Oligomerization of DNA replication regulatory protein RADX is essential to maintain replication fork stability.

Genome integrity requires complete and accurate DNA replication once per cell division cycle. Replication stress poses obstacles to this process that must be overcome to prevent replication fork collapse. An important regulator of replication fork stability is the RAD51 protein, which promotes replication fork reversal and protects nascent DNA strands from nuclease-mediated degradation. Many regulatory proteins control these RAD51 activities, including RADX, which binds both ssDNA and RAD51 at replication forks to ensure that fork reversal is confined to stalled forks. Many ssDNA-binding proteins function as hetero- or homo-oligomers. In this study, we addressed whether this is also the case for RADX. Using biochemical and genetic approaches, we found that RADX acts as a homo-oligomer to control replication fork stability. RADX oligomerizes using at least two different interaction surfaces, including one mapped to a C-terminal region. We demonstrate that mutations in this region prevent oligomerization and prevent RADX function in cells, and that addition of a heterologous dimerization domain to the oligomerization mutants restored their ability to regulate replication. Taken together, our results demonstrate that like many ssDNA-binding proteins, oligomerization is essential for RADX-mediated regulation of genome stability.

RADx-UP Testing Core: Access to COVID-19 Diagnostics in Community-Engaged Research with Underserved Populations.

Research on the COVID-19 pandemic revealed a disproportionate burden of COVID-19 infection and death among underserved populations and exposed low rates of SARS-CoV-2 testing in these communities. A landmark National Institutes of Health (NIH) funding initiative, the Rapid Acceleration of Diagnostics-Underserved Populations (RADx-UP) program, was developed to address the research gap in understanding the adoption of COVID-19 testing in underserved populations. This program is the single largest investment in health disparities and community-engaged research in the history of the NIH. The RADx-UP Testing Core (TC) provides community-based investigators with essential scientific expertise and guidance on COVID-19 diagnostics. This commentary describes the first 2 years of the TC's experience, highlighting the challenges faced and insights gained to safely and effectively deploy large-scale diagnostics for community-initiated research in underserved populations during a pandemic. The success of RADx-UP shows that community-based research to increase access and uptake of testing among underserved populations can be accomplished during a pandemic with tools, resources, and multidisciplinary expertise provided by a centralized testing-specific coordinating center. We developed adaptive tools to support individual testing strategies and frameworks for these diverse studies and ensured continuous monitoring of testing strategies and use of study data. In a rapidly evolving setting of tremendous uncertainty, the TC provided essential and real-time technical expertise to support safe, effective, and adaptive testing. The lessons learned go beyond this pandemic and can serve as a framework for rapid deployment of testing in response to future crises, especially when populations are affected inequitably.

Using the multiphase optimization strategy (MOST) framework to optimize an intervention to increase COVID-19 testing for Black and Latino/Hispanic frontline essential workers: A study protocol.

BACKGROUND: Among those at highest risk for COVID-19 exposure is the large population of frontline essential workers in occupations such food service, retail, personal care, and in-home health services, among whom Black and Latino/Hispanic persons are over-represented. For those not vaccinated and at risk for exposure to COVID-19, including frontline essential workers, regular (approximately weekly) COVID-19 testing is recommended. However, Black and Latino/Hispanic frontline essential workers in these occupations experience serious impediments to COVID-19 testing at individual/attitudinal- (e.g., lack of knowledge of guidelines), social- (e.g., social norms), and structural-levels of influence (e.g., poor access), and rates of testing for COVID-19 are insufficient. METHODS/DESIGN: The proposed community-engaged study uses the multiphase optimization strategy (MOST) framework and an efficient factorial design to test four candidate behavioral intervention components informed by an integrated conceptual model that combines critical race theory, harm reduction, and self-determination theory. They are A) motivational interview counseling, B) text messaging grounded in behavioral economics, C) peer education, and D) access to testing (via navigation to an appointment vs. a self-test kit). All participants receive health education on COVID-19. The specific aims are to: identify which components contribute meaningfully to improvement in the primary outcome, COVID-19 testing confirmed with documentary evidence, with the most effective combination of components comprising an "optimized" intervention that strategically balances effectiveness against affordability, scalability, and efficiency (Aim 1); identify mediators and moderators of the effects of components (Aim 2); and use a mixed-methods approach to explore relationships among COVID-19 testing and vaccination (Aim 3). Participants will be N = 448 Black and Latino/Hispanic frontline essential workers not tested for COVID-19 in the past six months and not fully vaccinated for COVID-19, randomly assigned to one of 16 intervention conditions, and assessed at 6- and 12-weeks post-baseline. Last, N = 50 participants will engage in qualitative in-depth interviews. DISCUSSION: This optimization trial is designed to yield an effective, affordable, and efficient behavioral intervention that can be rapidly scaled in community settings. Further, it will advance the literature on intervention approaches for social inequities such as those evident in the COVID-19 pandemic. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05139927 ; Registered on 11/29/2021. Protocol version 1.0. May 2, 2022, Version 1.0.

Design and Development of an Antigen Test for SARS-CoV-2 Nucleocapsid Protein to Validate the Viral Quality Assurance Panels.

The continuing mutability of the SARS-CoV-2 virus can result in failures of diagnostic assays. To address this, we describe a generalizable bioinformatics-to-biology pipeline developed for the calibration and quality assurance of inactivated SARS-CoV-2 variant panels provided to Radical Acceleration of Diagnostics programs (RADx)-radical program awardees. A heuristic genetic analysis based on variant-defining mutations demonstrated the lowest genetic variance in the Nucleocapsid protein (Np)-C-terminal domain (CTD) across all SARS-CoV-2 variants. We then employed the Shannon entropy method on (Np) sequences collected from the major variants, verifying the CTD with lower entropy (less prone to mutations) than other Np regions. Polyclonal and monoclonal antibodies were raised against this target CTD antigen and used to develop an Enzyme-linked immunoassay (ELISA) test for SARS-CoV-2. Blinded Viral Quality Assurance (VQA) panels comprised of UV-inactivated SARS-CoV-2 variants (XBB.1.5, BF.7, BA.1, B.1.617.2, and WA1) and distractor respiratory viruses (CoV 229E, CoV OC43, RSV A2, RSV B, IAV H1N1, and IBV) were assembled by the RADx-rad Diagnostics core and tested using the ELISA described here. The assay tested positive for all variants with high sensitivity (limit of detection: 1.72-8.78 ng/mL) and negative for the distractor virus panel. Epitope mapping for the monoclonal antibodies identified a 20 amino acid antigenic peptide on the Np-CTD that an in-silico program also predicted for the highest antigenicity. This work provides a template for a bioinformatics pipeline to select genetic regions with a low propensity for mutation (low Shannon entropy) to develop robust 'pan-variant' antigen-based assays for viruses prone to high mutational rates.

Factors Associated With Surveillance Testing in Individuals With COVID-19 Symptoms During the Last Leg of the Pandemic: Multivariable Regression Analysis.

Background: Rural underserved areas facing health disparities have unequal access to health resources. By the third and fourth waves of SARS-CoV-2 infections in the United States, COVID-19 testing had reduced, with more reliance on home testing, and those seeking testing were mostly symptomatic. Objective: This study identifies factors associated with COVID-19 testing among individuals who were symptomatic versus asymptomatic seen at a Rapid Acceleration of Diagnostics for Underserved Populations phase 2 (RADx-UP2) testing site in West Virginia. Methods: Demographic, clinical, and behavioral factors were collected via survey from tested individuals. Logistic regression was used to identify factors associated with the presence of individuals who were symptomatic seen at testing sites. Global tests for spatial autocorrelation were conducted to examine clustering in the proportion of symptomatic to total individuals tested by zip code. Bivariate maps were created to display geographic distributions between higher proportions of tested individuals who were symptomatic and social determinants of health. Results: Among predictors, the presence of a physical (adjusted odds ratio [aOR] 1.85, 95% CI 1.3-2.65) or mental (aOR 1.53, 95% CI 0.96-2.48) comorbid condition, challenges related to a place to stay/live (aOR 307.13, 95% CI 1.46-10,6372), no community socioeconomic distress (aOR 0.99, 95% CI 0.98-1.00), no challenges in getting needed medicine (aOR 0.01, 95% CI 0.00-0.82) or transportation (aOR 0.23, 95% CI 0.05-0.64), an interaction between community socioeconomic distress and not getting needed medicine (aOR 1.06, 95% CI 1.00-1.13), and having no community socioeconomic distress while not facing challenges related to a place to stay/live (aOR 0.93, 95% CI 0.87-0.99) were statistically associated with an individual being symptomatic at the first test visit. Conclusions: This study addresses critical limitations to the current COVID-19 testing literature, which almost exclusively uses population-level disease screening data to inform public health responses.

COVID-19 Test Us: A Case for Embedding Ethics and Regulatory Expertise.

A key aspect of the National Institutes of Health (NIH) funded Rapid Acceleration of Diagnostics (RADx) Tech program was an active Clinical Studies Core including Committees with unique expertise to facilitate the development and implementation of studies to test novel diagnostic devices for Covid-19. The Ethics and Human Subjects Oversight Team (EHSO) was tasked to provide ethics and regulatory expertise to stakeholders in the RADx Tech effort. The EHSO developed a set of Ethical Principles to guide the overall effort and provided consultation on a wide range of ethical and regulatory concerns. Having access to a pool of experts with ethical and regulatory knowledge who met weekly to tackle issues of importance to the investigators was critical to the overall success of the project.

RADx Tech Viability and Steering Panels: A Model for MedTech Translational Grant Review.

RADxSM Tech's mission is to rapidly accelerate deployment of SARS-CoV-2 tests and could not utilize typical grant application and review processes that can run 4 to 6 months. Instead, RADx Tech leveraged methodologies developed by CIMIT and utilized by POCTRN as described further in this special issue. RADx Tech uses a multi-stage review with two review panels, a Viability Panel and a Steering Panel, that are supported by subject matter experts and a Deep Dive team. Members of the panels have extensive commercialization and business experience in addition to scientific and technical knowledge. The Viability Panel is responsible for assessing whether the proposal is a good fit with the RADx Tech Program and whether it should be recommended to move into a Deep Dive. Less detailed information is requested in the application than a typical SBIR application since the application is refined and details added during the Deep Dive. The Steering Panel reviews the results from the Deep Dive and decides whether to recommend further funding. Everyone on the Viability Panel and Steering Panel reviews every application, thereby providing consistency and context for the reviewers. Utilization of an "assess, improve, and then select" process with review panels comprised of highly experienced review panel members has resulted in improved timing, efficiency, and effectiveness of reviews and has the potential to be extensible beyond RADx Tech.

The RADx Tech Test Verification Core and the ACME POCT in the Evaluation of COVID-19 Testing Devices: A Model for Progress and Change.

Faced with the COVID-19 pandemic, the US system for developing and testing technologies was challenged in unparalleled ways. This article describes the multi-institutional, transdisciplinary team of the "RADxSM Tech Test Verification Core" and its role in expediting evaluations of COVID-19 testing devices. Expertise related to aspects of diagnostic testing was coordinated to evaluate testing devices with the goal of significantly expanding the ability to mass screen Americans to preserve lives and facilitate the safe return to work and school. Focal points included: laboratory and clinical device evaluation of the limit of viral detection, sensitivity, and specificity of devices in controlled and community settings; regulatory expertise to provide focused attention to barriers to device approval and distribution; usability testing from the perspective of patients and those using the tests to identify and overcome device limitations, and engineering assessment to evaluate robustness of design including human factors, manufacturability, and scalability.

The Role of CoLab and GAITS in Enabling the RADx Tech Program.

The RADxSM Tech initiative required a massive mobilization of the biomedical community. It was chartered with the extremely ambitious goal of rapidly developing and deploying innovative tests to detect people infected with the SARS-CoV-2 virus. It needed to do so at a scale and with urgency to get the country back to daily activities such as school and work as soon as possible. It required forming and supporting a diversity of teams with members from around the country and beyond. These teams collaborated in complex workflows that needed to be carefully monitored and tracked. This paper describes the key elements of the secure, web-based infrastructure that was configured to enable the efficient and effective operation of RADx Tech's key processes and address its unique and urgent challenges. One such challenge was to manage the flow of applications through a multi-stage, interactive selection process (using the CoLab platform) and another was to support and facilitate the progress of projects selected for support and funding through an accelerated commercialization program (using the GAITS platform).

The RADx Tech Deep Dive and Work Package 1 Process.

The RADxSM Tech program was a unique funding and support mechanism to accelerate the market introduction of diagnostic tests for SARS-CoV-2, the virus that causes COVID-19. In addition to providing funding, the RADx Tech program provided unprecedented levels of non- monetary support. Applications were evaluated using a deep dive process which involved a 1- to 2-week intensive collaboration between the applicant and a team of experts from RADx Tech. The result of this deep dive was a very comprehensive understanding of the potential and risks associated with the proposed work, which was far beyond what can typically be understood in a written grant application. This detail allowed the deep dive team to provide a better-informed recommendation on how to proceed. In some instances, the recommendation was made to not fund the project; in other cases, the recommendation was made to provide the applicant with more funding or support to help maximize their probability of success. After the deep dive, the project moved to a Work Package 1 (WP1) phase that focused on further de-risking. The same RADx Tech team that conducted the deep dive also worked with the applicant through the WP1 phase of the program. This allowed for joint responsibility of the work with the common goal of rapid, successful product introduction.

The RADx Tech Deployment Core: A Model for Academic/Government Based Support of Large-Scale Manufacturing and Implementation of Medical Technologies.

This paper explores how the approach, process, and learnings of the RADxSM Tech Deployment Core in its support of manufacturing, deployment, and implementation of medical technologies is creating a replicable model for the future. Initially, the key construct of the RADx Tech Deployment Core was helping companies manufacture, commercialize, and develop a digital infrastructure for the purpose of SARS-CoV-2 testing and reporting. However, the team and RADx Tech leadership soon realized that the larger infrastructure to deploy testing in non-clinical environments was nonexistent and that wrap-around services were required to build the necessary bridge between manufacturing and end users. Furthermore, the unique communities that required testing (e.g., manufacturing plants, transportation hubs, K-12 schools, etc.) had different infrastructure requirements and outsized needs for education and support around testing plan implementation. The Deployment Core, therefore, quickly scaled a team to help to complete the picture and provide guidance to end users and ultimately help shape public policy around a useful data model.

RADX Modulates RAD51 Activity to Control Replication Fork Protection.

RAD51 promotes homologous recombination repair (HR) of double-strand breaks and acts during DNA replication to facilitate fork reversal and protect nascent DNA strands from nuclease digestion. Several additional HR proteins regulate fork protection by promoting RAD51 filament formation. Here, we show that RADX modulates stalled fork protection by antagonizing RAD51. Consequently, silencing RADX restores fork protection in cells deficient for BRCA1, BRCA2, FANCA, FANCD2, or BOD1L. Inactivating RADX prevents both MRE11- and DNA2-dependent fork degradation. Furthermore, RADX overexpression causes fork degradation that is dependent on these nucleases and fork reversal. The amount of RAD51 determines the fate of stalled replication forks, with more RAD51 required for fork protection than fork reversal. Finally, we find that RADX effectively competes with RAD51 for binding to single-stranded DNA, supporting a model in which RADX buffers RAD51 to ensure the right amount of reversal and protection to maintain genome stability.