Improved detection of DNA replication fork-associated proteins.

Innovative methods to retrieve proteins associated with actively replicating DNA have provided a glimpse into the molecular dynamics of replication fork stalling. We report that a combination of density-based replisome enrichment by isolating proteins on nascent DNA (iPOND2) and label-free quantitative mass spectrometry (iPOND2-DRIPPER) substantially increases both replication factor yields and the dynamic range of protein quantification. Replication protein abundance in retrieved nascent DNA is elevated up to 300-fold over post-replicative controls, and recruitment of replication stress factors upon fork stalling is observed at similar levels. The increased sensitivity of iPOND2-DRIPPER permits direct measurement of ubiquitination events without intervening retrieval of diglycine tryptic fragments of ubiquitin. Using this approach, we find that stalled replisomes stimulate the recruitment of a diverse cohort of DNA repair factors, including those associated with poly-K63-ubiquitination. Finally, we uncover the temporally controlled association of stalled replisomes with nuclear pore complex components and nuclear cytoskeleton networks.

Oncolytic virus-driven immune remodeling revealed in mouse medulloblastomas at single cell resolution.

Oncolytic viruses, modified for tumor-restricted infection, are a promising cancer immunotherapeutic, yet much remains to be understood about factors driving their activity and outcome in the tumor microenvironment. Here, we report that oncolytic herpes simplex virus C134, previously found to exert T cell-dependent efficacy in mouse models of glioblastoma, exerts T cell-independent efficacy in mouse models of medulloblastoma, indicating this oncolytic virus uses different mechanisms in different tumors. We investigated C134's behavior in mouse medulloblastomas, using single cell RNA sequencing to map C134-induced gene expression changes across cell types, timepoints, and medulloblastoma subgroup models at whole-transcriptome resolution. Our work details substantial oncolytic virus-induced transcriptional remodeling of medulloblastoma-infiltrating immune cells, 10 subpopulations of monocytes and macrophages collectively demonstrating M1-like responses to C134, and suggests C134 be investigated as a potential new therapy for medulloblastoma.

Bi-allelic MCM10 variants associated with immune dysfunction and cardiomyopathy cause telomere shortening.

Minichromosome maintenance protein 10 (MCM10) is essential for eukaryotic DNA replication. Here, we describe compound heterozygous MCM10 variants in patients with distinctive, but overlapping, clinical phenotypes: natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of MCM10-associated disease, we modeled these variants in human cell lines. MCM10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of MCM10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in MCM10-deficient cells require endonucleolytic processing by MUS81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic variants in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes observed in both NKD and RCM patients.

An Undergraduate Surgery Interest Group: Introducing Premedical Students to the Practice of Surgery.

PURPOSE: Early exposure and surgical mentorship can augment interest in surgery. We evaluate the effect and feasibility of offering education and mentorship opportunities in surgery to premedical students at our institution through an undergraduate surgery interest group (USIG). MATERIALS AND METHODS: We conducted a 1-year assessment of our USIG and reviewed its organizational structure, funding resources, media promotion, and educational activities. Our USIG hosted introductory-level surgical skills workshops, guest lectures by surgeons, and various facility tours. To assess participants' interests, as well as the influences on them to pursue a surgical profession, we analyzed pre- and post-event questionnaires. Similar questionnaires were completed by medical students in our medical student surgery interest group to compare any differences in perception between premedical and medical students. RESULTS: Our USIG currently has 378 undergraduate student members, with a higher proportion of women than in our medical student surgery interest group (P < 0.003). Neurosurgery was the most popular career choice among undergraduate participants. Participants reported the highest satisfaction with suturing and high-fidelity trauma surgery skills workshops. Undergraduate participants indicated that their intrinsic interest in the sciences is the highest motivation to pursue a surgical profession. Resident lifestyle and social obligations of a surgical career were actually positive influences for undergraduate participants; in contrast, medical students viewed those variables as negative factors. CONCLUSION: Our USIG was met with enthusiasm by premedical students and faculty alike. Participation strengthened premedical students' interest in pursuing surgery and increased their understanding of the surgical profession. Early mentorship may positively influence premedical students' perception of surgical careers. USIG is economically feasible and time-efficient; we encourage other academic institutions and educators to consider investing in similar interest groups.