transcripts for several cell-type specific and ubiquitously expressed genes at the single cell level in whole-mount zebrafish embryo. We also demonstrate that the method can be adapted to detect two different genes in individual cells simultaneously. The whole-mount smFISH method described in this report is expected to facilitate the study of noise in gene expression and its role in zebrafish, a vertebrate animal model relevant to human biology.