Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

Modulation of Biophysical Properties of Nucleocapsid Protein in the Mutant Spectrum of SARS-CoV-2.

Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also exhibiting functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.

Exosome complex components 1 and 2 are vital for early mammalian development.

Exosome Complex Components 1 and 2 (EXOSC1 and 2) are two proteins in the RNA Exosome complex whose main function is 5' → 3' RNA degradation and processing. The RNA exosome complex is comprised of nine subunits that form two separate components: the S1/KH cap and the PH-core. EXOSC1 and 2 are both part of the S1/KH cap and are involved in binding nascent RNA. As part of a systemic characterization of early lethal alleles produced by the Knockout Mouse Project, we have examined Exosc1 and Exosc2 homozygous null (mutant) embryos to determine developmental and molecular phenotypes of embryos lacking their functions. Our studies reveal that Exosc1 null embryos implant and form an egg cylinder but are developmentally delayed and fail to initiate gastrulation by embryonic day 7.5. In contrast, Exosc2 null embryos are lethal during peri-implantation stages, and while they do form a morphologically normal blastocyst at E3.5, they cannot be recovered at post-implantation stages. We show the absence of stage-specific developmental and altered lineage-specification in both Exosc1 and Exosc2 mutant embryos and conclude that these genes are essential for the successful progression through early mammalian development.

Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-) protein into ribonucleoprotein particles (RNPs), 38±10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining mutant proteins in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multi-valent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

Mapping the scientific literature on obstetrical and perinatal health among sexual and gender minoritised (SGM) childbearing people and their infants: a scoping review protocol.

INTRODUCTION: Sexual and gender minoritised (SGM) populations are disproportionately impacted by multilevel risk factors for obstetrical and perinatal outcomes, including structural (eg, stigma, discrimination, access to care) and individual risk factors (eg, partner violence, poor mental health, substance use). Emerging evidence shows SGM childbearing people have worse obstetrical outcomes and their infants have worse perinatal outcomes, when compared with their cisgender and heterosexual counterparts; this emerging evidence necessitates a comprehensive examination of existing literature on obstetrical and perinatal health among SGM people. The goal of this scoping review is to comprehensively map the extent, range and nature of scientific literature on obstetrical and perinatal physical health outcomes among SGM populations and their infants. We aim to summarise findings from existing literature, potentially informing clinical guidelines on perinatal care, as well as highlighting knowledge gaps and providing directions for future research. METHODS AND ANALYSIS: We will follow the Joanna Briggs Institute (JBI) scoping review framework and report findings according to the PRISMA Extension for Scoping Reviews (PRISMA-ScR) guidelines. We will conduct a broad systematic search in Medline/PubMed, Embase, CINAHL and Web of Science Core Collection. Eligible studies will include peer-reviewed, empirical, English-language publications pertaining to obstetrical and perinatal physical health outcomes of SGM people or their infants. No temporal or geographical limitations will be applied to the search. Studies conducted in all settings will be considered. Records will be managed, screened and extracted by two independent reviewers. Study characteristics, key findings and research gaps will be presented in tables and summarised narratively. ETHICS AND DISSEMINATION: Ethical approval is not required as primary data will not be collected. The findings of this scoping review will be disseminated through a peer-reviewed journal and conference presentations. PROTOCOL REGISTRATION: Open Science Framework https://osf.io/6fg4a/.

Interferon signaling promotes tolerance to chromosomal instability during metastatic evolution in renal cancer.

Molecular routes to metastatic dissemination are critical determinants of aggressive cancers. Through in vivo CRISPR-Cas9 genome editing, we generated somatic mosaic genetically engineered models that faithfully recapitulate metastatic renal tumors. Disruption of 9p21 locus is an evolutionary driver to systemic disease through the rapid acquisition of complex karyotypes in cancer cells. Cross-species analysis revealed that recurrent patterns of copy number variations, including 21q loss and dysregulation of the interferon pathway, are major drivers of metastatic potential. In vitro and in vivo genomic engineering, leveraging loss-of-function studies, along with a model of partial trisomy of chromosome 21q, demonstrated a dosage-dependent effect of the interferon receptor genes cluster as an adaptive mechanism to deleterious chromosomal instability in metastatic progression. This work provides critical knowledge on drivers of renal cell carcinoma progression and defines the primary role of interferon signaling in constraining the propagation of aneuploid clones in cancer evolution.

Ether phospholipids are required for mitochondrial reactive oxygen species homeostasis.

Mitochondria are hubs where bioenergetics, redox homeostasis, and anabolic metabolism pathways integrate through a tightly coordinated flux of metabolites. The contributions of mitochondrial metabolism to tumor growth and therapy resistance are evident, but drugs targeting mitochondrial metabolism have repeatedly failed in the clinic. Our study in pancreatic ductal adenocarcinoma (PDAC) finds that cellular and mitochondrial lipid composition influence cancer cell sensitivity to pharmacological inhibition of electron transport chain complex I. Profiling of patient-derived PDAC models revealed that monounsaturated fatty acids (MUFAs) and MUFA-linked ether phospholipids play a critical role in maintaining ROS homeostasis. We show that ether phospholipids support mitochondrial supercomplex assembly and ROS production; accordingly, blocking de novo ether phospholipid biosynthesis sensitized PDAC cells to complex I inhibition by inducing mitochondrial ROS and lipid peroxidation. These data identify ether phospholipids as a regulator of mitochondrial redox control that contributes to the sensitivity of PDAC cells to complex I inhibition.

A Novel Medical Student Elective Course in Lesbian, Gay, Bisexual, Transgender, Queer, Intersex, Asexual, and Sexually and Gender Diverse Health: Training Tomorrow's Physician-Leaders.

Purpose: Inadequate medical training in lesbian, gay, bisexual, transgender, queer, intersex, asexual, and sexually and gender diverse (LGBTQIA+) health care contributes to health disparities. This article reports on a novel 4-week elective course at Harvard Medical School that prepares students to become physician-leaders in LGBTQIA+ health care. Methods: The course engages students in multidisciplinary clinical placements, self-directed learning, and mentored scholarly projects, all specifically relevant to LGBTQIA+ health. The authors qualitatively reviewed student experiences, and quantitatively analyzed course evaluations. Results: Between 2016 and 2022, 30 students completed the course, and 12 scholarly projects resulted in a published peer-reviewed article authored by the student. Students who completed evaluations rated the course as excellent and reported increased interest and core knowledge in LGBTQIA+ health care. Conclusion: This novel program has demonstrated feasibility and may serve as a model for establishing other advanced medical student clinical and scholarly electives on LGBTQIA+ health care.

Leveraging Podcasts to Introduce Medical Students to the Broader Community of Health Care Professionals.

INTRODUCTION: Safe, patient-centered, and cost-effective care requires effective collaboration within interprofessional teams. Education programs for health care professionals are often siloed, providing students with limited interprofessional education (IPE) opportunities to learn from, with, and about other professions. Podcasts offer a novel approach to facilitate IPE, allowing for asynchronous conversations with interprofessional colleagues. METHODS: We developed four podcasts with various health care professionals for 135 preclinical medical students preparing to transition into clinical rotations. The podcasts were coupled with an hour-long interactive session with the podcast interviewees conducted via videoconference. The curriculum explored the distinct education paths, roles, and responsibilities of various health care disciplines. Strategies for communicating effectively with and learning from interprofessional team members were emphasized. RESULTS: There were 197 unique downloads of the podcasts, and 95 students attended the interactive session. Most students reported that the podcasts and follow-up live session enhanced their learning (100% and 98% of students who completed the postcurriculum survey, respectively). Responses to the postcurriculum survey revealed students learned strategies for engaging in productive interprofessional conversations, the importance of leveraging the distinct roles and responsibilities of diverse health professionals, the value of learning from other health professionals, and the use of respectful language. DISCUSSION: This IPE curriculum built around podcasts enhances medical student learning and represents an innovative approach to improving access to IPE in a virtual learning environment. This modality can be adapted to meet the needs of a wide spectrum of learners and can be coupled with in-person learning.

miR-9 modulates and predicts the response to radiotherapy and EGFR inhibition in HNSCC.

Radiotherapy (RT) plus the anti-EGFR monoclonal antibody Cetuximab (CTX) is an effective combination therapy for a subset of head and neck squamous cell carcinoma (HNSCC) patients. However, predictive markers of efficacy are missing, resulting in many patients treated with disappointing results and unnecessary toxicities. Here, we report that activation of EGFR upregulates miR-9 expression, which sustains the aggressiveness of HNSCC cells and protects from RT-induced cell death. Mechanistically, by targeting KLF5, miR-9 regulates the expression of the transcription factor Sp1 that, in turn, stimulates tumor growth and confers resistance to RT+CTX in vitro and in vivo. Intriguingly, high miR-9 levels have no effect on the sensitivity of HNSCC cells to cisplatin. In primary HNSCC, miR-9 expression correlated with Sp1 mRNA levels and high miR-9 expression predicted poor prognosis in patients treated with RT+CTX. Overall, we have discovered a new signaling axis linking EGFR activation to Sp1 expression that dictates the response to combination treatments in HNSCC. We propose that miR-9 may represent a valuable biomarker to select which HNSCC patients might benefit from RT+CTX therapy.

Medium-Chain Acyl-CoA Dehydrogenase Protects Mitochondria from Lipid Peroxidation in Glioblastoma.

Glioblastoma (GBM) is highly resistant to chemotherapies, immune-based therapies, and targeted inhibitors. To identify novel drug targets, we screened orthotopically implanted, patient-derived glioblastoma sphere-forming cells using an RNAi library to probe essential tumor cell metabolic programs. This identified high dependence on mitochondrial fatty acid metabolism. We focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids (MCFA), due to its consistently high score and high expression among models and upregulation in GBM compared with normal brain. Beyond the expected energetics impairment, MCAD depletion in primary GBM models induced an irreversible cascade of detrimental metabolic effects characterized by accumulation of unmetabolized MCFAs, which induced lipid peroxidation and oxidative stress, irreversible mitochondrial damage, and apoptosis. Our data uncover a novel protective role for MCAD to clear lipid molecules that may cause lethal cell damage, suggesting that therapeutic targeting of MCFA catabolism may exploit a key metabolic feature of GBM. SIGNIFICANCE: MCAD exerts a protective role to prevent accumulation of toxic metabolic by-products in glioma cells, actively catabolizing lipid species that would otherwise affect mitochondrial integrity and induce cell death. This work represents a first demonstration of a nonenergetic role for dependence on fatty acid metabolism in cancer.This article is highlighted in the In This Issue feature, p. 2659.

Sequential Administration of XPO1 and ATR Inhibitors Enhances Therapeutic Response in TP53-mutated Colorectal Cancer.

BACKGROUND & AIMS: Understanding the mechanisms by which tumors adapt to therapy is critical for developing effective combination therapeutic approaches to improve clinical outcomes for patients with cancer. METHODS: To identify promising and clinically actionable targets for managing colorectal cancer (CRC), we conducted a patient-centered functional genomics platform that includes approximately 200 genes and paired this with a high-throughput drug screen that includes 262 compounds in four patient-derived xenografts (PDXs) from patients with CRC. RESULTS: Both screening methods identified exportin 1 (XPO1) inhibitors as drivers of DNA damage-induced lethality in CRC. Molecular characterization of the cellular response to XPO1 inhibition uncovered an adaptive mechanism that limited the duration of response in TP53-mutated, but not in TP53-wild-type CRC models. Comprehensive proteomic and transcriptomic characterization revealed that the ATM/ATR-CHK1/2 axes were selectively engaged in TP53-mutant CRC cells upon XPO1 inhibitor treatment and that this response was required for adapting to therapy and escaping cell death. Administration of KPT-8602, an XPO1 inhibitor, followed by AZD-6738, an ATR inhibitor, resulted in dramatic antitumor effects and prolonged survival in TP53-mutant models of CRC. CONCLUSIONS: Our findings anticipate tremendous therapeutic benefit and support the further evaluation of XPO1 inhibitors, especially in combination with DNA damage checkpoint inhibitors, to elicit an enduring clinical response in patients with CRC harboring TP53 mutations.

Comprehensive Molecular Characterization Identifies Distinct Genomic and Immune Hallmarks of Renal Medullary Carcinoma.

Renal medullary carcinoma (RMC) is a highly lethal malignancy that mainly afflicts young individuals of African descent and is resistant to all targeted agents used to treat other renal cell carcinomas. Comprehensive genomic and transcriptomic profiling of untreated primary RMC tissues was performed to elucidate the molecular landscape of these tumors. We found that RMC was characterized by high replication stress and an abundance of focal copy-number alterations associated with activation of the stimulator of the cyclic GMP-AMP synthase interferon genes (cGAS-STING) innate immune pathway. Replication stress conferred a therapeutic vulnerability to drugs targeting DNA-damage repair pathways. Elucidation of these previously unknown RMC hallmarks paves the way to new clinical trials for this rare but highly lethal malignancy.

Pooled library screening with multiplexed Cpf1 library.

Capitalizing on the inherent multiplexing capability of AsCpf1, we developed a multiplexed, high-throughput screening strategy that minimizes library size without sacrificing gene targeting efficiency. We demonstrated that AsCpf1 can be used for functional genomics screenings and that an AsCpf1-based multiplexed library performs similarly as compared to currently available monocistronic CRISPR/Cas9 libraries, with only one vector required for each gene. We construct the smallest whole-genome CRISPR knock-out library, Mini-human, for the human genome (n = 17,032 constructs targeting 16,977 protein-coding genes), which performs favorably compared to conventional Cas9 libraries.

A network of human functional gene interactions from knockout fitness screens in cancer cells.

Genetic interactions mediate the emergence of phenotype from genotype. The systematic survey of genetic interactions in yeast showed that genes operating in the same biological process have highly correlated genetic interaction profiles, and this observation has been exploited to infer gene function in model organisms. Such assays of digenic perturbations in human cells are also highly informative, but are not scalable, even with CRISPR-mediated methods. As an alternative, we developed an indirect method of deriving functional interactions. We show that genes having correlated knockout fitness profiles across diverse, non-isogenic cell lines are analogous to genes having correlated genetic interaction profiles across isogenic query strains and similarly imply shared biological function. We constructed a network of genes with correlated fitness profiles across 276 high-quality CRISPR knockout screens in cancer cell lines into a "coessentiality network," with up to 500-fold enrichment for co-functional gene pairs, enabling strong inference of gene function and highlighting the modular organization of the cell.

p53 Is a Master Regulator of Proteostasis in SMARCB1-Deficient Malignant Rhabdoid Tumors.

Alterations in chromatin remodeling genes have been increasingly implicated in human oncogenesis. Specifically, the biallelic inactivation of the SWI/SNF subunit SMARCB1 results in the emergence of extremely aggressive pediatric malignancies. Here, we developed embryonic mosaic mouse models of malignant rhabdoid tumors (MRTs) that faithfully recapitulate the clinical-pathological features of the human disease. We demonstrated that SMARCB1-deficient malignancies exhibit dramatic activation of the unfolded protein response (UPR) and ER stress response via a genetically intact MYC-p19ARF-p53 axis. As a consequence, these tumors display an exquisite sensitivity to agents inducing proteotoxic stress and inhibition of the autophagic machinery. In conclusion, our findings provide a rationale for drug repositioning trials investigating combinations of agents targeting the UPR and autophagy in SMARCB1-deficient MRTs.

Modeling a novel hypothetical use of postal collection boxes as automated external defibrillator access points.

INTRODUCTION: Optimizing placement of Automated External Defibrillators (AED) can increase survival after an out-of-hospital cardiac arrest (OHCA). Using postal collection boxes (PCB) as locations for AEDs could potentially enhance accessibility and streamline maintenance. In this study, we modeled the hypothetical effects of deploying AEDs at PCB locations. HYPOTHESIS: We hypothesized that PCB-AEDs would increase AED coverage overall and in residential areas, and reduce the distance from OHCA to an AED. METHODS: AEDs in Pittsburgh, PA were identified by the University of Pittsburgh Resuscitation Logistics and Informatics Venture (n=747). PCB locations were obtained from the United States Postal Service (n=479). OHCA locations from 2009 to 2014 were obtained from the Pittsburgh site of the Resuscitation Outcomes Consortium. AED coverage assuming a » mile radius around each AED was estimated for known AEDs, PCB-AEDs (hypothetical AED locations), and known AEDs augmented by PCB-AEDs, both overall and for residential and non-residential zones. Linear distance from each OHCA to the nearest AED was calculated and compared between the sets. RESULTS: The set of known AEDs augmented with PCB-AEDs covered more of the city overall (55% vs 30%), as well as greater proportions of residential (62% vs 27%) and non-residential areas (45% vs 30%). The median distance from OHCA to AED was significantly shorter when known AEDs were augmented with PCB-AEDs (0.12mi vs 0.32mi; p=0.001). CONCLUSION: Augmenting existing publicly accessible AEDs with AEDs deployed at PCBs can increase AED spatial coverage in both residential and non-residential areas, and reduce the distance from AED to OHCA.

Mobile Health Physical Activity Intervention Preferences in Cancer Survivors: A Qualitative Study.

BACKGROUND: Cancer survivors are at an elevated risk for several negative health outcomes, but physical activity (PA) can decrease those risks. Unfortunately, adherence to PA recommendations among survivors is low. Fitness mobile apps have been shown to facilitate the adoption of PA in the general population, but there are limited apps specifically designed for cancer survivors. This population has unique needs and barriers to PA, and most existing PA apps do not address these issues. Moreover, incorporating user preferences has been identified as an important priority for technology-based PA interventions, but at present there is limited literature that serves to establish these preferences in cancer survivors. This is especially problematic given the high cost of app development and because the majority of downloaded apps fail to engage users over the long term. OBJECTIVE: The aim of this study was to take a qualitative approach to provide practical insight regarding this population's preferences for the features and messages of an app to increase PA. METHODS: A total of 35 cancer survivors each attended 2 focus groups; a moderator presented slide shows on potential app features and messages and asked open-ended questions to elicit participant preferences. All sessions were audio recorded and transcribed verbatim. Three reviewers independently conducted thematic content analysis on all transcripts, then organized and consolidated findings to identify salient themes. RESULTS: Participants (mean age 63.7, SD 10.8, years) were mostly female (24/35, 69%) and mostly white (25/35, 71%). Participants generally had access to technology and were receptive to engaging with an app to increase PA. Themes identified included preferences for (1) a casual, concise, and positive tone, (2) tools for personal goal attainment, (3) a prescription for PA, and (4) an experience that is tailored to the user. Participants reported wanting extensive background data collection with low data entry burden and to have a trustworthy source translate their personal data into individualized PA recommendations. They expressed a desire for app functions that could facilitate goal achievement and articulated a preference for a more private social experience. Finally, results indicated that PA goals might be best established in the context of personally held priorities and values. CONCLUSIONS: Many of the desired features identified are compatible with both empirically supported methods of behavior change and the relative strengths of an app as a delivery vehicle for behavioral intervention. Participating cancer survivors' preferences contrasted with many current standard practices for mobile app development, including value-based rather than numeric goals, private socialization in small groups rather than sharing with broader social networks, and interpretation of PA data rather than merely providing numerical data. Taken together, these insights may help increase the acceptability of theory-based mHealth PA interventions in cancer survivors.