Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

Fundamental immune-oncogenicity trade-offs define driver mutation fitness.

Missense driver mutations in cancer are concentrated in a few hotspots1. Various mechanisms have been proposed to explain this skew, including biased mutational processes2, phenotypic differences3-6 and immunoediting of neoantigens7,8; however, to our knowledge, no existing model weighs the relative contribution of these features to tumour evolution. We propose a unified theoretical 'free fitness' framework that parsimoniously integrates multimodal genomic, epigenetic, transcriptomic and proteomic data into a biophysical model of the rate-limiting processes underlying the fitness advantage conferred on cancer cells by driver gene mutations. Focusing on TP53, the most mutated gene in cancer1, we present an inference of mutant p53 concentration and demonstrate that TP53 hotspot mutations optimally solve an evolutionary trade-off between oncogenic potential and neoantigen immunogenicity. Our model anticipates patient survival in The Cancer Genome Atlas and patients with lung cancer treated with immunotherapy as well as the age of tumour onset in germline carriers of TP53 variants. The predicted differential immunogenicity between hotspot mutations was validated experimentally in patients with cancer and in a unique large dataset of healthy individuals. Our data indicate that immune selective pressure on TP53 mutations has a smaller role in non-cancerous lesions than in tumours, suggesting that targeted immunotherapy may offer an early prophylactic opportunity for the former. Determining the relative contribution of immunogenicity and oncogenic function to the selective advantage of hotspot mutations thus has important implications for both precision immunotherapies and our understanding of tumour evolution.

Neoantigen quality predicts immunoediting in survivors of pancreatic cancer.

Cancer immunoediting1 is a hallmark of cancer2 that predicts that lymphocytes kill more immunogenic cancer cells to cause less immunogenic clones to dominate a population. Although proven in mice1,3, whether immunoediting occurs naturally in human cancers remains unclear. Here, to address this, we investigate how 70 human pancreatic cancers evolved over 10 years. We find that, despite having more time to accumulate mutations, rare long-term survivors of pancreatic cancer who have stronger T cell activity in primary tumours develop genetically less heterogeneous recurrent tumours with fewer immunogenic mutations (neoantigens). To quantify whether immunoediting underlies these observations, we infer that a neoantigen is immunogenic (high-quality) by two features-'non-selfness'  based on neoantigen similarity to known antigens4,5, and 'selfness'  based on the antigenic distance required for a neoantigen to differentially bind to the MHC or activate a T cell compared with its wild-type peptide. Using these features, we estimate cancer clone fitness as the aggregate cost of T cells recognizing high-quality neoantigens offset by gains from oncogenic mutations. With this model, we predict the clonal evolution of tumours to reveal that long-term survivors of pancreatic cancer develop recurrent tumours with fewer high-quality neoantigens. Thus, we submit evidence that that the human immune system naturally edits neoantigens. Furthermore, we present a model to predict how immune pressure induces cancer cell populations to evolve over time. More broadly, our results argue that the immune system fundamentally surveils host genetic changes to suppress cancer.

Dataset for the life-cycle assessment of the Basque Y high-speed rail line in Spain.

This dataset presents a detailed description of the data and information used in the life-cycle assessment (LCA) of the Basque Y HSR line, which is a high-performance line for mixed traffic still under construction in 2023 (190 km). The LCI data presented in this paper support the original research carried out on whether the construction of the Basque Y HSR line infrastructure is justified in terms of reducing environmental impacts and energy consumption [1]. Life-cycle inventory (LCI) data related to the construction and maintenance phases of the infrastructure was collected using Google Earth tool following the information from stakeholder AHT gelditu [2], including the length of each item (bridges, tunnels, earthworks, railway tracks); and complemented with data obtained from the LCA carried out by Tuchschmid et al. [3]. LCI data associated with the operation phase of the infrastructure was built on passenger data for the years 2020, 2030, 2040 and 2049 available in ADIF [4], and freight data for the period of 2023-2050 available in the report by ADIF and the Basque Government [5]. Environmental impacts for transport modes were obtained from the ecoinvent v3.7 database [6,7] and processed with openLCA software [8]. Life-cycle impact assessment (LCIA) results gathered in the dataset include Global Warming (GWP100a), Cumulative Energy Demand and total emissions for PM10, SO2, NOX and NMVOC. Access to the explanation of these data allows any reader to reproduce the calculations of the main project and may be used as a baseline for future studies on transport economics too.

Distinct genomic and immunologic tumor evolution in germline TP53-driven breast cancers.

Pathogenic germline TP53 alterations cause Li-Fraumeni Syndrome (LFS), and breast cancer is the most common cancer in LFS females. We performed first of its kind multimodal analysis of LFS breast cancer (LFS-BC) compared to sporadic premenopausal BC. Nearly all LFS-BC underwent biallelic loss of TP53 with no recurrent oncogenic variants except ERBB2 (HER2) amplification. Compared to sporadic BC, in situ and invasive LFS-BC exhibited a high burden of short amplified aneuploid segments (SAAS). Pro-apoptotic p53 target genes BAX and TP53I3 failed to be up-regulated in LFS-BC as was seen in sporadic BC compared to normal breast tissue. LFS-BC had lower CD8+ T-cell infiltration compared to sporadic BC yet higher levels of proliferating cytotoxic T-cells. Within LFS-BC, progression from in situ to invasive BC was marked by an increase in chromosomal instability with a decrease in proliferating cytotoxic T-cells. Our study uncovers critical events in mutant p53-driven tumorigenesis in breast tissue.

Glossolomamagenticristatum (Gesneriaceae), a new species from the Cordillera Oriental of the Colombian Andes.

A narrowly endemic new species of Glossoloma is described from the Cordillera Oriental of the northern Andes, in the Colombian departments of Caquetá and Huila. Glossolomamagenticristatum J.L.Clark, D.Hoyos & Clavijo, sp. nov. differs from most other congeners by a habit that is usually epiphytic with elongate scandent subwoody shoots, the presence of a magenta corolla tube, and a creased calyx formed by tightly appressed adjacent lobes. A brief summary of Gesneriaceae diversity in the Colombia departments of Caquetá and Huila is discussed with an emphasis on the old highway between Florencia and Guadalupe. The conservation status of G.magenticristatum is assessed as Endangered (EN) based on IUCN Criteria.

ResumenSe describe una especie de Glossoloma endémica de la Cordillera Oriental de los Andes del norte, en los departamentos de Caquetá y Huila. Glossolomamagenticristatum J.L.Clark, D.Hoyos & Clavijo, sp. nov. se diferencia de otras especies en el género por el hábito usualmente epífito con ramas alargadas, escandentes y subleñosas, el tubo de la corola magenta, y un cáliz con los lóbulos estrechamente adpresos y con margen ondulada. Se presenta una breve síntesis de la diversidad de Gesneriaceae en los departamentos de Caquetá y Huila, con un énfasis en la vieja carretera entre Florencia y Guadalupe. Se evalúa el estado de conservación de G.magenticristatum y se propone que se debería categorizar como En Peligro (EN), de acuerdo a los criterios de la UICN.

Dengue Virus Infection Alters Inter-Endothelial Junctions and Promotes Endothelial-Mesenchymal-Transition-Like Changes in Human Microvascular Endothelial Cells.

Dengue virus (DENV) is a pathogenic arbovirus that causes human disease. The most severe stage of the disease (severe dengue) is characterized by vascular leakage, hypovolemic shock, and organ failure. Endothelial dysfunction underlies these phenomena, but the causal mechanisms of endothelial dysfunction are poorly characterized. This study investigated the role of c-ABL kinase in DENV-induced endothelial dysfunction. Silencing c-ABL with artificial miRNA or targeting its catalytic activity with imatinib revealed that c-ABL is required for the early steps of DENV infection. DENV-2 infection and conditioned media from DENV-infected cells increased endothelial expression of c-ABL and CRKII phosphorylation, promoted expression of mesenchymal markers, e.g., vimentin and N-cadherin, and decreased the levels of endothelial-specific proteins, e.g., VE-cadherin and ZO-1. These effects were reverted by silencing or inhibiting c-ABL. As part of the acquisition of a mesenchymal phenotype, DENV infection and treatment with conditioned media from DENV-infected cells increased endothelial cell motility in a c-ABL-dependent manner. In conclusion, DENV infection promotes a c-ABL-dependent endothelial phenotypic change that leads to the loss of intercellular junctions and acquisition of motility.

Correction: Escudero-Flórez et al. Dengue Virus Infection Alters Inter-Endothelial Junctions and Promotes Endothelial-Mesenchymal-Transition-like Changes in Human Microvascular Endothelial Cells. Viruses 2023, 15, 1437.

Ryan L [...].

Cancer cells co-evolve with retrotransposons to mitigate viral mimicry.

Overexpression of repetitive elements is an emerging hallmark of human cancers 1 . Diverse repeats can mimic viruses by replicating within the cancer genome through retrotransposition, or presenting pathogen-associated molecular patterns (PAMPs) to the pattern recognition receptors (PRRs) of the innate immune system 2-5 . Yet, how specific repeats affect tumor evolution and shape the tumor immune microenvironment (TME) in a pro- or anti-tumorigenic manner remains poorly defined. Here, we integrate whole genome and total transcriptome data from a unique autopsy cohort of multiregional samples collected in pancreatic ductal adenocarcinoma (PDAC) patients, into a comprehensive evolutionary analysis. We find that more recently evolved S hort I nterspersed N uclear E lements (SINE), a family of retrotransposable repeats, are more likely to form immunostimulatory double-strand RNAs (dsRNAs). Consequently, younger SINEs are strongly co-regulated with RIG-I like receptor associated type-I interferon genes but anti-correlated with pro-tumorigenic macrophage infiltration. We discover that immunostimulatory SINE expression in tumors is regulated by either L ong I nterspersed N uclear E lements 1 (LINE1/L1) mobility or ADAR1 activity in a TP53 mutation dependent manner. Moreover, L1 retrotransposition activity tracks with tumor evolution and is associated with TP53 mutation status. Altogether, our results suggest pancreatic tumors actively evolve to modulate immunogenic SINE stress and induce pro-tumorigenic inflammation. Our integrative, evolutionary analysis therefore illustrates, for the first time, how dark matter genomic repeats enable tumors to co-evolve with the TME by actively regulating viral mimicry to their selective advantage.

Perfecting antigen prediction.

Advances in genomics and precision measurement have continued to demonstrate the importance of the immune system across many disease types. At the heart of many emerging approaches to leverage these insights for precision immunotherapies is the computational antigen prediction problem. We propose a threefold approach to improving antigen predictions: further defining the geometry of the antigen landscape, incorporating the coupling of antigen recognition to other cellular processes, and diversifying the training sets used for models that predict immunogenicity.

The genotypes and phenotypes of missense mutations in the proline domain of the p53 protein.

The p53 protein is structurally and functionally divided into five domains. The proline-rich domain is localized at amino acids 55-100. 319 missense mutations were identified solely in the proline domain from human cancers. Six hotspot mutations were identified at amino acids 72, 73, 82, 84, 89, and 98. Codon 72 contains a polymorphism that changes from proline (and African descent) to arginine (with Caucasian descent) with increasing latitudes northward and is under natural selection for pigmentation and protection from UV light exposure. Cancers associated with mutations in the proline domain were considerably enriched for melanomas and skin cancers compared to mutations in other p53 domains. These hotspot mutations are enriched at UV mutational signatures disrupting amino acid signals for binding SH-3-containing proteins important for p53 function. Among the protein-protein interaction sites identified by hotspot mutations were MDM-2, a negative regulator of p53, XAF-1, promoting p53 mediated apoptosis, and PIN-1, a proline isomerase essential for structural folding of this domain.

Prolonged SARS-CoV-2 Infection in Patients with Lymphoid Malignancies.

Coronavirus disease 2019 (COVID-19) infection results in both acute mortality and persistent and/or recurrent disease in patients with hematologic malignancies, but the drivers of persistent infection in this population are unknown. We found that B-cell lymphomas were at particularly high risk for persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positivity. Further analysis of these patients identified discrete risk factors for initial disease severity compared with disease chronicity. Active therapy and diminished T-cell counts were drivers of acute mortality in COVID-19-infected patients with lymphoma. Conversely, B cell-depleting therapy was the primary driver of rehospitalization for COVID-19. In patients with persistent SARS-CoV-2 positivity, we observed high levels of viral entropy consistent with intrahost viral evolution, particularly in patients with impaired CD8+ T-cell immunity. These results suggest that persistent COVID-19 infection is likely to remain a risk in patients with impaired adaptive immunity and that additional therapeutic strategies are needed to enable viral clearance in this high-risk population. SIGNIFICANCE: We describe the largest cohort of persistent symptomatic COVID-19 infection in patients with lymphoid malignancies and identify B-cell depletion as the key immunologic driver of persistent infection. Furthermore, we demonstrate ongoing intrahost viral evolution in patients with persistent COVID-19 infection, particularly in patients with impaired CD8+ T-cell immunity.This article is highlighted in the In This Issue feature, p. 1.

Systemic and Oligo-Acquired Resistance to PD-(L)1 Blockade in Lung Cancer.

PURPOSE: Clinical patterns and the associated optimal management of acquired resistance to PD-(L)1 blockade are poorly understood. EXPERIMENTAL DESIGN: All cases of metastatic lung cancer treated with PD-(L)1 blockade at Memorial Sloan Kettering were reviewed. In acquired resistance (complete/partial response per RECIST, followed by progression), clinical patterns were distinguished as oligo (OligoAR ≤ 3 lesions of disease progression) or systemic (sAR). We analyzed the relationships between patient characteristics, burden/location of disease, outcomes, and efficacy of therapeutic interventions. RESULTS: Of 1,536 patients, 312 (20%) had an initial response and 143 developed AR (9% overall, 46% of responders). OligoAR was the most common pattern (80/143, 56%). Baseline tumor mutational burden, depth of response, and duration of response were significantly increased in oligoAR compared with sAR (P < 0.001, P = 0.03, P = 0.04, respectively), whereas baseline PD-L1 and tumor burden were similar. Post-progression, oligoAR was associated with improved overall survival (median 28 months vs. 10 months, P < 0.001) compared with sAR. Within oligoAR, post-progression survival was greater among patients treated with locally-directed therapy (e.g., radiation, surgery; HR, 0.41; P = 0.039). Fifty-eight percent of patients with oligoAR treated with locally-directed therapy alone are progression-free at last follow-up (median 16 months), including 13 patients who are progression-free more than 2 years after local therapy. CONCLUSIONS: OligoAR is a common and distinct pattern of acquired resistance to PD-(L)1 blockade compared with sAR. OligoAR is associated with improved post-progression survival and some cases can be effectively managed with local therapies with durable benefit.

Calreticulin mutant myeloproliferative neoplasms induce MHC-I skewing, which can be overcome by an optimized peptide cancer vaccine.

The majority of JAK2V617F-negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin (CALR), resulting in a common carboxyl-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in patients with CALRMUT MPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALRMUT MPN from two independent cohorts. We observed that MHC-I alleles that present CALRMUT neoepitopes with high affinity are underrepresented in patients with CALRMUT MPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALRMUT MPN would not efficiently respond to a CALRMUT fragment cancer vaccine but would when immunized with a modified CALRMUT heteroclitic peptide vaccine approach. We found that heteroclitic CALRMUT peptides specifically designed for the MHC-I alleles of patients with CALRMUT MPN efficiently elicited a CALRMUT cross-reactive CD8+ T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALRMUT native peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8+ T cell response to the CALRMUT fragment upon immunization with a CALRMUT heteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide-based cancer vaccines in patients with CALRMUT MPN.

Dataset for the life cycle assessment of the high speed rail network in Spain.

A life cycle assessment (LCA) of the Spanish high speed rail (HSR) network in service in 2016 (2583 km) was conducted. Life cycle inventory (LCI) data related to the construction and maintenance phases of the infrastructure was collected using Google Earth tool, and complemented with data obtained from the LCA carried out by Tuchschmid et al. [1]. LCI data associated with the operation phase of the infrastructure was built on available fragmentary data on passenger movements for the year 2016 [2-4], processed with a python algorithm to estimate the transport service provided by the infrastructure. Environmental impacts for transport modes were obtained from Ecoinvent v3.7 database [5,6] and processed with openLCA software [7]. Life cycle impact assessment (LCIA) results gathered in the dataset include Global Warming (GWP100a), Cumulative Energy Demand and total emissions for PM10, SO2, NOX and NMVOC. This dataset presents a detailed description of the Spanish HSR network, including the length of each item (bridges, tunnels, earthworks, railway tracks), and a robust estimation of passenger transport over the infrastructure for year 2016. The LCI data presented in this paper support the original research done on whether the construction of Spanish HSR network infrastructure is justified in terms of reducing environmental impacts and energy consumption [8], and may be used as a baseline for future studies on transport economics.

Radiation therapy enhances immunotherapy response in microsatellite stable colorectal and pancreatic adenocarcinoma in a phase II trial.

Overcoming intrinsic resistance to immune checkpoint blockade for microsatellite stable (MSS) colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) remains challenging. We conducted a single-arm, non-randomized, phase II trial (NCT03104439) combining radiation, ipilimumab and nivolumab to treat patients with metastatic MSS CRC (n = 40) and PDAC (n = 25) with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. The primary endpoint was disease control rate (DCR) by intention to treat. DCRs were 25% for CRC (ten of 40; 95% confidence interval (CI), 13-41%) and 20% for PDAC (five of 25; 95% CI, 7-41%). In the per-protocol analysis, defined as receipt of radiation, DCR was 37% (ten of 27; 95% CI, 19-58%) in CRC and 29% (five of 17; 95% CI, 10-56%) in PDAC. Pretreatment biopsies revealed low tumor mutational burden for all samples but higher numbers of natural killer (NK) cells and expression of the HERVK repeat RNA in patients with disease control. This study provides proof of concept of combining radiation with immune checkpoint blockade in immunotherapy-resistant cancers.

Response of Leptomeningeal Metastases in EGFR-Mutated Non-Small-Cell Lung Cancer to Afatinib in the Absence of Radiotherapy.

Palliative radiotherapy is currently the medical standard of care for non-small-cell lung cancer (NSCLC) patients with symptomatic CNS and leptomeningeal disease. We report the case of a 62-year-old male patient with EGFR mutation (del19+) NSLC with symptomatic lymph node, bone, CNS, and leptomeningeal metastases. Taking into account on one hand the response to tyrosine kinase inhibitors (TKIs) and on the other hand the short- to medium-term side effects of radiotherapy and the lack of timely availability in our healthcare system, the patient was treated with afatinib (40 mg daily) and exhibited a rapid response with improvement of neurological symptoms. The patient presented partial response of extracranial, CNS, and leptomeningeal lesions at 3, 6, and 12 months of treatment, currently completing 16 months of progression-free survival despite presenting mild dermatological and gastrointestinal toxicities. Afatinib is an effective and safe option in patients with NSLC EGFR mutation del19+ with CNS and leptomeningeal compromise avoiding or delaying radiotherapy and its side effects, especially in countries where there is a lack of access to this kind of therapy.

A multiple indicator solution approach to endogeneity in discrete-choice models for environmental valuation.

Endogeneity is an often neglected issue in empirical applications of discrete choice modelling despite its severe consequences in terms of inconsistent parameter estimation and biased welfare measures. This article analyses the performance of the multiple indicator solution method to deal with endogeneity arising from omitted explanatory variables in discrete choice models for environmental valuation. We also propose and illustrate a factor analysis procedure for the selection of the indicators in practice. Additionally, the performance of this method is compared with the recently proposed hybrid choice modelling framework. In an empirical application we find that the multiple indicator solution method and the hybrid model approach provide similar results in terms of welfare estimates, although the multiple indicator solution method is more parsimonious and notably easier to implement. The empirical results open a path to explore the performance of this method when endogeneity is thought to have a different cause or under a different set of indicators.

Incorporating environmental attitudes in discrete choice models: an exploration of the utility of the awareness of consequences scale.

Environmental economists are increasingly interested in better understanding how people cognitively organise their beliefs and attitudes towards environmental change in order to identify key motives and barriers that stimulate or prevent action. In this paper, we explore the utility of a commonly used psychometric scale, the awareness of consequences (AC) scale, in order to better understand stated choices. The main contribution of the paper is that it provides a novel approach to incorporate attitudinal information into discrete choice models for environmental valuation: firstly, environmental attitudes are incorporated using a reinterpretation of the classical AC scale recently proposed by Ryan and Spash (2012); and, secondly, attitudinal data is incorporated as latent variables under a hybrid choice modelling framework. This novel approach is applied to data from a survey conducted in the Basque Country (Spain) in 2008 aimed at valuing land-use policies in a Natura 2000 Network site. The results are relevant to policy-making because choice models that are able to accommodate underlying environmental attitudes may help in designing more effective environmental policies.