Illuminating Genetic Mysteries of the Dead Sea Scrolls.

The discovery of the 2,000-year-old Dead Sea Scrolls had an incomparable impact on the historical understanding of Judaism and Christianity. "Piecing together" scroll fragments is like solving jigsaw puzzles with an unknown number of missing parts. We used the fact that most scrolls are made from animal skins to "fingerprint" pieces based on DNA sequences. Genetic sorting of the scrolls illuminates their textual relationship and historical significance. Disambiguating the contested relationship between Jeremiah fragments supplies evidence that some scrolls were brought to the Qumran caves from elsewhere; significantly, they demonstrate that divergent versions of Jeremiah circulated in parallel throughout Israel (ancient Judea). Similarly, patterns discovered in non-biblical scrolls, particularly the Songs of the Sabbath Sacrifice, suggest that the Qumran scrolls represent the broader cultural milieu of the period. Finally, genetic analysis divorces debated fragments from the Qumran scrolls. Our study demonstrates that interdisciplinary approaches enrich the scholar's toolkit.

Fundamental Biological Features of Spaceflight: Advancing the Field to Enable Deep-Space Exploration.

Research on astronaut health and model organisms have revealed six features of spaceflight biology that guide our current understanding of fundamental molecular changes that occur during space travel. The features include oxidative stress, DNA damage, mitochondrial dysregulation, epigenetic changes (including gene regulation), telomere length alterations, and microbiome shifts. Here we review the known hazards of human spaceflight, how spaceflight affects living systems through these six fundamental features, and the associated health risks of space exploration. We also discuss the essential issues related to the health and safety of astronauts involved in future missions, especially planned long-duration and Martian missions.

Comprehensive Multi-omics Analysis Reveals Mitochondrial Stress as a Central Biological Hub for Spaceflight Impact.

Spaceflight is known to impose changes on human physiology with unknown molecular etiologies. To reveal these causes, we used a multi-omics, systems biology analytical approach using biomedical profiles from fifty-nine astronauts and data from NASA's GeneLab derived from hundreds of samples flown in space to determine transcriptomic, proteomic, metabolomic, and epigenetic responses to spaceflight. Overall pathway analyses on the multi-omics datasets showed significant enrichment for mitochondrial processes, as well as innate immunity, chronic inflammation, cell cycle, circadian rhythm, and olfactory functions. Importantly, NASA's Twin Study provided a platform to confirm several of our principal findings. Evidence of altered mitochondrial function and DNA damage was also found in the urine and blood metabolic data compiled from the astronaut cohort and NASA Twin Study data, indicating mitochondrial stress as a consistent phenotype of spaceflight.

The Space Omics and Medical Atlas (SOMA) and international astronaut biobank.

Spaceflight induces molecular, cellular and physiological shifts in astronauts and poses myriad biomedical challenges to the human body, which are becoming increasingly relevant as more humans venture into space1-6. Yet current frameworks for aerospace medicine are nascent and lag far behind advancements in precision medicine on Earth, underscoring the need for rapid development of space medicine databases, tools and protocols. Here we present the Space Omics and Medical Atlas (SOMA), an integrated data and sample repository for clinical, cellular and multi-omic research profiles from a diverse range of missions, including the NASA Twins Study7, JAXA CFE study8,9, SpaceX Inspiration4 crew10-12, Axiom and Polaris. The SOMA resource represents a more than tenfold increase in publicly available human space omics data, with matched samples available from the Cornell Aerospace Medicine Biobank. The Atlas includes extensive molecular and physiological profiles encompassing genomics, epigenomics, transcriptomics, proteomics, metabolomics and microbiome datasets, which reveal some consistent features across missions, including cytokine shifts, telomere elongation and gene expression changes, as well as mission-specific molecular responses and links to orthologous, tissue-specific mouse datasets. Leveraging the datasets, tools and resources in SOMA can help to accelerate precision aerospace medicine, bringing needed health monitoring, risk mitigation and countermeasure data for upcoming lunar, Mars and exploration-class missions.

Molecular and physiological changes in the SpaceX Inspiration4 civilian crew.

Human spaceflight has historically been managed by government agencies, such as in the NASA Twins Study1, but new commercial spaceflight opportunities have opened spaceflight to a broader population. In 2021, the SpaceX Inspiration4 mission launched the first all-civilian crew to low Earth orbit, which included the youngest American astronaut (aged 29), new in-flight experimental technologies (handheld ultrasound imaging, smartwatch wearables and immune profiling), ocular alignment measurements and new protocols for in-depth, multi-omic molecular and cellular profiling. Here we report the primary findings from the 3-day spaceflight mission, which induced a broad range of physiological and stress responses, neurovestibular changes indexed by ocular misalignment, and altered neurocognitive functioning, some of which match those of long-term spaceflight2, but almost all of which did not differ from baseline (pre-flight) after return to Earth. Overall, these preliminary civilian spaceflight data suggest that short-duration missions do not pose a significant health risk, and moreover present a rich opportunity to measure the earliest phases of adaptation to spaceflight in the human body at anatomical, cellular, physiological and cognitive levels. Finally, these methods and results lay the foundation for an open, rapidly expanding biomedical database for astronauts3, which can inform countermeasure development for both private and government-sponsored space missions.

The spatial landscape of lung pathology during COVID-19 progression.

Recent studies have provided insights into the pathology of and immune response to COVID-191-8. However, a thorough investigation of the interplay between infected cells and the immune system at sites of infection has been lacking. Here we use high-parameter imaging mass cytometry9 that targets the expression of 36 proteins to investigate the cellular composition and spatial architecture of acute lung injury in humans (including injuries derived from SARS-CoV-2 infection) at single-cell resolution. These spatially resolved single-cell data unravel the disordered structure of the infected and injured lung, alongside the distribution of extensive immune infiltration. Neutrophil and macrophage infiltration are hallmarks of bacterial pneumonia and COVID-19, respectively. We provide evidence that SARS-CoV-2 infects predominantly alveolar epithelial cells and induces a localized hyperinflammatory cell state that is associated with lung damage. We leverage the temporal range of fatal outcomes of COVID-19 in relation to the onset of symptoms, which reveals increased macrophage extravasation and increased numbers of mesenchymal cells and fibroblasts concomitant with increased proximity between these cell types as the disease progresses-possibly as a result of attempts to repair the damaged lung tissue. Our data enable us to develop a biologically interpretable landscape of lung pathology from a structural, immunological and clinical standpoint. We use this landscape to characterize the pathophysiology of the human lung from its macroscopic presentation to the single-cell level, which provides an important basis for understanding COVID-19 and lung pathology in general.

Distinct and opposite effects of leukemogenic Idh and Tet2 mutations in hematopoietic stem and progenitor cells.

Mutations in IDH1, IDH2, and TET2 are recurrently observed in myeloid neoplasms. IDH1 and IDH2 encode isocitrate dehydrogenase isoforms, which normally catalyze the conversion of isocitrate to α-ketoglutarate (α-KG). Oncogenic IDH1/2 mutations confer neomorphic activity, leading to the production of D-2-hydroxyglutarate (D-2-HG), a potent inhibitor of α-KG-dependent enzymes which include the TET methylcytosine dioxygenases. Given their mutual exclusivity in myeloid neoplasms, IDH1, IDH2, and TET2 mutations may converge on a common oncogenic mechanism. Contrary to this expectation, we observed that they have distinct, and even opposite, effects on hematopoietic stem and progenitor cells in genetically engineered mice. Epigenetic and single-cell transcriptomic analyses revealed that Idh2R172K and Tet2 loss-of-function have divergent consequences on the expression and activity of key hematopoietic and leukemogenic regulators. Notably, chromatin accessibility and transcriptional deregulation in Idh2R172K cells were partially disconnected from DNA methylation alterations. These results highlight unanticipated divergent effects of IDH1/2 and TET2 mutations, providing support for the optimization of genotype-specific therapies.

PRMT5 supports multiple oncogenic pathways in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy with an overall poor prognosis, particularly for patients that progress on targeted therapies. Novel, more durable treatment options are needed for patients with MCL. Protein arginine methyltransferase 5 (PRMT5) is overexpressed in MCL and plays an important oncogenic role in this disease via epigenetic and posttranslational modification of cell cycle regulators, DNA repair genes, components of prosurvival pathways, and RNA splicing regulators. The mechanism of targeting PRMT5 in MCL remains incompletely characterized. Here, we report on the antitumor activity of PRMT5 inhibition in MCL using integrated transcriptomics of in vitro and in vivo models of MCL. Treatment with a selective small-molecule inhibitor of PRMT5, PRT-382, led to growth arrest and cell death and provided a therapeutic benefit in xenografts derived from patients with MCL. Transcriptional reprograming upon PRMT5 inhibition led to restored regulatory activity of the cell cycle (p-RB/E2F), apoptotic cell death (p53-dependent/p53-independent), and activation of negative regulators of B-cell receptor-PI3K/AKT signaling (PHLDA3, PTPROt, and PIK3IP1). We propose pharmacologic inhibition of PRMT5 for patients with relapsed/refractory MCL and identify MTAP/CDKN2A deletion and wild-type TP53 as biomarkers that predict a favorable response. Selective targeting of PRMT5 has significant activity in preclinical models of MCL and warrants further investigation in clinical trials.

Haplotype diversity and sequence heterogeneity of human telomeres.

Telomeres are regions of repetitive nucleotide sequences capping the ends of eukaryotic chromosomes that protect against deterioration, and whose lengths can be correlated with age and adverse health risk factors. Yet, given their length and repetitive nature, telomeric regions are not easily reconstructed from short-read sequencing, thus making telomere sequencing, mapping, and variant resolution challenging problems. Recently, long-read sequencing, with read lengths measuring in hundreds of kilobase pairs, has made it possible to routinely read into telomeric regions and inspect their sequence structure. Here, we describe a framework for extracting telomeric reads from whole-genome single-molecule sequencing experiments, including de novo identification of telomere repeat motifs and repeat types, and also describe their sequence variation. We find that long, complex telomeric stretches and repeats can be accurately captured with long-read sequencing, observe extensive sequence heterogeneity of human telomeres, discover and localize noncanonical telomere sequence motifs (both previously reported, as well as novel), and validate them in short-read sequence data. These data reveal extensive intra- and inter-population diversity of repeats in telomeric haplotypes, reveal higher paternal inheritance of telomeric variants, and represent the first motif composition maps of multi-kilobase-pair human telomeric haplotypes across three distinct ancestries (Ashkenazi, Chinese, and Utah), which can aid in future studies of genetic variation, aging, and genome biology.

SARS-CoV-2 genomic diversity and the implications for qRT-PCR diagnostics and transmission.

The COVID-19 pandemic has sparked an urgent need to uncover the underlying biology of this devastating disease. Though RNA viruses mutate more rapidly than DNA viruses, there are a relatively small number of single nucleotide polymorphisms (SNPs) that differentiate the main SARS-CoV-2 lineages that have spread throughout the world. In this study, we investigated 129 RNA-seq data sets and 6928 consensus genomes to contrast the intra-host and inter-host diversity of SARS-CoV-2. Our analyses yielded three major observations. First, the mutational profile of SARS-CoV-2 highlights intra-host single nucleotide variant (iSNV) and SNP similarity, albeit with differences in C > U changes. Second, iSNV and SNP patterns in SARS-CoV-2 are more similar to MERS-CoV than SARS-CoV-1. Third, a significant fraction of insertions and deletions contribute to the genetic diversity of SARS-CoV-2. Altogether, our findings provide insight into SARS-CoV-2 genomic diversity, inform the design of detection tests, and highlight the potential of iSNVs for tracking the transmission of SARS-CoV-2.

Investigation of product-derived lymphoma following infusion of piggyBac-modified CD19 chimeric antigen receptor T cells.

We performed a phase 1 clinical trial to evaluate outcomes in patients receiving donor-derived CD19-specific chimeric antigen receptor (CAR) T cells for B-cell malignancy that relapsed or persisted after matched related allogeneic hemopoietic stem cell transplant. To overcome the cost and transgene-capacity limitations of traditional viral vectors, CAR T cells were produced using the piggyBac transposon system of genetic modification. Following CAR T-cell infusion, 1 patient developed a gradually enlarging retroperitoneal tumor due to a CAR-expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection, in an asymptomatic patient, of a second CAR T-cell tumor in thoracic para-aortic lymph nodes. Analysis of the first lymphoma showed a high transgene copy number, but no insertion into typical oncogenes. There were also structural changes such as altered genomic copy number and point mutations unrelated to the insertion sites. Transcriptome analysis showed transgene promoter-driven upregulation of transcription of surrounding regions despite insulator sequences surrounding the transgene. However, marked global changes in transcription predominantly correlated with gene copy number rather than insertion sites. In both patients, the CAR T-cell-derived lymphoma progressed and 1 patient died. We describe the first 2 cases of malignant lymphoma derived from CAR gene-modified T cells. Although CAR T cells have an enviable record of safety to date, our results emphasize the need for caution and regular follow-up of CAR T recipients, especially when novel methods of gene transfer are used to create genetically modified immune therapies. This trial was registered at www.anzctr.org.au as ACTRN12617001579381.

Betacoronavirus-specific alternate splicing.

Viruses can subvert a number of cellular processes including splicing in order to block innate antiviral responses, and many viruses interact with cellular splicing machinery. SARS-CoV-2 infection was shown to suppress global mRNA splicing, and at least 10 SARS-CoV-2 proteins bind specifically to one or more human RNAs. Here, we investigate 17 published experimental and clinical datasets related to SARS-CoV-2 infection, datasets from the betacoronaviruses SARS-CoV and MERS, as well as Streptococcus pneumonia, HCV, Zika virus, Dengue virus, influenza H3N2, and RSV. We show that genes showing differential alternative splicing in SARS-CoV-2 have a similar functional profile to those of SARS-CoV and MERS and affect a diverse set of genes and biological functions, including many closely related to virus biology. Additionally, the differentially spliced transcripts of cells infected by coronaviruses were more likely to undergo intron-retention, contain a pseudouridine modification, and have a smaller number of exons as compared with differentially spliced transcripts in the control groups. Viral load in clinical COVID-19 samples was correlated with isoform distribution of differentially spliced genes. A significantly higher number of ribosomal genes are affected by differential alternative splicing and gene expression in betacoronavirus samples, and the betacoronavirus differentially spliced genes are depleted for binding sites of RNA-binding proteins. Our results demonstrate characteristic patterns of differential splicing in cells infected by SARS-CoV-2, SARS-CoV, and MERS. The alternative splicing changes observed in betacoronaviruses infection potentially modify a broad range of cellular functions, via changes in the functions of the products of a diverse set of genes involved in different biological processes.

Insect genome content phylogeny and functional annotation of core insect genomes.

Twenty-one fully sequenced and well annotated insect genomes were examined for genome content in a phylogenetic context. Gene presence/absence matrices and phylogenetic trees were constructed using several phylogenetic criteria. The role of e-value on phylogenetic analysis and genome content characterization is examined using scaled e-value cutoffs and a single linkage clustering approach to orthology determination. Previous studies have focused on the role of gene loss in terminals in the insect tree of life. The present study examines several common ancestral nodes in the insect tree. We suggest that the common ancestors of major insect groups like Diptera, Hymenoptera, Hemiptera and Holometabola experience more gene gain than gene loss. This suggests that as major insect groups arose, their genomic repertoire expanded through gene duplication (segmental duplications), followed by contraction by gene loss in specific terminal lineages. In addition, we examine the functional significance of the loss and gain of genes in the divergence of some of the major insect groups.

In silico hybridization enables transcriptomic illumination of the nature and evolution of Myxozoa.

BACKGROUND: The Myxozoa, a group of oligocellular, obligate endoparasites, has long been poorly understood in an evolutionary context. Recent genome-level sequencing techniques such as RNA-seq have generated large amounts of myxozoan sequence data, providing valuable insight into their evolutionary history. However, sequences from host tissue contamination are present in next-generation sequencing reactions of myxozoan tissue, and differentiating between the two has been inadequately addressed. In order to shed light on the genetic underpinnings of myxozoan biology, assembled contigs generated from these studies that derived from the myxozoan must be decoupled from transcripts derived from host tissue and other contamination. This study describes a pipeline for categorization of transcripts asmyxozoan based on similarity searching with known host and parasite sequences, explores the extent to which host contamination is present in previously existing myxozoan datasets, and implements this pipeline on a newly sequenced transcriptome of Myxobolus pendula, a parasite of the common creek chub gill arch. METHODS: The insilico hybridization pipeline uses iterative BLAST searching and database-driven e-value comparison to categorize transcripts as deriving from host, parasite, or other contamination. Functional genetic analysis of M. pendula was conducted using further BLAST searching, Hidden Markov Modeling, and sequence alignment and phylogenetic reconstruction. RESULTS: Three RNA libraries of encysted M. pendula plasmodia were sequenced and subjected to the method. Nearly half of the final set of contiguous assembly sequences (47.3 %) was identified as putative myxozoan transcripts. Putative contamination was also identified in at least 1/3(rd) of previously published myxozoan transcripts. The set of M. pendula transcripts was mined for a range of biologically insightful genes, including taxonomically restricted nematocyst structural proteins and nematocyst proteins identified through mass tandem spectrometry of other cnidarians. Several novel findings emerged, including a fourth myxozoan minicollagen gene, putative myxozoan toxin proteins,and extracellular matrix glycoproteins. CONCLUSIONS: This study serves as a model for the handling of next-generation myxozoan sequence. The need for careful categorization was demonstrated in both previous and new sets of myxozoan sequences. The final set of confidently assigned myxozoan transcripts can be mined for any biologically relevant gene or gene family without spurious misidentification of host contamination as a myxozoan homolog. As exemplified by M. pendula, the repertoire of myxozoan polar capsules may be more complex than previously thought, with an additional minicollagen homolog and putative expression of toxin proteins.

Resistance to PRMT5-targeted therapy in mantle cell lymphoma.

ABSTRACT: Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma, and patients who relapse on targeted therapies have poor prognosis. Protein arginine methyltransferase 5 (PRMT5), an enzyme essential for B-cell transformation, drives multiple oncogenic pathways and is overexpressed in MCL. Despite the antitumor activity of PRMT5 inhibition (PRT-382/PRT-808), drug resistance was observed in a patient-derived xenograft (PDX) MCL model. Decreased survival of mice engrafted with these PRMT5 inhibitor-resistant cells vs treatment-naive cells was observed (P = .005). MCL cell lines showed variable sensitivity to PRMT5 inhibition. Using PRT-382, cell lines were classified as sensitive (n = 4; 50% inhibitory concentration [IC50], 20-140 nM) or primary resistant (n = 4; 340-1650 nM). Prolonged culture of sensitive MCL lines with drug escalation produced PRMT5 inhibitor-resistant cell lines (n = 4; 200-500 nM). This resistant phenotype persisted after prolonged culture in the absence of drug and was observed with PRT-808. In the resistant PDX and cell line models, symmetric dimethylarginine reduction was achieved at the original PRMT5 inhibitor IC50, suggesting activation of alternative resistance pathways. Bulk RNA sequencing of resistant cell lines and PDX relative to sensitive or short-term-treated cells, respectively, highlighted shared upregulation of multiple pathways including mechanistic target of rapamycin kinase [mTOR] signaling (P < 10-5 and z score > 0.3 or < 0.3). Single-cell RNA sequencing analysis demonstrated a strong shift in global gene expression, with upregulation of mTOR signaling in resistant PDX MCL samples. Targeted blockade of mTORC1 with temsirolimus overcame the PRMT5 inhibitor-resistant phenotype, displayed therapeutic synergy in resistant MCL cell lines, and improved survival of a resistant PDX.

Towards geospatially-resolved public-health surveillance via wastewater sequencing.

Wastewater is a geospatially- and temporally-linked microbial fingerprint of a given population, making it a potentially valuable tool for tracking public health across locales and time. Here, we integrate targeted and bulk RNA sequencing (N = 2238 samples) to track the viral, bacterial, and functional content over geospatially distinct areas within Miami Dade County, USA, from 2020-2022. We used targeted amplicon sequencing to track diverse SARS-CoV-2 variants across space and time, and we found a tight correspondence with positive PCR tests from University students and Miami-Dade hospital patients. Additionally, in bulk metatranscriptomic data, we demonstrate that the bacterial content of different wastewater sampling locations serving small population sizes can be used to detect putative, host-derived microorganisms that themselves have known associations with human health and diet. We also detect multiple enteric pathogens (e.g., Norovirus) and characterize viral diversity across sites. Moreover, we observed an enrichment of antimicrobial resistance genes (ARGs) in hospital wastewater; antibiotic-specific ARGs correlated to total prescriptions of those same antibiotics (e.g Ampicillin, Gentamicin). Overall, this effort lays the groundwork for systematic characterization of wastewater that can potentially influence public health decision-making.

Genome and clonal hematopoiesis stability contrasts with immune, cfDNA, mitochondrial, and telomere length changes during short duration spaceflight.

Background: The Inspiration4 (I4) mission, the first all-civilian orbital flight mission, investigated the physiological effects of short-duration spaceflight through a multi-omic approach. Despite advances, there remains much to learn about human adaptation to spaceflight's unique challenges, including microgravity, immune system perturbations, and radiation exposure. Methods: To provide a detailed genetics analysis of the mission, we collected dried blood spots pre-, during, and post-flight for DNA extraction. Telomere length was measured by quantitative PCR, while whole genome and cfDNA sequencing provided insight into genomic stability and immune adaptations. A robust bioinformatic pipeline was used for data analysis, including variant calling to assess mutational burden. Result: Telomere elongation occurred during spaceflight and shortened after return to Earth. Cell-free DNA analysis revealed increased immune cell signatures post-flight. No significant clonal hematopoiesis of indeterminate potential (CHIP) or whole-genome instability was observed. The long-term gene expression changes across immune cells suggested cellular adaptations to the space environment persisting months post-flight. Conclusion: Our findings provide valuable insights into the physiological consequences of short-duration spaceflight, with telomere dynamics and immune cell gene expression adapting to spaceflight and persisting after return to Earth. CHIP sequencing data will serve as a reference point for studying the early development of CHIP in astronauts, an understudied phenomenon as previous studies have focused on career astronauts. This study will serve as a reference point for future commercial and non-commercial spaceflight, low Earth orbit (LEO) missions, and deep-space exploration.

Longitudinal multi-omics analysis of host microbiome architecture and immune responses during short-term spaceflight.

Maintenance of astronaut health during spaceflight will require monitoring and potentially modulating their microbiomes. However, documenting microbial shifts during spaceflight has been difficult due to mission constraints that lead to limited sampling and profiling. Here we executed a six-month longitudinal study to quantify the high-resolution human microbiome response to three days in orbit for four individuals. Using paired metagenomics and metatranscriptomics alongside single-nuclei immune cell profiling, we characterized time-dependent, multikingdom microbiome changes across 750 samples and 10 body sites before, during and after spaceflight at eight timepoints. We found that most alterations were transient across body sites; for example, viruses increased in skin sites mostly during flight. However, longer-term shifts were observed in the oral microbiome, including increased plaque-associated bacteria (for example, Fusobacteriota), which correlated with immune cell gene expression. Further, microbial genes associated with phage activity, toxin-antitoxin systems and stress response were enriched across multiple body sites. In total, this study reveals in-depth characterization of microbiome and immune response shifts experienced by astronauts during short-term spaceflight and the associated changes to the living environment, which can help guide future missions, spacecraft design and space habitat planning.

Single-cell multi-ome and immune profiles of the Inspiration4 crew reveal conserved, cell-type, and sex-specific responses to spaceflight.

Spaceflight induces an immune response in astronauts. To better characterize this effect, we generated single-cell, multi-ome, cell-free RNA (cfRNA), biochemical, and hematology data for the SpaceX Inspiration4 (I4) mission crew. We found that 18 cytokines/chemokines related to inflammation, aging, and muscle homeostasis changed after spaceflight. In I4 single-cell multi-omics data, we identified a "spaceflight signature" of gene expression characterized by enrichment in oxidative phosphorylation, UV response, immune function, and TCF21 pathways. We confirmed the presence of this signature in independent datasets, including the NASA Twins Study, the I4 skin spatial transcriptomics, and 817 NASA GeneLab mouse transcriptomes. Finally, we observed that (1) T cells showed an up-regulation of FOXP3, (2) MHC class I genes exhibited long-term suppression, and (3) infection-related immune pathways were associated with microbiome shifts. In summary, this study reveals conserved and distinct immune disruptions occurring and details a roadmap for potential countermeasures to preserve astronaut health.