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Background: Health literacy is important in chronic conditions, such as kidney transplantation. Understanding patients' health literacy profiles can assist tailoring follow-up and educational programmes to the health literacy needs of vulnerable kidney transplant recipients. This approach enabled us to cluster patients according to their profiles of challenges and strengths in different health literacy domains., Objectives: This study aimed to identify different health literacy profiles within kidney transplant recipients and what characterized the different profiles., Design: Cross-sectional study., Participants: One hundred ninety-five kidney transplanted recipients were included., Measurements: We used the self-reported Health Literacy Questionnaire and analyzed using Ward's method (hierarchical cluster approach). We also collected background characteristics and clinical variables, including psychological distress (Hopkins Symptoms Checklist) and perceived health status (visual analogue scale, EuroQol-5D)., Results: The analysis revealed four clusters with substantial differences in health literacy profiles. One cluster's patients had the most challenges in all health literacy domains constituting 24% of the sample. Compared to the other three clusters, this cluster was associated with shorter duration of kidney disease, higher number of patients in dialysis before transplantation, higher percentage of male patients, lower number of kidneys from living donors, higher number of patients not working and higher representation of psychological distress. All four clusters reported the most challenges in the same domain: the ability to critically appraise health information., Conclusion: In kidney transplant recipients, profiling clusters with the Health Literacy Questionnaire and Ward's method aids in identifying health literacy needs in vulnerable groups, enabling transplant professionals to offer tailored health literacy support., (© 2024 The Author(s). Journal of Renal Care published by John Wiley & Sons Ltd on behalf of European Dialysis & Transplant Nurses Association/European Renal Care Association.)

The human vascular system, comprising endothelial cells (ECs) and mural cells, covers a vast surface area in the body, providing a critical interface between blood and tissue environments. Functional differences exist across specific vascular beds, but their molecular determinants across tissues remain largely unknown. In this study, we integrated single-cell transcriptomics data from 19 human organs and tissues and defined 42 vascular cell states from approximately 67,000 cells (62 donors), including angiotypic transitional signatures along the arterial endothelial axis from large to small caliber vessels. We also characterized organotypic populations, including splenic littoral and blood-brain barrier ECs, thus clarifying the molecular profiles of these important cell states. Interrogating endothelial-mural cell molecular crosstalk revealed angiotypic and organotypic communication pathways related to Notch, Wnt, retinoic acid, prostaglandin and cell adhesion signaling. Transcription factor network analysis revealed differential regulation of downstream target genes in tissue-specific modules, such as those of FOXF1 across multiple lung vascular subpopulations. Additionally, we make mechanistic inferences of vascular drug targets within different vascular beds. This open-access resource enhances our understanding of angiodiversity and organotypic molecular signatures in human vascular cells, and has therapeutic implications for vascular diseases across tissues., Competing Interests: Competing interests: In the past 3 years, S.A.T. has consulted for or been a member of scientific advisory boards (SABs) at Qiagen, OMass Therapeutics, Xaira Therapeutics and ForeSite Labs, and a non-executive director of 10x Genomics. She is a co-founder and equity holder of TransitionBio and Ensocell, and a part-time employee at GSK. R.E. is a co-founder and equity holder in Ensocell. The other authors declare no competing interests., (© 2024. The Author(s).)

Background: In organ donation from deceased donors, the interaction between the donor's relatives and intensive care personnel is an important factor. The organ donation (OD) process is complex, and patients' relatives play a vital role. Intensive care professionals need knowledge about how relatives perceive and experience the process to create a caring environment and support them throughout. Therefore, this collaborative project aims to explore both relatives' and intensive care personnel's perspectives of care in deceased organ donation in Scandinavia., Objective: This study aims to (1) investigate donor relatives' satisfaction and ICU personnel's perception of their own professional competence and (2) explore donor relatives' and ICU personnel's experiences in the OD process to design for care and support in OD., Methods: This protocol outlines a Scandinavian (Norway, Sweden, and Denmark) project, including 4 work packages. Work package 1 started in 2023 with the translation and validation of the Family Satisfaction in the Intensive Care Unit questionnaire into a Danish version and the translation of the Professional Competence in Organ Donation Questionnaire into a Swedish and Danish version. A cross-sectional survey measuring Scandinavian relatives' perception of support in and satisfaction with the organ donation process and a cross-sectional survey measuring Scandinavian intensive care personnel's competence in organ donation are the foundation for work package 2 (2024). The data from both surveys will be analyzed using descriptive and comparative analysis. The results will inform the interview guides in qualitative studies (work packages 3 and 4). Participants in the quantitative study will be invited to participate in in-depth interviews. In work package 3, in-depth interviews will be conducted to illuminate relatives' experiences in the organ donation process. The interviews will be analyzed using thematic analysis, according to Braun and Clarke. In work package 4 (2025-2026), 1 qualitative design study will be conducted to illuminate ICU personnel's experiences. Furthermore, the results from work packages 2 and 3 will inform the development of specific programs for care, support, and communication in the organ donation process., Results: The project was funded by the Norwegian Organ Donor Foundation in 2022 and Scandiatransplant in 2023. The Norwegian Nurses Organisation supports the project by funding a PhD student. The PhD student was employed by the University in Agder in May 2024., Conclusions: This project will provide new knowledge that will assist us in designing and establishing programs for care, support, and donor relatives' involvement in OD processes., International Registered Report Identifier (irrid): PRR1-10.2196/55643., (©Kathe B Meyer, Gudrun Rohde, Gro Frivold. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 11.11.2024.)

The gastrointestinal tract is a multi-organ system crucial for efficient nutrient uptake and barrier immunity. Advances in genomics and a surge in gastrointestinal diseases 1,2 has fuelled efforts to catalogue cells constituting gastrointestinal tissues in health and disease 3 . Here we present systematic integration of 25 single-cell RNA sequencing datasets spanning the entire healthy gastrointestinal tract in development and in adulthood. We uniformly processed 385 samples from 189 healthy controls using a newly developed automated quality control approach (scAutoQC), leading to a healthy reference atlas with approximately 1.1 million cells and 136 fine-grained cell states. We anchor 12 gastrointestinal disease datasets spanning gastrointestinal cancers, coeliac disease, ulcerative colitis and Crohn's disease to this reference. Utilizing this 1.6 million cell resource (gutcellatlas.org), we discover epithelial cell metaplasia originating from stem cells in intestinal inflammatory diseases with transcriptional similarity to cells found in pyloric and Brunner's glands. Although previously linked to mucosal healing 4 , we now implicate pyloric gland metaplastic cells in inflammation through recruitment of immune cells including T cells and neutrophils. Overall, we describe inflammation-induced changes in stem cells that alter mucosal tissue architecture and promote further inflammation, a concept applicable to other tissues and diseases., Competing Interests: Competing interests: S.A.T. is a scientific advisory board member of ForeSite Labs, OMass Therapeutics, a co-founder and equity holder of TransitionBio and EnsoCell Therapeutics, a non-executive director of 10x Genomics and a part-time employee of GlaxoSmithKline. R.E. is an equity holder in EnsoCell. P.K. has consulted for AstraZeneca, UCB, Biomunex and Infinitopes. N.M.P reports consulting fees from Infinitopes. J.S.-R. reports funding from GSK, Pfizer and Sanofi and fees/honoraria from Travere Therapeutics, Stadapharm, Astex, Owkin, Pfizer, Moderna and Grunenthal. A.S. is the recipient of research grants from Roche-Genentech, Abbvie, GSK, Scipher Medicine, Pfizer, Alimentiv, Boehringer Ingelheim and Agomab and has received consulting fees from Genentech, GSK, Pfizer, HotSpot Therapeutics, Alimentiv, Agomab, Goodgut and Orikine. R.E. and S.A.T are inventors on the patent GB2412853.0 filed in the UK, some components of which are related to this work. All other authors declare no competing interests., (© 2024. The Author(s).)

Human embryonic bone and joint formation is determined by coordinated differentiation of progenitors in the nascent skeleton. The cell states, epigenetic processes and key regulatory factors that underlie lineage commitment of these cells remain elusive. Here we applied paired transcriptional and epigenetic profiling of approximately 336,000 nucleus droplets and spatial transcriptomics to establish a multi-omic atlas of human embryonic joint and cranium development between 5 and 11 weeks after conception. Using combined modelling of transcriptional and epigenetic data, we characterized regionally distinct limb and cranial osteoprogenitor trajectories across the embryonic skeleton and further described regulatory networks that govern intramembranous and endochondral ossification. Spatial localization of cell clusters in our in situ sequencing data using a new tool, ISS-Patcher, revealed mechanisms of progenitor zonation during bone and joint formation. Through trajectory analysis, we predicted potential non-canonical cellular origins for human chondrocytes from Schwann cells. We also introduce SNP2Cell, a tool to link cell-type-specific regulatory networks to polygenic traits such as osteoarthritis. Using osteolineage trajectories characterized here, we simulated in silico perturbations of genes that cause monogenic craniosynostosis and implicate potential cell states and disease mechanisms. This work forms a detailed and dynamic regulatory atlas of bone and cartilage maturation and advances our fundamental understanding of cell-fate determination in human skeletal development., Competing Interests: Competing interests: C.D.B. is a founder of Mestag Therapeutics. S.A.T. is a scientific advisory board member of ForeSite Labs, OMass Therapeutics, Qiagen, a co-founder and equity holder of TransitionBio and EnsoCell Therapeutics, a non-executive director of 10x Genomics and a part-time employee of GlaxoSmithKline. Sarah also holds equity in TransitionBio and options for equity in Element Biosciences, where she is a former SAB member.  All other authors declare no competing interests., (© 2024. The Author(s).)

Over the last decade, single-cell genomics has revealed remarkable heterogeneity and plasticity of cell types in the lungs and airways. The challenge now is to understand how these cell types interact in three-dimensional space to perform lung functions, facilitating airflow and gas exchange while simultaneously providing barrier function to avoid infection. An explosion in novel spatially resolved gene expression technologies, coupled with computational tools that harness machine learning and deep learning, now promise to address this challenge. Here, we review the most commonly used spatial analysis workflows, highlighting their advantages and limitations, and outline recent developments in machine learning and artificial intelligence that will augment how we interpret spatial data. Together these technologies have the potential to transform our understanding of the respiratory system in health and disease, and we showcase studies in lung development, COVID-19, lung cancer, and fibrosis where spatially resolved transcriptomics is already providing novel insights.

Single-cell data analysis can infer dynamic changes in cell populations, for example across time, space or in response to perturbation, thus deriving pseudotime trajectories. Current approaches comparing trajectories often use dynamic programming but are limited by assumptions such as the existence of a definitive match. Here we describe Genes2Genes, a Bayesian information-theoretic dynamic programming framework for aligning single-cell trajectories. It is able to capture sequential matches and mismatches of individual genes between a reference and query trajectory, highlighting distinct clusters of alignment patterns. Across both real world and simulated datasets, it accurately inferred alignments and demonstrated its utility in disease cell-state trajectory analysis. In a proof-of-concept application, Genes2Genes revealed that T cells differentiated in vitro match an immature in vivo state while lacking expression of genes associated with TNF signaling. This demonstrates that precise trajectory alignment can pinpoint divergence from the in vivo system, thus guiding the optimization of in vitro culture conditions., (© 2024. The Author(s).)

Here we propose "molecular connectomics" to link molecular and morphological cell features in three dimensions across scales, using machine learning and artificial intelligence to reveal emergent properties of complex biological systems., Competing Interests: In the past 3 years, S.A.T. has received remuneration for scientific advisory board membership from Sanofi, GlaxoSmithKline, Foresite Labs and Qiagen. S.A.T. is a co-founder and holds equity in Transition Bio and Ensocell. From 8 January 2024, S.A.T. is a part-time employee of GlaxoSmithKline., (Copyright: © 2024 Megas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The COVID-19 pandemic is an ongoing global health threat, yet our understanding of the dynamics of early cellular responses to this disease remains limited 1 . Here in our SARS-CoV-2 human challenge study, we used single-cell multi-omics profiling of nasopharyngeal swabs and blood to temporally resolve abortive, transient and sustained infections in seronegative individuals challenged with pre-Alpha SARS-CoV-2. Our analyses revealed rapid changes in cell-type proportions and dozens of highly dynamic cellular response states in epithelial and immune cells associated with specific time points and infection status. We observed that the interferon response in blood preceded the nasopharyngeal response. Moreover, nasopharyngeal immune infiltration occurred early in samples from individuals with only transient infection and later in samples from individuals with sustained infection. High expression of HLA-DQA2 before inoculation was associated with preventing sustained infection. Ciliated cells showed multiple immune responses and were most permissive for viral replication, whereas nasopharyngeal T cells and macrophages were infected non-productively. We resolved 54 T cell states, including acutely activated T cells that clonally expanded while carrying convergent SARS-CoV-2 motifs. Our new computational pipeline Cell2TCR identifies activated antigen-responding T cells based on a gene expression signature and clusters these into clonotype groups and motifs. Overall, our detailed time series data can serve as a Rosetta stone for epithelial and immune cell responses and reveals early dynamic responses associated with protection against infection., (© 2024. The Author(s).)

Single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq) has emerged as a powerful tool for dissecting regulatory landscapes and cellular heterogeneity. However, an exploration of systemic biases among scATAC-seq technologies has remained absent. In this study, we benchmark the performance of eight scATAC-seq methods across 47 experiments using human peripheral blood mononuclear cells (PBMCs) as a reference sample and develop PUMATAC, a universal preprocessing pipeline, to handle the various sequencing data formats. Our analyses reveal significant differences in sequencing library complexity and tagmentation specificity, which impact cell-type annotation, genotype demultiplexing, peak calling, differential region accessibility and transcription factor motif enrichment. Our findings underscore the importance of sample extraction, method selection, data processing and total cost of experiments, offering valuable guidance for future research. Finally, our data and analysis pipeline encompasses 169,000 PBMC scATAC-seq profiles and a best practices code repository for scATAC-seq data analysis, which are freely available to extend this benchmarking effort to future protocols., (© 2023. The Author(s).)

Objective: This study aimed to evaluate the effect of a new health communication intervention focusing on knowledge management skills on health literacy and medication adherence during the first year following kidney transplantation., Methods: We randomized 195 patients during 2020-2021, to either intervention- or control group. Questionnaires were completed at baseline and at 12 months post-transplantation with a 12-month response rate of 84%. Health literacy was measured by the multidimensional Health Literacy Questionnaire (HLQ) instrument. Medication adherence was measured by the self-reported questionnaire (BAASIS©)., Results: Results showed that the intervention group had a significant increase in 2 HLQ domains compared to the control group capturing the "ability to appraise health information" Domain 5, (p-value = 0.002) and the "ability to navigate the healthcare system" Domain 7, (p-value <0.04). The effect sizes of SRM were 0.49 (Domain 5) and 0.33 (Domain 7). Medication adherence was comparable in the groups at any measure points., Conclusions: This study contributes to important knowledge about how a health communication intervention focusing on knowledge translation using motivational interviewing techniques positively strengthens health literacy in kidney transplant recipients., Practical Implications: Current patient education practice may benefit from focusing on knowledge translation in combination with motivational interview technique., Competing Interests: Declaration of Competing Interest The authors have no relevant financial or non-financial conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: To investigate health literacy (HL) and digital health literacy (eHL) among patients hospitalised in surgical and medical wards using a cluster analysis approach., Design: Cross-sectional study using Ward's hierarchical clustering method to measure cluster adequacy by evaluating distances between cluster centroids (a measure of cohesion). Different distances produced different cluster solutions., Setting and Participants: The study was conducted at a Norwegian university hospital. A total of 260 hospitalised patients were enrolled between 24 May and 6 June 2021., Data Collection: All data were collected by self-reported questionnaires. Data on HL and eHL were collected by the Health Literacy Questionnaire (HLQ) and the eHealth Literacy Questionnaire (eHLQ). We also collected data on background characteristics, health status and patient diagnosis., Results: We found six HLQ clusters to be the best solution of the sample, identifying substantial diversity in HL strengths and challenges. Two clusters, representing 21% of the total sample, reported the lowest HLQ scores in eight of nine HLQ domains. Compared with the other clusters, these two contained the highest number of women, as well as the patients with the highest mean age, a low level of education and the lowest proportion of being employed. One of these clusters also represented patients with the lowest health status score. We identified six eHL clusters, two of which represented 31% of the total sample with the lowest eHLQ scores in five of seven eHLQ domains, with background characteristics comparable to patients in the low-scoring HLQ clusters., Conclusions: This study provides new, nuanced knowledge about HL and eHL profiles in different clusters of patients hospitalised in surgical and medical wards. With such data, healthcare professionals can take into account vulnerable patients' HL needs and tailor information and communication accordingly., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Multisystem inflammatory syndrome in children is a post-infectious presentation SARS-CoV-2 associated with expansion of the T cell receptor Vβ21.3+ T-cell subgroup. Here we apply muti-single cell omics to compare the inflammatory process in children with acute respiratory COVID-19 and those presenting with non SARS-CoV-2 infections in children. Here we show that in Multi-Inflammatory Syndrome in Children (MIS-C), the natural killer cell and monocyte population demonstrate heightened CD95 (Fas) and Interleuking 18 receptor expression. Additionally, TCR Vβ21.3+ CD4+ T-cells exhibit skewed differentiation towards T helper 1, 17 and regulatory T cells, with increased expression of the co-stimulation receptors ICOS, CD28 and interleukin 18 receptor. We observe no functional evidence for NLRP3 inflammasome pathway overactivation, though MIS-C monocytes show elevated active caspase 8. This, coupled with raised IL18 mRNA expression in CD16- NK cells on single cell RNA sequencing analysis, suggests interleukin 18 and CD95 signalling may trigger activation of TCR Vβ21.3+ T-cells in MIS-C, driven by increased IL-18 production from activated monocytes and CD16- Natural Killer cells., (© 2024. The Author(s).)

Children infected with SARS-CoV-2 rarely progress to respiratory failure. However, the risk of mortality in infected people over 85 years of age remains high. Here we investigate differences in the cellular landscape and function of paediatric (<12 years), adult (30-50 years) and older adult (>70 years) ex vivo cultured nasal epithelial cells in response to infection with SARS-CoV-2. We show that cell tropism of SARS-CoV-2, and expression of ACE2 and TMPRSS2 in nasal epithelial cell subtypes, differ between age groups. While ciliated cells are viral replication centres across all age groups, a distinct goblet inflammatory subtype emerges in infected paediatric cultures and shows high expression of interferon-stimulated genes and incomplete viral replication. In contrast, older adult cultures infected with SARS-CoV-2 show a proportional increase in basaloid-like cells, which facilitate viral spread and are associated with altered epithelial repair pathways. We confirm age-specific induction of these cell types by integrating data from in vivo COVID-19 studies and validate that our in vitro model recapitulates early epithelial responses to SARS-CoV-2 infection., (© 2024. The Author(s).)

Skeletal muscle aging is a key contributor to age-related frailty and sarcopenia with substantial implications for global health. Here we profiled 90,902 single cells and 92,259 single nuclei from 17 donors to map the aging process in the adult human intercostal muscle, identifying cellular changes in each muscle compartment. We found that distinct subsets of muscle stem cells exhibit decreased ribosome biogenesis genes and increased CCL2 expression, causing different aging phenotypes. Our atlas also highlights an expansion of nuclei associated with the neuromuscular junction, which may reflect re-innervation, and outlines how the loss of fast-twitch myofibers is mitigated through regeneration and upregulation of fast-type markers in slow-twitch myofibers with age. Furthermore, we document the function of aging muscle microenvironment in immune cell attraction. Overall, we present a comprehensive human skeletal muscle aging resource ( https://www.muscleageingcellatlas.org/ ) together with an in-house mouse muscle atlas to study common features of muscle aging across species., (© 2024. The Author(s).)

Background: Lung cancer is the leading cause of cancer-related death in the world. In contrast to many other cancers, a direct connection to modifiable lifestyle risk in the form of tobacco smoke has long been established. More than 50% of all smoking-related lung cancers occur in former smokers, 40% of which occur more than 15 years after smoking cessation. Despite extensive research, the molecular processes for persistent lung cancer risk remain unclear. We thus set out to examine whether risk stratification in the clinic and in the general population can be improved upon by the addition of genetic data and to explore the mechanisms of the persisting risk in former smokers., Methods: We analysed transcriptomic data from accessible airway tissues of 487 subjects, including healthy volunteers and clinic patients of different smoking statuses. We developed a computational model to assess smoking-associated gene expression changes and their reversibility after smoking is stopped, comparing healthy subjects to clinic patients with and without lung cancer., Results: We find persistent smoking-associated immune alterations to be a hallmark of the clinic patients. Integrating previous GWAS data using a transcriptional network approach, we demonstrate that the same immune- and interferon-related pathways are strongly enriched for genes linked to known genetic risk factors, demonstrating a causal relationship between immune alteration and lung cancer risk. Finally, we used accessible airway transcriptomic data to derive a non-invasive lung cancer risk classifier., Conclusions: Our results provide initial evidence for germline-mediated personalized smoke injury response and risk in the general population, with potential implications for managing long-term lung cancer incidence and mortality., (© 2024. The Author(s).)

Background: Clinical practice guidelines emphasize shared decision-making for kidney replacement treatment, yet little is known about the influence of cultural differences on that process. We undertook a retrospective chart review to explore the process and timing of dialysis decision making and initiation in Chinese American patients to provide quality kidney care for this population., Design: Participants received outpatient care at Tufts Medical Center and dialysis at Dialysis Clinic, Inc. Boston or Somerville, MA from 2001-2021. Clinic chart review sourced demographic, clinical, and end-of-life care information from 180 participants (82 Chinese American, 98 other) from stage 4 chronic kidney disease (CKD) and dialysis initiation., Results: Chinese American participants were older (mean 70 vs. 59, p < 0.0001), less likely to speak English (12% vs. 87%, p < 0.0001), and used interpreter services more (80% vs. 11%, p < 0.0001). Chinese American participants had more visits (median 14 vs. 10, p = 0.005); were more often accompanied by family members (75% vs. 40%, p < 0.001); and had significantly lower rates of healthcare proxy documentation (35% vs. 55%, p = 0.006). There was no statistical difference in months between first CKD 4 visit and first dialysis. Both groups started dialysis at the same average eGFR and with similar rates of permanent dialysis access. Chinese American participants had significantly lower serum albumin at dialysis initiation (mean 3.3 g/dL vs 3.7 g/dL, p = 0.0003). Documentation reflected a low number of conversations about non-dialytic care, end-of-life planning, or palliative care in both groups across all visits., Conclusion: The time between CKD 4 and dialysis initiation was the same in both groups, suggesting a similar overall outcome of care. Chart documentation suggests that Chinese American participants had a significantly higher number of visits with nephrologists where discussion about dialysis was noted and were more likely to have a family member present at the visit. Fewer Chinese American participants completed healthcare proxies. Among all study participants, healthcare proxy, code status, and palliative care discussions were reported less frequently than expected. These findings highlight opportunities for collaboration between palliative care clinicians and nephrologists., (© 2024. The Author(s).)

Assessment of single-cell gene expression (single-cell RNA sequencing) and adaptive immune receptor (AIR) sequencing (scVDJ-seq) has been invaluable in studying lymphocyte biology. Here we introduce Dandelion, a computational pipeline for scVDJ-seq analysis. It enables the application of standard V(D)J analysis workflows to single-cell datasets, delivering improved V(D)J contig annotation and the identification of nonproductive and partially spliced contigs. We devised a strategy to create an AIR feature space that can be used for both differential V(D)J usage analysis and pseudotime trajectory inference. The application of Dandelion improved the alignment of human thymic development trajectories of double-positive T cells to mature single-positive CD4/CD8 T cells, generating predictions of factors regulating lineage commitment. Dandelion analysis of other cell compartments provided insights into the origins of human B1 cells and ILC/NK cell development, illustrating the power of our approach. Dandelion is available at https://www.github.com/zktuong/dandelion ., (© 2023. The Author(s).)

Harmonizing cell types across the single-cell community and assembling them into a common framework is central to building a standardized Human Cell Atlas. Here, we present CellHint, a predictive clustering tree-based tool to resolve cell-type differences in annotation resolution and technical biases across datasets. CellHint accurately quantifies cell-cell transcriptomic similarities and places cell types into a relationship graph that hierarchically defines shared and unique cell subtypes. Application to multiple immune datasets recapitulates expert-curated annotations. CellHint also reveals underexplored relationships between healthy and diseased lung cell states in eight diseases. Furthermore, we present a workflow for fast cross-dataset integration guided by harmonized cell types and cell hierarchy, which uncovers underappreciated cell types in adult human hippocampus. Finally, we apply CellHint to 12 tissues from 38 datasets, providing a deeply curated cross-tissue database with ∼3.7 million cells and various machine learning models for automatic cell annotation across human tissues., Competing Interests: Declaration of interests S.A.T. is a remunerated member of the Scientific Advisory Board, Foresite Labs, and Qiagen. She is co-founder and equity holder of Transition Bio., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Studies of human lung development have focused on epithelial and mesenchymal cell types and function, but much less is known about the developing lung immune cells, even though the airways are a major site of mucosal immunity after birth. An unanswered question is whether tissue-resident immune cells play a role in shaping the tissue as it develops in utero. Here, we profiled human embryonic and fetal lung immune cells using scRNA-seq, smFISH, and immunohistochemistry. At the embryonic stage, we observed an early wave of innate immune cells, including innate lymphoid cells, natural killer cells, myeloid cells, and lineage progenitors. By the canalicular stage, we detected naive T lymphocytes expressing high levels of cytotoxicity genes and the presence of mature B lymphocytes, including B-1 cells. Our analysis suggests that fetal lungs provide a niche for full B cell maturation. Given the presence and diversity of immune cells during development, we also investigated their possible effect on epithelial maturation. We found that IL-1β drives epithelial progenitor exit from self-renewal and differentiation to basal cells in vitro. In vivo, IL-1β-producing myeloid cells were found throughout the lung and adjacent to epithelial tips, suggesting that immune cells may direct human lung epithelial development.

Cell type-specific differential gene expression analyses based on single-cell transcriptome datasets are sensitive to the presence of cell-free mRNA in the droplets containing single cells. This so-called ambient RNA contamination may differ between samples obtained from patients and healthy controls. Current ambient RNA correction methods were not developed specifically for single-cell differential gene expression (sc-DGE) analyses and might therefore not sufficiently correct for ambient RNA-derived signals. Here, we show that ambient RNA levels are highly sample-specific. We found that without ambient RNA correction, sc-DGE analyses erroneously identify transcripts originating from ambient RNA as cell type-specific disease-associated genes. We therefore developed a computationally lean and intuitive correction method, Fast Correction for Ambient RNA (FastCAR), optimized for sc-DGE analysis of scRNA-Seq datasets generated by droplet-based methods including the 10XGenomics Chromium platform. FastCAR uses the profile of transcripts observed in libraries that likely represent empty droplets to determine the level of ambient RNA in each individual sample, and then corrects for these ambient RNA gene expression values. FastCAR can be applied as part of the data pre-processing and QC in sc-DGE workflows comparing scRNA-Seq data in a health versus disease experimental design. We compared FastCAR with two methods previously developed to remove ambient RNA, SoupX and CellBender. All three methods identified additional genes in sc-DGE analyses that were not identified in the absence of ambient RNA correction. However, we show that FastCAR performs better at correcting gene expression values attributed to ambient RNA, resulting in a lower frequency of false-positive observations. Moreover, the use of FastCAR in a sc-DGE workflow increases the cell-type specificity of sc-DGE analyses across disease conditions., (© 2023. The Author(s).)

Aims: To perform the first psychometric analysis of the Norwegian version of the eHLQ using confirmative factor analysis (CFA) procedures in a population of patients admitted to hospital using a cross-sectional design. The eHLQ consists of 35 items capturing the 7-dimensional eHealth Literacy Framework (eHLF) which describes users' attributes, user's interaction with technologies and user's experience with digital health systems., Methods: The 7 independent scales of the eHLQ was translated from Danish and culturally adapted into the Norwegian language following a standardised protocol. Assessment of construct validity of the eHLQ was undertaken using data from a cross-sectional survey of 260 patients hospitalized at a Norwegian University Hospital in the Oslo area during a two-week period in June 2021. The analysis included using correlation analysis (Pearsons R), internal consistency (Cronbach's alpha) and confirmatory factor analysis (CFA)., Results: All factor loadings were high to acceptable (i.e. > 0.6), except for five items which had somewhat lower loadings. Regarding internal consistency, alpha ranged from 0.73 to 0.90. For optimal CFA fit for the different scale models, correlated residuals were required for five of the seven scales. Overall our analysis shows an intermediate fit of the orginal construct. Scale intercorrelations were all below 0.8, indicating an overall acceptable discriminant validity between the 7 dimensions., Conclusions: The results from the CFA analysis indicate that for almost all 7 eHLQ scales, an acceptable model fit was achieved. The 260 hospitalized patients included in this study represented a variety of diagnoses, recruited from a geographically limited area. Further studies on psychometric properties of the Norwegian version of eHLQ in larger samples, diverse settings and by using more comprehensive approaches are warranted., (© 2023. The Author(s).)

Joint analysis of single-cell genomics data from diseased tissues and a healthy reference can reveal altered cell states. We investigate whether integrated collections of data from healthy individuals (cell atlases) are suitable references for disease-state identification and whether matched control samples are needed to minimize false discoveries. We demonstrate that using a reference atlas for latent space learning followed by differential analysis against matched controls leads to improved identification of disease-associated cells, especially with multiple perturbed cell types. Additionally, when an atlas is available, reducing control sample numbers does not increase false discovery rates. Jointly analyzing data from a COVID-19 cohort and a blood cell atlas, we improve detection of infection-related cell states linked to distinct clinical severities. Similarly, we studied disease states in pulmonary fibrosis using a healthy lung atlas, characterizing two distinct aberrant basal states. Our analysis provides guidelines for designing disease cohort studies and optimizing cell atlas use., (© 2023. The Author(s).)

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.

Single-cell technologies have transformed our understanding of human tissues. Yet, studies typically capture only a limited number of donors and disagree on cell type definitions. Integrating many single-cell datasets can address these limitations of individual studies and capture the variability present in the population. Here we present the integrated Human Lung Cell Atlas (HLCA), combining 49 datasets of the human respiratory system into a single atlas spanning over 2.4 million cells from 486 individuals. The HLCA presents a consensus cell type re-annotation with matching marker genes, including annotations of rare and previously undescribed cell types. Leveraging the number and diversity of individuals in the HLCA, we identify gene modules that are associated with demographic covariates such as age, sex and body mass index, as well as gene modules changing expression along the proximal-to-distal axis of the bronchial tree. Mapping new data to the HLCA enables rapid data annotation and interpretation. Using the HLCA as a reference for the study of disease, we identify shared cell states across multiple lung diseases, including SPP1 + profibrotic monocyte-derived macrophages in COVID-19, pulmonary fibrosis and lung carcinoma. Overall, the HLCA serves as an example for the development and use of large-scale, cross-dataset organ atlases within the Human Cell Atlas., (© 2023. The Author(s).)

Common genetic variants across individuals modulate the cellular response to pathogens and are implicated in diverse immune pathologies, yet how they dynamically alter the response upon infection is not well understood. Here, we triggered antiviral responses in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-sequencing. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), a statistical approach designed to identify nonlinear dynamic genetic effects across transcriptional trajectories of cells. This approach identified 1,275 expression quantitative trait loci (local false discovery rate 10%) that manifested during the responses, many of which were colocalized with susceptibility loci identified by genome-wide association studies of infectious and autoimmune diseases, including the OAS1 splicing quantitative trait locus in a COVID-19 susceptibility locus. In summary, our analytical approach provides a unique framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution., (© 2023. The Author(s).)

Rationale: Severe asthma and chronic obstructive pulmonary disease (COPD) share common pathophysiological traits such as relative corticosteroid insensitivity. We recently published three transcriptome-associated clusters (TACs) using hierarchical analysis of the sputum transcriptome in asthmatics from the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort comprising one Th2-high inflammatory signature (TAC1) and two Th2-low signatures (TAC2 and TAC3)., Objective: We examined whether gene expression signatures obtained in asthma can be used to identify the subgroup of patients with COPD with steroid sensitivity., Methods: Using gene set variation analysis, we examined the distribution and enrichment scores (ES) of the 3 TACs in the transcriptome of bronchial biopsies from 46 patients who participated in the Groningen Leiden Universities Corticosteroids in Obstructive Lung Disease COPD study that received 30 months of treatment with inhaled corticosteroids (ICS) with and without an added long-acting β-agonist (LABA). The identified signatures were then associated with longitudinal clinical variables after treatment. Differential gene expression and cellular convolution were used to define key regulated genes and cell types., Measurements and Main Results: Bronchial biopsies in patients with COPD at baseline showed a wide range of expression of the 3 TAC signatures. After ICS±LABA treatment, the ES of TAC1 was significantly reduced at 30 months, but those of TAC2 and TAC3 were unaffected. A corticosteroid-sensitive TAC1 signature was developed from the TAC1 ICS-responsive genes. This signature consisted of mast cell-specific genes identified by single-cell RNA-sequencing and positively correlated with bronchial biopsy mast cell numbers following ICS±LABA. Baseline levels of gene transcription correlated with the change in RV/TLC %predicted following 30-month ICS±LABA., Conclusion: Sputum-derived transcriptomic signatures from an asthma cohort can be recapitulated in bronchial biopsies of patients with COPD and identified a signature of airway mast cells as a predictor of corticosteroid responsiveness., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Rationale: Obesity affects 40% of U.S. adults, is associated with a proinflammatory state, and presents a significant risk factor for the development of severe coronavirus disease (COVID-19). To date, there is limited information on how obesity might affect immune cell responses in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Objectives: To determine the impact of obesity on respiratory tract immunity in COVID-19 across the human lifespan. Methods: We analyzed single-cell transcriptomes from BAL in three ventilated adult cohorts with ( n  = 24) or without ( n  = 9) COVID-19 from nasal immune cells in children with ( n  = 14) or without ( n  = 19) COVID-19, and from peripheral blood mononuclear cells in an independent adult COVID-19 cohort ( n  = 42), comparing obese and nonobese subjects. Measurements and Main Results: Surprisingly, we found that obese adult subjects had attenuated lung immune or inflammatory responses in SARS-CoV-2 infection, with decreased expression of IFN-α, IFN-γ, and TNF-α (tumor necrosis factor α) response gene signatures in almost all lung epithelial and immune cell subsets, and lower expression of IFNG and TNF in specific lung immune cells. Peripheral blood immune cells in an independent adult cohort showed a similar but less marked reduction in type-I IFN and IFNγ response genes, as well as decreased serum IFNα, in obese patients with SARS-CoV-2. Nasal immune cells from obese children with COVID-19 also showed reduced enrichment of IFN-α and IFN-γ response genes. Conclusions: These findings show blunted tissue immune responses in obese patients with COVID-19, with implications for treatment stratification, supporting the specific application of inhaled recombinant type-I IFNs in this vulnerable subset.

Variation in lung alveolar development is strongly linked to disease susceptibility. However, underlying cellular and molecular mechanisms are difficult to study in humans. We have identified an alveolar-fated epithelial progenitor in human fetal lungs, which we grow as self-organizing organoids that model key aspects of cell lineage commitment. Using this system, we have functionally validated cell-cell interactions in the developing human alveolar niche, showing that Wnt signaling from differentiating fibroblasts promotes alveolar-type-2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. We identify a Wnt-NKX2.1 axis controlling alveolar differentiation. Moreover, we show that differential binding of NKX2.1 coordinates alveolar maturation, allowing us to model the effects of human genetic variation in NKX2.1 on alveolar differentiation. Our organoid system recapitulates key aspects of human fetal lung stem cell biology allowing mechanistic experiments to determine the cellular and molecular regulation of human development and disease., Competing Interests: Declaration of interests S.A.T. is a member of the scientific advisory board for the following companies: Biogen, Foresite Labs, GSK, Qiagen, CRG Barcelona, Jax Labs, SciLife Lab, and Allen Institute. She is a consultant for Genentech and Roche. She is co-founder of Transition Bio and a member of the Board., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Single-cell transcriptomics has allowed unprecedented resolution of cell types/states in the human lung, but their spatial context is less well defined. To (re)define tissue architecture of lung and airways, we profiled five proximal-to-distal locations of healthy human lungs in depth using multi-omic single cell/nuclei and spatial transcriptomics (queryable at lungcellatlas.org ). Using computational data integration and analysis, we extend beyond the suspension cell paradigm and discover macro and micro-anatomical tissue compartments including previously unannotated cell types in the epithelial, vascular, stromal and nerve bundle micro-environments. We identify and implicate peribronchial fibroblasts in lung disease. Importantly, we discover and validate a survival niche for IgA plasma cells in the airway submucosal glands (SMG). We show that gland epithelial cells recruit B cells and IgA plasma cells, and promote longevity and antibody secretion locally through expression of CCL28, APRIL and IL-6. This new 'gland-associated immune niche' has implications for respiratory health., (© 2022. The Author(s).)

Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8 + T cell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target., Competing Interests: Declaration of interests In the past 3 years, S.A.T. has consulted for Roche and Genentech and is a Scientific Advisory Board member of Qiagen, Foresite labs, Biogen, and GSK, as well as a consultant and equity holder as co-founder of Transition Bio., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

We present a multiomic cell atlas of human lung development that combines single-cell RNA and ATAC sequencing, high-throughput spatial transcriptomics, and single-cell imaging. Coupling single-cell methods with spatial analysis has allowed a comprehensive cellular survey of the epithelial, mesenchymal, endothelial, and erythrocyte/leukocyte compartments from 5-22 post-conception weeks. We identify previously uncharacterized cell states in all compartments. These include developmental-specific secretory progenitors and a subtype of neuroendocrine cell related to human small cell lung cancer. Our datasets are available through our web interface (https://lungcellatlas.org). To illustrate its general utility, we use our cell atlas to generate predictions about cell-cell signaling and transcription factor hierarchies which we rigorously test using organoid models., Competing Interests: Declaration of interests S.A.T. is a member of the Scientific Advisory Board for the following companies: Biogen, Foresite Labs, GSK, Qiagen, CRG Barcelona, Jax Labs, SciLife Lab, and Allen Institute. She is a consultant for Genentech and Roche. She is co-founder of Transition Bio and a member of the Board. Z.K.T. has received consulting fees from Synteny Biotechnologies for activities unrelated to this work., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The balance between self-renewal and differentiation in human foetal lung epithelial progenitors controls the size and function of the adult organ. Moreover, progenitor cell gene regulation networks are employed by both regenerating and malignant lung cells, where modulators of their effects could potentially be of therapeutic value. Details of the molecular networks controlling human lung progenitor self-renewal remain unknown. We performed the first CRISPRi screen in primary human lung organoids to identify transcription factors controlling progenitor self-renewal. We show that SOX9 promotes proliferation of lung progenitors and inhibits precocious airway differentiation. Moreover, by identifying direct transcriptional targets using Targeted DamID, we place SOX9 at the centre of a transcriptional network, which amplifies WNT and RTK signalling to stabilise the progenitor cell state. In addition, the proof-of-principle CRISPRi screen and Targeted DamID tools establish a new workflow for using primary human organoids to elucidate detailed functional mechanisms underlying normal development and disease., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Rationale & Objective: High-dose influenza vaccine provides better protection against influenza infection in older adults than standard-dose vaccine. We compared vaccine seroresponse among hemodialysis patients over a period of 4 months after administration of high-dose trivalent inactivated (HD-IIV3), standard-dose quadrivalent inactivated (SD-IIV4), or quadrivalent recombinant quadrivalent (RIV4) influenza vaccine., Study Design: Prospective observational study., Setting & Participants: Patients at 4 hemodialysis clinics who received influenza vaccine., Exposure: Type of influenza vaccine., Outcome: Hemagglutination inhibition (HI) titers were measured at baseline and at 1, 2, 3, and 4 months after vaccination. The primary outcome was seroprotection rates at HI titers of at least 1:40 and at least 1:160 (antibody levels providing protection from infection in approximately 50% and 95% of immunocompetent individuals, respectively) at 1, 2, 3, and 4 months after vaccination., Analytical Approach: We calculated geometric mean titer as well as seroprotection and seroconversion rates. Adjusted generalized linear models with additional trend analyses were performed to evaluate the association between vaccine type and outcomes., Results: 254 hemodialysis patients were vaccinated against influenza with HD-IIV3 (n = 141), SD-IIV4 (n = 36), or RIV4 (n = 77). A robust initial seroresponse to influenza A strains was observed after all 3 vaccines. Geometric mean titer and seroprotection (HI titer ≥1:160) rates against influenza A strains were higher and more sustained with HD-IIV3 than SD-IIV4 or RIV4. More than 80% of patients vaccinated with HD-IIV3 were seroprotected (HI titer ≥1:160) at month 4 (P < 0.001), whereas, among patients vaccinated with SD-IIV4 or RIV4, seroprotection rates were similar to those at baseline. Seroprotection rates were lower against B strains for all vaccines., Limitations: Because of the use of observational data, bias from unmeasured confounders may exist. Some age subgroups were small in number. Clinical outcome data were not available., Conclusions: Hemodialysis patients exhibited high seroprotection rates after all 3 influenza vaccines. The seroresponse waned more slowly with HD-IIV3 compared with SD-IIV4 and RIV4 vaccines., (Copyright © 2022 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

The Human Cell Atlas (HCA) consortium aims to establish an atlas of all organs in the healthy human body at single-cell resolution to increase our understanding of basic biological processes that govern development, physiology and anatomy, and to accelerate diagnosis and treatment of disease. The Lung Biological Network of the HCA aims to generate the Human Lung Cell Atlas as a reference for the cellular repertoire, molecular cell states and phenotypes, and cell-cell interactions that characterise normal lung homeostasis in healthy lung tissue. Such a reference atlas of the healthy human lung will facilitate mapping the changes in the cellular landscape in disease. The discovAIR project is one of six pilot actions for the HCA funded by the European Commission in the context of the H2020 framework programme. discovAIR aims to establish the first draft of an integrated Human Lung Cell Atlas, combining single-cell transcriptional and epigenetic profiling with spatially resolving techniques on matched tissue samples, as well as including a number of chronic and infectious diseases of the lung. The integrated Human Lung Cell Atlas will be available as a resource for the wider respiratory community, including basic and translational scientists, clinical medicine, and the private sector, as well as for patients with lung disease and the interested lay public. We anticipate that the Human Lung Cell Atlas will be the founding stone for a more detailed understanding of the pathogenesis of lung diseases, guiding the design of novel diagnostics and preventive or curative interventions., Competing Interests: Conflict of interest: F.J. Theis reports personal fees and nonfinancial support from Cellarity and Dermagnostix, during the conduct of the study. J. Schniering was supported by an ERS/EU RESPIRE4 Marie Skłlodowska-Curie Postdoctoral Fellowship. J. Lundeberg reports personal fees from 10x Genomics, outside the submitted work. P. Powell and J. Denning are employees of the European Lung Foundation. W. Timens reports personal fees from Merck Sharp Dohme and Bristol-Myers-Squibb, outside the submitted work. M. Nilsson reports personal fees from 10x Genomics, outside the submitted work. G.H. Koppelman reports grants from the Netherlands Lung Foundation, GlaxoSmithKline, Vertex, TEVA, UBBO EMMIUS Foundation and European Union (H2020 grant), outside the submitted work, and has participated in advisory board meetings to GlaxoSmithKline and PURE-IMS, outside the submitted work. M. van den Berge reports research grants paid to UMCG from GlaxoSmithKline, Genentech, Roche and Novartis, outside the submitted work. M.C. Nawijn reports grants from the European Commission, Chan Zuckerberg Initiative and Netherlands Lung Foundation, during the conduct of the study; grants from GlaxoSmithKline, outside the submitted work. All other authors do not have a conflict of interest., (Copyright ©The authors 2022.)

Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. We surveyed the immune compartment of 16 tissues from 12 adult donors by single-cell RNA sequencing and VDJ sequencing generating a dataset of ~360,000 cells. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of finely phenotyped immune cell types, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. Our multitissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis, and antigen receptor sequencing.

The SARS-CoV-2 Omicron BA.1 variant emerged in 2021 1 and has multiple mutations in its spike protein 2 . Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways 3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis., (© 2022. The Author(s).)