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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 observe 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. Like other types of RNA viruses, the genetic material of SARS-CoV-2 (the agent responsible for COVID-19) is formed of an RNA molecule which is prone to accumulating mutations. This gives SARS-CoV-2 the ability to evolve quickly, and often to remain one step ahead of treatments. Understanding how these mutations shape the behavior of RNA viruses is therefore crucial to keep diseases such as COVID-19 under control. The gene that codes for the protein that ‘packages’ the genetic information inside SARS-CoV-2 is particularly prone to mutations. This nucleocapsid (N) protein participates in many key processes during the life cycle of the virus, including potentially interfering with the immune response. Exactly how the physical properties of the N-Protein are impacted by the mutations in its genetic sequence remains unclear. To investigate this question, Nguyen et al. predicted the various biophysical properties of different regions of the N-protein based on a computer-based analysis of SARS-CoV-2 genetic databases. This allowed them to determine if specific protein regions were positively or negatively charged in different mutants. The analyses showed that some domains exhibited great variability in their charge between protein variants – reflecting the fact that the corresponding genetic sequences showed high levels of plasticity. Other regions remained conserved, however, including across related coronaviruses. Nguyen et al. also conducted biochemical experiments on a range of N-proteins obtained from clinically relevant SARS-CoV-2 variants. Their results highlighted the importance of protein segments with no fixed three-dimensional structure. Mutations in the related sequences created high levels of variation in the physical properties of these ‘intrinsically disordered’ regions, which had wide-ranging consequences. Some of these genetic changes even gave individual N-proteins the ability to interact with each other in a completely new way. These results shed new light on the relationship between genetic mutations and the variable physical properties of RNA virus proteins. Nguyen et al. hope that this knowledge will eventually help to develop more effective treatments for viral infections.

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. AsCpf1 is an alternative nuclease to Cas9 for CRISPR mediated genome engineering. Here the authors demonstrate functional genomic screens with AsCpf1 that minimize library size with no loss in gene targeting efficiency.

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. Cancer cells can be dependent on mitochondrial respiration to survive. Here, in pancreatic cancer cells, the authors show that monounsaturated fatty acids-linked ether lipids maintain mitochondrial redox homeostasis and modulate sensitivity to inhibition to electron transport chain complex I.

OBJECTIVES To reduce health inequities for lesbian, gay, bisexual, transgender, queer, intersex, asexual, and all sexually and gender diverse (LGBTQIA+) people, healthcare professionals need increased access to education and training resources on LGBTQIA + health. Web-based, asynchronous, electronic learning (e-learning) resources are critical for expanding the availability of LGBTQIA + health programs. This article presents the design and utilization outcomes of a novel e-learning platform for engaging healthcare professionals in LGBTQIA + health online continuing education. METHODS As of December 2022, the e-learning platform consisted of 293 resources within 17 topic domains. Modalities included: learning modules, recorded webinars, publications, videos, and toolkits. We conducted a descriptive analysis of the e-learning platform's website traffic and user engagement data. Google Universal Analytics and event tracking were used to measure website traffic, user locations, and publication downloads. Learning module and webinar completions were exported from the learning management system and run as frequencies. RESULTS Between January 1, 2020, and December 31, 2022, over 650,000 people from all U.S. states, 182 countries, and 31 territories visited the website. Platform users downloaded publications 66,225 times, and completed 29,351 learning modules and 24,654 webinars. CONCLUSION The broad reach and high user engagement of the e-learning platform indicate acceptability of web-based, asynchronous online continuing education in LGBTQIA + health, and suggest that this platform is filling a need in health professional education. Remote, online learning opportunities may be especially important in jurisdictions with bans on medical care for transgender and gender diverse youth. Future growth of the platform, paired with in-person and other online learning opportunities, has the potential to reduce gaps in LGBTQIA + health training, and mitigate LGBTQIA + health inequities.

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. Synopsis The combination therapy of Radiotherapy + Cetuximab (RT + CTX) is currently used for the treatment of HNSCC. Its lower toxicity compared to chemotherapy makes it the primary choice for fragile patients. This study identifies miR‐9 as a biomarker of RT + CTX responsiveness and explains why miR‐9 may be especially relevant in TP53 mutated HNSCC. In HNSCC cells, miR‐9 expression is regulated by EGFR activity. High miR‐9 expression confers to HNSCC cells higher tumorigenic activity and resistance to RT + CTX, predicting shorter survival of HNSCC patients treated with RT + CTX. miR‐9 targets the transcription factor KLF5 by binding its 3’UTR. In TP53 mutated context, miR‐9‐mediated silencing of KLF5 causes activation of SP1 that drives tumour progression program of HNSCC and mediates response to therapies. Graphical Abstract The combination therapy of Radiotherapy + Cetuximab (RT + CTX) is currently used for the treatment of HNSCC. Its lower toxicity compared to chemotherapy makes it the primary choice for fragile patients. This study identifies miR‐9 as a biomarker of RT + CTX responsiveness and explains why miR‐9 may be especially relevant in TP53 mutated HNSCC.

Background Evidence suggests sexual and gender minoritized (SGM) childbearing individuals and their infants experience more adverse obstetric and perinatal outcomes compared to their cisgender, heterosexual counterparts. This study aimed to comprehensively map obstetric and perinatal physical health literature among SGM populations and their infants and identify knowledge gaps. Methods PubMed, Embase, CINAHL, and Web of Science Core Collection were systematically searched to identify published studies reporting obstetric and perinatal outcomes in SGM individuals or their infants. Study characteristics, sample characteristics, and outcome findings were systematically extracted and analyzed. Results Our search yielded 8,740 records; 55 studies (1981–2023) were included. Sexual orientation was measured by self-identification (72%), behavior (55%), and attraction (9%). Only one study captured all three dimensions. Inconsistent measures of sexual orientation and gender identity (SOGI) were common, and 68% conflated sex and gender. Most (85%) focused on sexual minorities, while 31% addressed gender minorities. Demographic measures employed varied widely and were inconsistent; 35% lacked race/ethnicity data, and 44% lacked socioeconomic data. Most studies (78%) examined outcomes among SGM individuals, primarily focusing on morbidity and pregnancy outcomes. Pregnancy termination was most frequently studied, while pregnancy and childbirth complications (e.g., gestational hypertension, postpartum hemorrhage) were rarely examined. Evidence of disparities were mixed. Infant outcomes were investigated in 60% of the studies, focusing on preterm birth and low birthweight. Disparities were noted among different sexual orientation and racial/ethnic groups. Qualitative insights highlighted how stigma and discriminatory care settings can lead to adverse pregnancy and birth outcomes. Conclusions Frequent conflation of sex and gender and a lack of standardized SOGI measures hinder the comparison and synthesis of existing evidence. Nuanced sociodemographic data should be collected to understand the implications of intersecting identities. Findings on perinatal health disparities were mixed, highlighting the need for standardized SOGI measures and comprehensive sociodemographic data. The impact of stigma and discriminatory care on adverse outcomes underscores the need for inclusive healthcare environments. Future research should address these gaps; research on SGM perinatal outcomes remains urgently lacking. Trial registration The review protocol was developed a priori in February 2023, registered on Open Science Framework ( https://doi.org/10.17605/OSF.IO/5DQV4 ) and published in BMJ Open ( https://bmjopen.bmj.com/content/13/11/e075443 ).

IntroductionSexual 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 analysisWe 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 disseminationEthical 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 registrationOpen Science Framework https://osf.io/6fg4a/.

BackgroundUveal melanoma (UM) is a rare tumor characterized by mutually exclusive activating mutations in GNAQ in GNA11, followed by secondary events in BAP1, SF3B1 and EIF1AX. Notably, a large subset of patients presents with copy-loss of chromosome 3 (monosomy 3), which is highly associated with metastatic progression in late-stage disease. Monosomy 3 tumors demonstrate a marked resistance to chemotherapy, targeted therapeutics, and immunotherapy, despite successes observed in cutaneous melanoma.Informing on the biology underlying chromosome 3 copy-loss, and its impact on the tumor microenvironment, is critical towards directing future efforts in targeted therapeutics and immunotherapy. Current limited insight can be attributed to both lack of: (a) preclinical models, and (b) in-depth characterization of paired primary and metastatic tumors in patients.MethodsTo address this, we first induced chromosome 3 copy-loss through CRISPR-based centromere targeting in a well characterized Disomy 3 UM cell line. Disomy 3 (D3) and Monosomy 3 (M3) clones derived from these efforts have enabled us to develop patient derived xenograft (PDX) models to compare D3 and M3 behavior in paired primary and metastatic settings.Additionally, we identified and collected a range of match-paired (primary and metastatic) clinical UM samples across multiple patients.Leveraging these unique samples, we applied spatial transcriptomics to inform on tumor intrinsic and extrinsic features of progressive UM, identifying gene signatures of disease advancement and deconvoluting the evolving tumor microenvironment.ResultsInvestigation of metastatic UM tumor heterogeneity in our models enables us to characterize unique features of phenotypically transformed clones (e.g. depigmentation and growth advantage) (figure 1). We also adapted existing methodology to infer chromosomal copy number events from spatial transcriptomics data to our PDX system, overcoming the lack of same-species microenvironment controls. We complement our preclinical analyses with an investigation of spatial heterogeneity in a patient cohort of paired primary and metastatic tumors (figure 2). Leveraging single-cell deconvolution in this paired dataset, we captured unique immune microenvironments in primary vs. metastatic tumors. Additionally, we integrated our preclinical tumor intrinsic signatures to pair differential gene expression signatures of tumor sub-clones with differential immune cell populations.ConclusionsOur methodology allows for deep characterization of sub-clonal heterogeneity in primary and metastatic settings and informs on the unique microenvironmental heterogeneity underlying invasiveness and outgrowth of M3 tumors. More broadly, comparing these preclinical and patient tumors provides an opportunity to expand on our knowledge of metastatic disease drivers and derive prognostic signatures associated with poor survival and lack of response to immunotherapy.Abstract 1498 Figure 1Spatial Transcriptomics Informs on Liver Outgrowth Phenotypes in Engineered UM Preclinical Models. (A) Spatial slides of two Disomy 3 tumors in the mouse liver and matched clustering following the removal of mouse background demonstrated with differential pigrnentahon and tumor size. (B) Corresponding copy number inference of the D3.5_2 slide demonstrates our ability to identify subclones based on copy number heterogeneity. (C) Spatial slides of two Monosomy 3 tumors in the mouse liver and matched clustering following the removal of mouse background. (D) Corresponding copy number inference of the M3.8_2 slide unable to accurately capture copy number in the absence of microenvironment as a normal control demonstrated by chromosome 3 being inferred as copy neutralAbstract 1498 Figure 2Spatial Transcriptomics on Uveal Melanoma Clinical Samples — Paired Primary Eye and Liver Metastases. (A) Spatial slide with matched primary eye and liver sections from Patient #1. (B) Spatial slide with matched primary eye and liver sections from Patient #2. (C) Predicted cell types observed in matched primary and liver tumors of Patient #1. (D) Predicted cell types in matched primary and liver tumors of Patient #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 observe 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. Like other types of RNA viruses, the genetic material of SARS-CoV-2 (the agent responsible for COVID-19) is formed of an RNA molecule which is prone to accumulating mutations. This gives SARS-CoV-2 the ability to evolve quickly, and often to remain one step ahead of treatments. Understanding how these mutations shape the behavior of RNA viruses is therefore crucial to keep diseases such as COVID-19 under control. The gene that codes for the protein that ‘packages’ the genetic information inside SARS-CoV-2 is particularly prone to mutations. This nucleocapsid (N) protein participates in many key processes during the life cycle of the virus, including potentially interfering with the immune response. Exactly how the physical properties of the N-Protein are impacted by the mutations in its genetic sequence remains unclear. To investigate this question, Nguyen et al. predicted the various biophysical properties of different regions of the N-protein based on a computer-based analysis of SARS-CoV-2 genetic databases. This allowed them to determine if specific protein regions were positively or negatively charged in different mutants. The analyses showed that some domains exhibited great variability in their charge between protein variants – reflecting the fact that the corresponding genetic sequences showed high levels of plasticity. Other regions remained conserved, however, including across related coronaviruses. Nguyen et al. also conducted biochemical experiments on a range of N-proteins obtained from clinically relevant SARS-CoV-2 variants. Their results highlighted the importance of protein segments with no fixed three-dimensional structure. Mutations in the related sequences created high levels of variation in the physical properties of these ‘intrinsically disordered’ regions, which had wide-ranging consequences. Some of these genetic changes even gave individual N-proteins the ability to interact with each other in a completely new way. These results shed new light on the relationship between genetic mutations and the variable physical properties of RNA virus proteins. Nguyen et al. hope that this knowledge will eventually help to develop more effective treatments for viral infections.

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.