An intronic RNA element modulates Factor VIII exon-16 splicing.

Pathogenic variants in the human Factor VIII (F8) gene cause Hemophilia A (HA). Here, we investigated the impact of 97 HA-causing single-nucleotide variants on the splicing of 11 exons from F8. For the majority of F8 exons, splicing was insensitive to the presence of HA-causing variants. However, splicing of several exons, including exon-16, was impacted by variants predicted to alter exonic splicing regulatory sequences. Using exon-16 as a model, we investigated the structure-function relationship of HA-causing variants on splicing. Intriguingly, RNA chemical probing analyses revealed a three-way junction structure at the 3'-end of intron-15 (TWJ-3-15) capable of sequestering the polypyrimidine tract. We discovered antisense oligonucleotides (ASOs) targeting TWJ-3-15 partially rescue splicing-deficient exon-16 variants by increasing accessibility of the polypyrimidine tract. The apical stem loop region of TWJ-3-15 also contains two hnRNPA1-dependent intronic splicing silencers (ISSs). ASOs blocking these ISSs also partially rescued splicing. When used in combination, ASOs targeting both the ISSs and the region sequestering the polypyrimidine tract, fully rescue pre-mRNA splicing of multiple HA-linked variants of exon-16. Together, our data reveal a putative RNA structure that sensitizes F8 exon-16 to aberrant splicing.

Rescue of blood coagulation Factor VIII exon-16 mis-splicing by antisense oligonucleotides.

The human Factor VIII ( F8 ) protein is essential for the blood coagulation cascade and specific F8 mutations cause the rare bleeding disorder Hemophilia A (HA). Here, we investigated the impact of HA-causing single-nucleotide mutations on F8 pre-mRNA splicing. We found that 14/97 (∼14.4%) coding sequence mutations tested in our study induced exon skipping. Splicing patterns of 4/11 (∼36.4%) F8 exons tested were especially sensitive to the presence of common disease-causing mutations. RNA-chemical probing analyses revealed a three-way junction structure at the 3' end of intron 15 (TWJ-3-15). TWJ-3-15 sequesters the polypyrimidine tract, a key determinant of 3' splice site strength. Using exon-16 of the F8 gene as a model, we designed specific antisense oligonucleotides (ASOs) that target TWJ-3-15 and identified three that promote the splicing of F8 exon-16. Interaction of TWJ-3-15 with ASOs increases accessibility of the polypyrimidine tract and inhibits the binding of hnRNPA1-dependent splicing silencing factors. Moreover, ASOs targeting TWJ-3-15 rescue diverse splicing-sensitive HA-causing mutations, most of which are distal to the 3' splice site being impacted. The TWJ-3-15 structure and its effect on mRNA splicing provide a model for HA etiology in patients harboring specific F8 mutations and provide a framework for precision RNA-based HA therapies.

An N-Cadherin 2 expressing epithelial cell subpopulation predicts response to surgery, chemotherapy and immunotherapy in bladder cancer.

Neoadjuvant chemotherapy (NAC) prior to surgery and immune checkpoint therapy (ICT) have revolutionized bladder cancer management. However, stratification of patients that would benefit most from these modalities remains a major clinical challenge. Here, we combine single nuclei RNA sequencing with spatial transcriptomics and single-cell resolution spatial proteomic analysis of human bladder cancer to identify an epithelial subpopulation with therapeutic response prediction ability. These cells express Cadherin 12 (CDH12, N-Cadherin 2), catenins, and other epithelial markers. CDH12-enriched tumors define patients with poor outcome following surgery with or without NAC. In contrast, CDH12-enriched tumors exhibit superior response to ICT. In all settings, patient stratification by tumor CDH12 enrichment offers better prediction of outcome than currently established bladder cancer subtypes. Molecularly, the CDH12 population resembles an undifferentiated state with inherently aggressive biology including chemoresistance, likely mediated through progenitor-like gene expression and fibroblast activation. CDH12-enriched cells express PD-L1 and PD-L2 and co-localize with exhausted T-cells, possibly mediated through CD49a (ITGA1), providing one explanation for ICT efficacy in these tumors. Altogether, this study describes a cancer cell population with an intriguing diametric response to major bladder cancer therapeutics. Importantly, it also provides a compelling framework for designing biomarker-guided clinical trials.

The Effects of Immersion and Real-World Distractions on Virtual Social Interactions.

This study explores the independent and joint effects of immersion and real-world distractions (a ringing cell phone) on cognitive (i.e., recognition and recall), affective valence, and interpersonal outcomes (i.e., interpersonal liking and communication satisfaction) as well as general feelings of presence (social presence and telepresence) during a virtual experience. Participants interacted with a virtual agent in an immersive virtual environment or nonimmersive virtual environment under three different levels of real-world distractions (i.e., no distraction, passively being exposed to the sound of a ringing cell phone, and actively responding to ringing cell phone). Increased immersion had a positive effect on telepresence, but a negative effect on recognition and recall; immersion did not have a significant effect on social presence. Real-world distractions had a negative effect on recognition, recall, and social presence, but did not affect telepresence or affective valence. Participants who were actively distracted performed more poorly on the recall measure and reported lower levels of social presence than their passively distracted counterparts. These findings suggest that (a) increased immersion will not uniformly improve social virtual reality experiences and (b) more research is needed on whether and how real-world events should be integrated into virtual environments.

A Systematic Review of Social Presence: Definition, Antecedents, and Implications.

Social presence, or the feeling of being there with a "real" person, is a crucial component of interactions that take place in virtual reality. This paper reviews the concept, antecedents, and implications of social presence, with a focus on the literature regarding the predictors of social presence. The article begins by exploring the concept of social presence, distinguishing it from two other dimensions of presence-telepresence and self-presence. After establishing the definition of social presence, the article offers a systematic review of 233 separate findings identified from 152 studies that investigate the factors (i.e., immersive qualities, contextual differences, and individual psychological traits) that predict social presence. Finally, the paper discusses the implications of heightened social presence and when it does and does not enhance one's experience in a virtual environment.

Addisonian pigmentation of the oral mucosa.

Cutaneous pigmentation is a hallmark of Addison disease. When present, the hyperpigmentation generally localizes to sun-exposed surfaces. This case highlights a less well-recognized cutaneous feature that is pathognomonic for the disease: oral mucous membrane hyperpigmentation. We describe this unique type of discoloration in detail and contrast it with other forms of oral pigmentation.