Extracellular vesicles (EVs) actively participate in intercellular communication and pathological processes. Studying the molecular signatures of EVs is the key to reveal their biological functions and clinical values, which, however, is greatly hindered by their sub-100-nm dimensions, the low quantities of biomolecules each EV carries, and the large population heterogeneity. Here, we report the single-EV Flow Cytometry Analysis technique that realizes single EV counting and phenotyping in a conventional flow cytometer for the first time, enabled by Target-Initiated Engineering (TIE) of DNA nanostructures on each EV. By illuminating multiple markers on single EVs, we reveal statistically significant differences among the molecular signatures of EVs originated from several breast cancer cell lines, and successfully recognize the cancer cell-derived EVs among the heterogeneous EV populations. Thus, our approach holds great potential for various biological and biomedical applications.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the second leading cause of cancer mortality worldwide. Heterogeneity of clinical conditions contributes to the complex management of care for patients with advanced HCC. Recently, the treatment landscape for advanced HCC has expanded rapidly, with the additional FDA approvals of several oral tyrosine kinase inhibitors (lenvatinib, regorafenib, and cabozantinib), as well as immunotherapies such as immune check point inhibitors (nivolumab and pembrolizumab) and the monoclonal IgG1 antibody, ramucirumab. This expansion has generated a need for novel treatment sequencing strategies in this patient population. In light of these developments, an evaluation of the impact of FDA-approved therapeutics on patient-centered outcomes such as health-related quality of life (HRQoL) is warranted. An increased understanding of HRQoL in patients included in advanced HCC clinical trials could potentially help physician decision-making for treatment sequencing in patients with advanced HCC.

IMPORTANCE National guidelines recommend against continuous pulse oximetry use for hospitalized children with bronchiolitis who are not receiving supplemental oxygen, yet guidelinediscordant use remains high. OBJECTIVES To evaluate deimplementation outcomes of educational outreach and audit and feedback strategies aiming to reduce guideline-discordant continuous pulse oximetry use in children hospitalized with bronchiolitis who are not receiving supplemental oxygen. DESIGN, SETTING, AND PARTICIPANTS A nonrandomized clinical single-group deimplementation trial was conducted in 14 non-intensive care units in 5 freestanding children's hospitals and 1 community hospital from December 1, 2019, through March 14, 2020, among 847 nurses and physicians caring for hospitalized children with bronchiolitis who were not receiving supplemental oxygen. INTERVENTIONS Educational outreach focused on communicating details of the existing guidelines and evidence. Audit and feedback strategies included 2 formats: (1) weekly aggregate data feedback to multidisciplinary teams with review of unit-level and hospital-level use of continuous pulse oximetry, and (2) real-time 1:1 feedback to clinicians when guideline-discordant continuous pulse oximetry use was discovered during in-person data audits. MAIN OUTCOMES AND MEASURES Clinician ratings of acceptability, appropriateness, feasibility, and perceived safety were assessed using a questionnaire. Guideline-discordant continuous pulse oximetry use in hospitalized children was measured using direct observation of a convenience sample of patients with bronchiolitis who were not receiving supplemental oxygen. RESULTS A total of 847 of 1193 eligible clinicians (695 women [82.1%]) responded to a Likert scalebased questionnaire (71% response rate). Most respondents rated the deimplementation strategies of education and audit and feedback as acceptable (education, 435 of 474 [92%]; audit and feedback, 615 of 664 [93%]), appropriate (education, 457 of 474 [96%]; audit and feedback, 622 of 664 [94%]), feasible (education, 424 of 474 [89%]; audit and feedback, 557 of 664 [84%]), and safe (803 of 847 [95%]). Sites collected 1051 audit observations (range, 47-403 per site) on 709 unique patient admissions (range, 31-251 per site) during a 3.5-month period of continuous pulse oximetry use in children with bronchiolitis not receiving supplemental oxygen, which were compared with 579 observations (range, 57-154 per site) from the same hospitals during the baseline 4-month period (prior season) to determine whether the strategies were associated with a reduction in use. (continued) Key Points Question Are audit and feedback strategies and educational outreach associated with clinician perceptions of the feasibility, acceptability, appropriateness, and safety of continuous pulse oximetry use in children hospitalized with bronchiolitis who are not receiving supplemental oxygen (guideline-discordant use)? Findings In this 6-hospital single-group nonrandomized clinical trial, 847 nurses and physicians highl...

MicroRNAs (miRNAs) are small RNAs that bind to mRNA targets and regulate their translation. A functional study of miRNAs and exploration of their utility as disease markers require miRNA extraction from biological samples, which contain large amounts of interfering compounds for downstream RNA identification and quantification. The most common extraction methods employ silica columns or the TRIzol reagent but give out low recovery for small RNAs probably due to their short strand lengths. Herein, we fabricated the titanium dioxide nanofibers using electrospinning to facilitate miRNA extraction and developed the optimal buffer conditions to improve miRNA recovery from biological matrices of cell lysate and serum. We found that our TiO fibers could obtain a recovery of 18.0 ± 3.6% for miRNA fibers while carrying out the extraction in the more complex medium of cell lysate, much higher than the 0.02 ± 0.0001% recovery from the commercial kit. The much improved extraction of miRNAs from our fibers could be originated from the strong coordination between TiO and RNA's phosphate backbone. In addition, the binding, washing, and elution buffers judiciously developed in the present study can achieve selective extraction of small RNA shorter than 500 nucleotides in length. Our results demonstrate that TiO nanofibers can work as a valuable tool for extraction of miRNAs from biological samples with high recovery. Graphical abstract Schematic for extraction of small RNAs using TiO nanofibers.

Extracellular vesicles (EVs) actively participate in intercellular communication and pathological processes. Studying the molecular signatures of EVs is key to reveal their biological functions and clinical values,w hich, however, is greatly hindered by their sub-100 nm dimensions,t he low quantities of biomolecules each EV carries,a nd the large population heterogeneity.N ow,s ingle-EV flow cytometry analysis is introduced to realizes ingle EV counting and phenotyping in aconventional flowcytometer for the first time, enabled by target-initiated engineering (TIE) of DNAn anostructures on each EV.B yi lluminating multiple markers on single EVs,s tatistically significant differences are revealed among the molecular signatures of EVs originating from several breast cancer cell lines,and the cancer cell-derived EVs among the heterogeneous EV populations are successfully recognized. Thus,o ur approachh olds great potential for various biological and biomedical applications.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.