RTF2 controls replication repriming and ribonucleotide excision at the replisome.

DNA replication through a challenging genomic landscape is coordinated by the replisome, which must adjust to local conditions to provide appropriate replication speed and respond to lesions that hinder its progression. We have previously shown that proteasome shuttle proteins, DNA Damage Inducible 1 and 2 (DDI1/2), regulate Replication Termination Factor 2 (RTF2) levels at stalled replisomes, allowing fork stabilization and restart. Here, we show that during unperturbed replication, RTF2 regulates replisome localization of RNase H2, a heterotrimeric enzyme that removes RNA from RNA-DNA heteroduplexes. RTF2, like RNase H2, is essential for mammalian development and maintains normal replication speed. However, persistent RTF2 and RNase H2 at stalled replication forks prevent efficient replication restart, which is dependent on PRIM1, the primase component of DNA polymerase α-primase. Our data show a fundamental need for RTF2-dependent regulation of replication-coupled ribonucleotide removal and reveal the existence of PRIM1-mediated direct replication restart in mammalian cells.

RTF2 controls replication repriming and ribonucleotide excision at the replisome.

Genetic information is duplicated via the highly regulated process of DNA replication. The machinery coordinating this process, the replisome, encounters many challenges, including replication fork-stalling lesions that threaten the accurate and timely transmission of genetic information. Cells have multiple mechanisms to repair or bypass lesions that would otherwise compromise DNA replication1,2. We have previously shown that proteasome shuttle proteins, DNA Damage Inducible 1 and 2 (DDI1/2) function to regulate Replication Termination Factor 2 (RTF2) at the stalled replisome, allowing for replication fork stabilization and restart3. Here we show that RTF2 regulates replisome localization of RNase H2, a heterotrimeric enzyme responsible for removing RNA in the context of RNA-DNA heteroduplexes4-6. We show that during unperturbed DNA replication, RTF2, like RNase H2, is required to maintain normal replication fork speeds. However, persistent RTF2 and RNase H2 at stalled replication forks compromises the replication stress response, preventing efficient replication restart. Such restart is dependent on PRIM1, the primase component of DNA polymerase α-primase. Our data show a fundamental need for regulation of replication-coupled ribonucleotide incorporation during normal replication and the replication stress response that is achieved through RTF2. We also provide evidence for PRIM1 function in direct replication restart following replication stress in mammalian cells.

A Medical Student-Run Telehealth Primary Care Clinic During the COVID-19 Pandemic: Maintaining Care for the Underserved.

BACKGROUND: In this report, we outline our approach to implementing a hybrid in-person and virtual clinic model at a student-run free clinic (SRFC) during the COVID-19 pandemic. Individuals of low socioeconomic status (SES) are at an increased risk for COVID-19 infection and severe clinical outcomes. It is unclear if telehealth is a viable continuity of care enabler for the underserved. METHODS: The Weill Cornell Community Clinic (WCCC) implemented a novel telehealth clinic model to serve uninsured patients in May 2020. A phone survey of was conducted to assess WCCC patients access to technology needed for telehealth visits (eg, personal computers, smartphones). Patient no-show rates were retrospectively assessed for both in-person (pre-pandemic) and hybrid continuity of care models. RESULTS: The phone survey found that 90% of WCCC patients had access to technology needed for telehealth visits. In the 8 months following implementation of the hybrid model, telehealth and in-person no-show rates were 11% (14/128) and 15% (10/67) respectively; the combined hybrid no-show rate was 12% (24/195). For comparison, the in-person 2019 no-show rate was 23% (84/367). This study aligns with previous reports that telehealth improves patient attendance. CONCLUSION: Literature on the transition of SRFCs from in-person to telehealth care delivery models is limited. At the WCCC, the reduction in no-show rates supports the feasibility and benefits of adopting telehealth for the delivery of care to underserved patient populations. We believe the hybrid telehealth model described here is a viable model for other student run free clinics to increase access to care in low SES communities.

Peptide-TLR-7/8a conjugate vaccines chemically programmed for nanoparticle self-assembly enhance CD8 T-cell immunity to tumor antigens.

Personalized cancer vaccines targeting patient-specific neoantigens are a promising cancer treatment modality; however, neoantigen physicochemical variability can present challenges to manufacturing personalized cancer vaccines in an optimal format for inducing anticancer T cells. Here, we developed a vaccine platform (SNP-7/8a) based on charge-modified peptide-TLR-7/8a conjugates that are chemically programmed to self-assemble into nanoparticles of uniform size (~20 nm) irrespective of the peptide antigen composition. This approach provided precise loading of diverse peptide neoantigens linked to TLR-7/8a (adjuvant) in nanoparticles, which increased uptake by and activation of antigen-presenting cells that promote T-cell immunity. Vaccination of mice with SNP-7/8a using predicted neoantigens (n = 179) from three tumor models induced CD8 T cells against ~50% of neoantigens with high predicted MHC-I binding affinity and led to enhanced tumor clearance. SNP-7/8a delivering in silico-designed mock neoantigens also induced CD8 T cells in nonhuman primates. Altogether, SNP-7/8a is a generalizable approach for codelivering peptide antigens and adjuvants in nanoparticles for inducing anticancer T-cell immunity.

Impact of Polymer-TLR-7/8 Agonist (Adjuvant) Morphology on the Potency and Mechanism of CD8 T Cell Induction.

Small molecule Toll-like receptor-7 and -8 agonists (TLR-7/8a) can be used as vaccine adjuvants to induce CD8 T cell immunity but require formulations that prevent systemic toxicity and focus adjuvant activity in lymphoid tissues. Here, we covalently attached TLR-7/8a to polymers of varying composition, chain architecture and hydrodynamic behavior (∼300 nm submicrometer particles, ∼10 nm micelles and ∼4 nm flexible random coils) and evaluated how these parameters of polymer-TLR-7/8a conjugates impact adjuvant activity in vivo. Attachment of TLR-7/8a to any of the polymer compositions resulted in a nearly 10-fold reduction in systemic cytokines (toxicity). Moreover, both lymph node cytokine production and the magnitude of CD8 T cells induced against protein antigen increased with increasing polymer-TLR-7/8a hydrodynamic radius, with the submicrometer particle inducing the highest magnitude responses. Notably, CD8 T cell responses induced by polymer-TLR-7/8a were dependent on CCR2+ monocytes and IL-12, whereas responses by a small molecule TLR-7/8a that unexpectedly persisted in vaccine-site draining lymph nodes (T1/2 = 15 h) had less dependence on monocytes and IL-12 but required Type I IFNs. This study shows how modular properties of synthetic adjuvants can be chemically programmed to alter immunity in vivo through distinct immunological mechanisms.

A small molecule regulator of tissue transglutaminase conformation inhibits the malignant phenotype of cancer cells.

The protein crosslinking enzyme tissue transglutaminase (tTG) is an acyltransferase which catalyzes transamidation reactions between two proteins, or between a protein and a polyamine. It is frequently overexpressed in several different types of human cancer cells, where it has been shown to contribute to their growth, survival, and invasiveness. tTG is capable of adopting two distinct conformational states: a protein crosslinking active ("open") state, and a GTP-bound, crosslinking inactive ("closed") state. We have previously shown that the ectopic expression of mutant forms of tTG, which constitutively adopt the open conformation, are toxic to cells. This raises the possibility that strategies directed toward causing tTG to maintain an open state could potentially provide a therapeutic benefit for cancers in which tTG is highly expressed. Here, we report the identification of a small molecule, TTGM 5826, which stabilizes the open conformation of tTG. Treatment of breast and brain cancer cell lines, as well as glioma stem cells, with this molecule broadly inhibits their transformed phenotypes. Thus, TTGM 5826 represents the lead compound for a new class of small molecules that promote the toxicity of cancer cells by stabilizing the open state of tTG.