Long-Term Characteristics of Human-Derived Biliary Organoids under a Single Continuous Culture Condition

Organoids have been used to investigate the three-dimensional (3D) organization and function of their respective organs. These self-organizing 3D structures offer a distinct advantage over traditional two-dimensional (2D) culture techniques by creating a more physiologically relevant milieu to study complex biological systems. The goal of this study was to determine the feasibility of establishing organoids from various pediatric liver diseases and characterize the long-term evolution of cholangiocyte organoids (chol-orgs) under a single continuous culture condition. We established chol-orgs from 10 different liver conditions and characterized their multicellular organization into complex epithelial structures through budding, merging, and lumen formation. Immunofluorescent staining, electron microscopy, and single-nucleus RNA (snRNA-seq) sequencing confirmed the cholangiocytic nature of the chol-orgs. There were significant cell population differences in the transcript profiles of two-dimensional and organoid cultures based on snRNA-seq. Our study provides an approach for the generation and long-term maintenance of chol-orgs from various pediatric liver diseases under a single continuous culture condition

Barriers to recruitment to an orthopaedic randomized controlled trial comparing two surgical procedures for ankle arthritis

Aims A multicentre, randomized, clinician-led, pragmatic, parallel-group orthopaedic trial of two surgical procedures was set up to obtain high-quality evidence of effectiveness. However, the trial faced recruitment challenges and struggled to maintain recruitment rates over 30%, although this is not unusual for surgical trials. We conducted a qualitative study with the aim of gathering information about recruitment practices to identify barriers to patient consent and participation to an orthopaedic trial. Methods We collected 11 audio recordings of recruitment appointments and interviews of research team members (principal investigators and research nurses) from five hospitals involved in recruitment to an orthopaedic trial. We analyzed the qualitative data sets thematically with the aim of identifying aspects of informed consent and information provision that was either unclear, disrupted, or hindered trial recruitment. Results Recruiters faced four common obstacles when recruiting to a surgical orthopaedic trial: patient preferences for an intervention; a complex recruitment pathway; various logistical issues; and conflicting views on equipoise. Clinicians expressed concerns that the trial may not show significant differences in the treatments, validating their equipoise. However, they experienced role conflicts due to their own preference and perceived patient preference for an intervention arm. Conclusion This study provided initial information about barriers to recruitment to an orthopaedic randomized controlled trial. We shared these findings in an all-site investigators’ meeting and encouraged researchers to find solutions to identified barriers; this led to the successful completion of recruitment. Complex trials may benefit for using of a mixed-methods approach to mitigate against recruitment failure, and to improve patient participation and informed consent

DNAJB1-PRKACA in HEK293T cells induces LINC00473 overexpression that depends on PKA signaling.

Fibrolamellar carcinoma (FLC) is a primary liver cancer that most commonly arises in adolescents and young adults in a background of normal liver tissue and has a poor prognosis due to lack of effective chemotherapeutic agents. The DNAJB1-PRKACA gene fusion (DP) has been reported in the majority of FLC tumors; however, its oncogenic mechanisms remain unclear. Given the paucity of cellular models, in particular FLC tumor cell lines, we hypothesized that engineering the DP fusion gene in HEK293T cells would provide insight into the cellular effects of the fusion gene. We used CRISPR/Cas9 to engineer HEK293T clones expressing DP fusion gene (HEK-DP) and performed transcriptomic, proteomic, and mitochondrial studies to characterize this cellular model. Proteomic analysis of DP interacting partners identified mitochondrial proteins as well as proteins in other subcellular compartments. HEK-DP cells demonstrated significantly elevated mitochondrial fission, which suggests a role for DP in altering mitochondrial dynamics. Transcriptomic analysis of HEK-DP cells revealed a significant increase in LINC00473 expression, similar to what has been observed in primary FLC samples. LINC00473 overexpression was reversible with siRNA targeting of PRKACA as well as pharmacologic targeting of PKA and Hsp40 in HEK-DP cells. Therefore, our model suggests that LINC00473 is a candidate marker for DP activity