STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity.

Transcription is a dynamic process. To detect the dynamic relationship among protein clusters of RNA polymerase II and coactivators, gene loci, and transcriptional activity, we insert an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site. By optimizing the individual elements, we develop the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNA polymerase II and BRD4 are observed proximal to the transcription start site of Nanog when the gene is transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 tend to be located near the transcription start site, even without transcription activity. Thus, the STREAMING-tag system reveals the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding transcriptional regulation in living cells.

The Role of miR-217-5p in the Puromycin Aminonucleoside-Induced Morphological Change of Podocytes.

Podocytes, alternatively called glomerular epithelial cells, are terminally differentiated cells that wrap around glomerular capillaries and function as a part of the glomerular filtration barrier in the kidney. Therefore, podocyte injury with morphological alteration and detachment from glomerular capillaries leads to severe proteinuria and subsequent renal failure through glomerulosclerosis. Previous RNA sequencing analysis of primary rat podocytes exposed to puromycin aminonucleoside (PAN), a well-known experimental model of injured podocytes, identified several transcripts as being aberrantly expressed. However, how the expression of these transcripts is regulated remains unclear. MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally inhibit the expression of their target transcripts. In this study, using small RNA sequencing analysis, miR-217-5p was identified as the most upregulated transcript in PAN-treated rat podocytes. MiR-217-5p overexpression in E11 podocyte cells led to shrunken cells with abnormal actin cytoskeletons. Consistent with these changes in cell morphology, gene ontology (GO) enrichment analysis showed that interactive GO terms related to cell morphogenesis were enriched with the predicted targets of miR-217-5p. Of the predicted targets highly downregulated by PAN, Myosin 1d (Myo1d) is a nonmuscle myosin predicted to be involved in actin filament organization and thought to play a role in podocyte morphogenesis and injury. We demonstrated that miR-217-5p targets Myo1d by luciferase assays, qRT-PCR, and Western blotting. Furthermore, we showed that miR-217-5p was present in urine from PAN- but not saline-administrated rats. Taken together, our data suggest that miR-217-5p may serve as a therapeutic target and a biomarker for podocyte injury.

Association between the types of bystander cardiopulmonary resuscitation and the survival with good neurologic outcome of preschool pediatric out-of-hospital cardiac arrest cases in Japan: A propensity score matching analysis using an extended nationwide database.

Background: Pediatric out-of-hospital cardiac arrests (OHCAs) are frequently associated with a respiratory etiology. Despite the high proportion of preschool children with OHCAs, very few studies on this special population exist. This study characterizes the epidemiologic features of preschool pediatric OHCAs and analyzes the advantage of conventional (ventilations with chest compressions) bystander cardiopulmonary resuscitation (CPR) over compression-only bystander CPR (BCPR) on the one-month post-event neurological status of the patient. Methods: Japanese nationwide databases for all ambulance transport events and OHCAs occurring during a 4-year period between 2016 and 2019 were combined, totalling 3,608 patient events. Children ≤6-years-old were included; physician- and EMS-witnessed events, no prehospital resuscitation effort events, and neonatal patient events were excluded. Neurologically favorable 1-month survival rates were compared among groups using univariate and multivariate analyses before and after propensity score matching. Results: From the combined database, 2,882 pediatric OHCAs meeting selection criteria were categorized as no BCPR (984), compression-only BCPR (1,428), and conventional BCPR (470). The proportion of bystander-witnessed cases was low (22.3%). Most OHCA witnesses were family members (88.5%), and most OHCAs occurred at home (88.0%). The neurologically favorable 1-month survival rates were: no BCPR 2.4%, compression only, 3.2%, and conventional 6.6% (P < 0.01). Multivariate logistic regression analysis before and after matching showed that conventional BCPR was associated with higher neurologically favorable 1-month survival than compression-only BCPR. Subgroup analyses after matching demonstrated that conventional BCPR was associated with better outcomes in nonmedical (adjusted odds ratio; 95% confidence interval, 2.83; 1.09-7.32) and unwitnessed OHCA cases (3.42; 1.09-10.8). Conclusions: Conventional CPR is rarely performed by bystanders in preschool pediatric OHCA. However, conventional BCPR results in neurologically favorable outcomes in nonmedical and unwitnessed cases.