Circulating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detection, prognosis and monitoring of cancer. They can exist in the bloodstream incorporated into extracellular vesicles (EVs) and ribonucleoprotein complexes. However, it is still debated if EVs contain biologically meaningful amounts of miRNAs and may provide a better source of miRNA biomarkers than whole plasma. The aim of this study was to systematically compare the diagnostic potential of prostate cancer-associated miRNAs in whole plasma and in plasma EVs. RNA was isolated from whole plasma and plasma EV samples from a well characterised cohort of 50 patient with prostate cancer (PC) and 22 patients with benign prostatic hyperplasia (BPH). Nine miRNAs known to have a diagnostic potential for PC in cell-free blood were quantified by RT-qPCR and the relative quantities were compared between patients with PC and BPH and between PC patients with Gleason score ≥ 8 and ≤6. Only a small fraction of the total cell-free miRNA was recovered from the plasma EVs, however the EV-incorporated and whole plasma cell-free miRNA profiles were clearly different. Four of the miRNAs analysed showed a diagnostic potential in our patient cohort. MiR-375 could differentiate between PC and BPH patients when analysed in the whole plasma, while miR-200c-3p and miR-21-5p performed better when analysed in plasma EVs. EV-incorporated but not whole plasma Let-7a-5p level could distinguish PC patients with Gleason score ≥ 8 vs ≤6. This study demonstrates that for some miRNA biomarkers EVs provide a more consistent source of RNA than whole plasma, while other miRNAs show better diagnostic performance when tested in the whole plasma.

Extracellular vesicles are small membrane-bound structures that are released by cells and play important roles in intercellular communication garnering significant attention in scientific society recently due to their potential as diagnostic and therapeutic tools. However, separating EVs from large-volume samples remains a challenge due to their small size and low concentration. In this manuscript, we presented a novel method for separating polystyrene beads as control and extracellular vesicles from large sample volumes using bifurcated asymmetric field flow fractionation in PDMS-free microfluidic devices. Separation characteristics were evaluated using the control system of polystyrene bead mix, which offers up to 3.7X enrichment of EV-sized beads. Furthermore, in the EV-sample from bioreactor culture media, we observed a notable population distribution shift of extracellular vesicles. Herein presented novel PDMS-free microfluidic device fabrication protocol resulted in devices with reduced EV-loss compared to size-exclusion columns. This method represented an improvement over the current state of the art in terms of EV separation from large sample volumes through the use of novel field flow fractionation design.

Extracellular vesicles (EV) have many attributes important for biomedicine; however, current EV isolation methods require long multi-step protocols that generally involve bulky equipment that cannot be easily translated to clinics. Our aim was to design a new cyclic olefin copolymer–off-stoichiometry thiol-ene (COC–OSTE) asymmetric flow field fractionation microfluidic device that could isolate EV from high-volume samples in a simple and efficient manner. We tested the device with large volumes of urine and conditioned cell media samples, and compared it with the two most commonly used EV isolation methods. Our device was able to separate particles by size and buoyancy, and the attained size distribution was significantly smaller than other methods. This would allow for targeting EV size fractions of interest in the future. However, the results were sample dependent, with some samples showing significant improvement over the current EV separation methods. We present a novel design for a COC–OSTE microfluidic device, based on bifurcating asymmetric flow field-flow fractionation (A4F) technology, which is able to isolate EV from large volume samples in a simple, continuous-flow manner. Its potential to be mass-manufactured increases the chances of implementing EV isolation in a clinical or industry-friendly setting, which requires high repeatability and throughput.

Study question: Does the oestrogen receptor isoform, ER46, contribute to regulation of endometrial function? Summary answer: ER46 is expressed in endometrial tissues during the proliferative and secretory phases and is the predominant ERα isoform in first trimester decidua. ER46 is abundantly expressed in uterine NK (uNK) cells and localised to the cell membrane. Activation of ER46 regulates the function of human uNK cells by increasing cell motility. What is known already: Oestrogens acting via their cognate receptors are essential regulators of endometrial function and play key roles in establishment of pregnancy. ER46 is a 46kDa truncated isoform of full length ERα (ER66, encoded by ESR1 ) that contains both ligand and DNA binding domains. Expression of ER46 in human endometrium has not been investigated previously. ER46 is located at the cell membrane of peripheral blood leukocytes and mediates rapid responses to oestrogens. UNK cells are a phenotypically distinct (CD56 ^bright CD16 ^- ) population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. We have shown that oestrogens stimulate rapid increases in uNK cell motility. Previous characterisation of uNK cells suggests they are ER66-negative but expression of ER46 has not been characterised. We hypothesise that uNK cells express ER46 and that rapid responses to oestrogens are mediated via this receptor. Study design, size, duration: This laboratory-based study used primary human endometrial (n=24) and decidual tissue biopsies (n=30) as well as uNK cells which were freshly isolated from first trimester human decidua (n=18). Participants/materials, setting, methods: Primary human endometrial and first trimester decidual tissue biopsies were collected using methods approved by the local institutional ethics committee (LREC/05/51104/12 and LREC/10/51402/59). The expression of oestrogen receptors (ER66, ER46 and ERβ) was assessed by qPCR, western blot and immunohistochemistry. Uterine Natural Killer (uNK) cells were isolated from first trimester human decidua by magnetic bead sorting. Cell motility of uNK cells was measured by live cell imaging: cells were treated with oestradiol (E2)-BSA (10nM equivalent), the ERβ-selective agonist 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; 10nM) or vehicle control (DMSO). Main results and the role of chance: ER46 was detected in proliferative and secretory phase tissues and was the predominant ERα isoform in first trimester decidua samples. Immunohistochemistry revealed ER46 was co-localised with ER66 in cell nuclei during the proliferative phase but detected in both the cytoplasm and cell membrane of stromal cells in the secretory phase and in decidua. Triple immunofluorescence staining of decidua tissues identified expression of ER46 in the cell membrane of CD56-positive uNK cells which were otherwise ER66-negative. Profiling of isolated uNK cells confirmed expression ER46 and localised ER46 protein to the cell membrane. Functional analysis of isolated uNK cells using live cell imaging demonstrated that activation of ER46 with E2-BSA significantly increased uNK cell motility. Limitations, reasons for caution: Expression patterns in endometrial tissue was only determined using samples from proliferative and secretory phases. Assessment of first trimester decidua samples was from a range of gestational ages which may have precluded insights into gestation specific changes in these tissues. Our results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in situ . Wider implications of the findings: E2 is an essential regulator of reproductive competence. This study provides the first evidence for expression of ER46 in human endometrium and decidua of early pregnancy. We describe a mechanism for regulating the function of human uNK cells via expression of ER46 and demonstrate that selective targeting with E2-BSA regulates uNK cell motility. These novel findings identify a role for ER46 in human endometrium and provide unique insight into the importance of membrane-initiated signalling in modulating the impact of E2 on uNK cell function in women. Study funding/competing interest(s): These studies were supported by MRC Programme Grants G1100356/1 and MR/N024524/1 to PTKS. HODC was supported by MRC grant G1002033.

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

Background Netherton syndrome (NS) is a rare congenital barrier disorder caused by pathogenic variation in SPINK5, resulting in severe epidermal dysfunction and immune dysregulation. Although dupilumab is increasingly used in NS, molecular data describing treatment-associated tissue responses remain limited, particularly in genetically complex presentations. Methods We investigated treatment-associated molecular trajectories during IL-4Rα blockade in two siblings with NS carrying heterozygous compound SPINK5 (c.2468dup & c.2243 A > G) and FLG (p.Arg501*) variants. Longitudinal whole-exome sequencing and paired bulk RNA sequencing of peripheral blood and lesional skin were performed before and after six months of dupilumab therapy. Transcriptomic changes were evaluated using effect-size– and rank-based approaches and contextualized through integration with publicly available NS reference datasets. Results Across both compartments, post-treatment transcriptional profiles exhibit coordinated shifts toward healthy-control–like expression states. Blood transcriptomes show reduced inflammatory, metabolic, and proliferative signatures alongside modulation of FURIN and increase expression of PAPPA, LPAR5, CELSR1, and ARHGEF19. Skin transcriptomes transition from neurosensory and follicular programs toward lipid metabolic organization, keratinocyte differentiation, and cornified-envelope–associated pathways. Cross-compartment integration highlights shared treatment-associated transcripts, including ADAM23, and candidate markers of immune–epithelial recalibration. Conclusions Together, this exploratory dual-compartment longitudinal analysis provides rare molecular insight into biologic treatment response in a genetically complex NS context and establishes a foundational reference for future genotype-informed and biomarker-oriented studies.