Diagnosing destabilization risk in global land carbon sinks.

Global net land carbon uptake or net biome production (NBP) has increased during recent decades1. Whether its temporal variability and autocorrelation have changed during this period, however, remains elusive, even though an increase in both could indicate an increased potential for a destabilized carbon sink2,3. Here, we investigate the trends and controls of net terrestrial carbon uptake and its temporal variability and autocorrelation from 1981 to 2018 using two atmospheric-inversion models, the amplitude of the seasonal cycle of atmospheric CO2 concentration derived from nine monitoring stations distributed across the Pacific Ocean and dynamic global vegetation models. We find that annual NBP and its interdecadal variability increased globally whereas temporal autocorrelation decreased. We observe a separation of regions characterized by increasingly variable NBP, associated with warm regions and increasingly variable temperatures, lower and weaker positive trends in NBP and regions where NBP became stronger and less variable. Plant species richness presented a concave-down parabolic spatial relationship with NBP and its variability at the global scale whereas nitrogen deposition generally increased NBP. Increasing temperature and its increasing variability appear as the most important drivers of declining and increasingly variable NBP. Our results show increasing variability of NBP regionally that can be mostly attributed to climate change and that may point to destabilization of the coupled carbon-climate system.

Systemized approach to equipping medical students with naloxone: a student-driven initiative to combat the opioid crisis.

BACKGROUND: Naloxone is an effective and safe opioid reversal medication now approved by the U.S. Food and Drug Administration (FDA) for use with or without a prescription. Despite this, naloxone dissemination lags at a time when U.S. opioid-related mortality expands. The authors proposed distributing naloxone to all U.S. medical students using established statewide standing prescription orders for naloxone, eliminating the financial burden of over-the-counter costs on students and streamlining workflow for the pharmacy. By focusing naloxone distribution on medical students, we are able to capitalize on a group that is already primed on healthcare intervention, while also working to combat stigma in the emerging physician workforce. METHODS: Beginning August 2022, the authors established a partnership between Harvard Medical School (HMS) and the outpatient pharmacy at Brigham and Women's Hospital (BWH) to facilitate access to naloxone for HMS medical students. BWH developed a HIPAA-secure electronic form to collect individual prescription information. BWH pharmacists processed submissions daily, integrating the naloxone prescription requests into their workflow for in-person pick-up or mail-order delivery. The electronic form was disseminated to medical students through a required longitudinal addiction medicine curriculum, listserv messaging, and an extracurricular harm reduction workshop. RESULTS: Over the 2022-2023 academic year, 63 medical students obtained naloxone kits (two doses per kit) through this collaboration. CONCLUSIONS: We propose that medical schools advocate for a hospital pharmacy-initiated workflow focused on convenience and accessibility to expand naloxone access to medical students as a strategy to strengthen the U.S. emergency response and prevention efforts aimed at reducing opioid-related morbidity and mortality. Expansion of our program to BWH internal medicine residents increased our distribution to over 110 healthcare workers, and efforts to expand the program to other BWH training programs and clinical sites such as the emergency department and outpatient infectious disease clinics are underway. With more than 90,000 medical students in the U.S., we believe that widespread implementation of targeted naloxone training and distribution to this population is an accessible approach to combating the public health crisis of opioid-related overdoses.

Genome sequence of the progenitor of the wheat D genome Aegilops tauschii.

Aegilops tauschii is the diploid progenitor of the D genome of hexaploid wheat (Triticum aestivum, genomes AABBDD) and an important genetic resource for wheat. The large size and highly repetitive nature of the Ae. tauschii genome has until now precluded the development of a reference-quality genome sequence. Here we use an array of advanced technologies, including ordered-clone genome sequencing, whole-genome shotgun sequencing, and BioNano optical genome mapping, to generate a reference-quality genome sequence for Ae. tauschii ssp. strangulata accession AL8/78, which is closely related to the wheat D genome. We show that compared to other sequenced plant genomes, including a much larger conifer genome, the Ae. tauschii genome contains unprecedented amounts of very similar repeated sequences. Our genome comparisons reveal that the Ae. tauschii genome has a greater number of dispersed duplicated genes than other sequenced genomes and its chromosomes have been structurally evolving an order of magnitude faster than those of other grass genomes. The decay of colinearity with other grass genomes correlates with recombination rates along chromosomes. We propose that the vast amounts of very similar repeated sequences cause frequent errors in recombination and lead to gene duplications and structural chromosome changes that drive fast genome evolution.

A SNP-based genetic dissection of versatile traits in bread wheat (Triticum aestivum L.).

The continuous increase in global population prompts increased wheat production. Future wheat (Triticum aestivum L.) breeding will heavily rely on dissecting molecular and genetic bases of wheat yield and related traits which is possible through the discovery of quantitative trait loci (QTLs) in constructed populations, such as recombinant inbred lines (RILs). Here, we present an evaluation of 92 RILs in a bi-parental RIL mapping population (the International Triticeae Mapping Initiative Mapping Population [ITMI/MP]) using newly generated phenotypic data in 3-year experiments (2015), older phenotypic data (1997-2009), and newly created single nucleotide polymorphism (SNP) marker data based on 92 of the original RILs to search for novel and stable QTLs. Our analyses of more than 15 unique traits observed in multiple experiments included analyses of 46 traits in three environments in the USA, 69 traits in eight environments in Germany, 149 traits in 10 environments in Russia, and 28 traits in four environments in India (292 traits in 25 environments) with 7584 SNPs (292 × 7584 = 2 214 528 data points). A total of 874 QTLs were detected with limit of detection (LOD) scores of 2.01-3.0 and 432 QTLs were detected with LOD > 3.0. Moreover, 769 QTLs could be assigned to 183 clusters based on the common markers and relative proximity of related QTLs, indicating gene-rich regions throughout the A, B, and D genomes of common wheat. This upgraded genotype-phenotype information of ITMI/MP can assist breeders and geneticists who can make crosses with suitable RILs to improve or investigate traits of interest.

Introgression of perennial growth habit from Lophopyrum elongatum into wheat.

KEY MESSAGE: A locus for perennial growth was mapped on Lophopyrum elongatum chromosome arm 4ES and introgressed into the wheat genome. Evidence was obtained that in addition to chromosome 4E, other L. elongatum chromosomes control perennial growth. Monocarpy versus polycarpy is one of the fundamental developmental dichotomies in flowering plants. Advances in the understanding of the genetic basis of this dichotomy are important for basic biological reasons and practically for genetic manipulation of growth development in economically important plants. Nine wheat introgression lines (ILs) harboring germplasm of the Lophopyrum elongatum genome present in the octoploid amphiploid Triticum aestivum cv. Chinese Spring (subgenomes AABBDD) × L. elongatum (genomes EE) were selected from a population of ILs developed earlier. These ILs were employed here in genomic analyses of post-sexual cycle regrowth (PSCR), which is a component of polycarpy in caespitose L. elongatum. Analyses of disomic substitution (DS) lines confirmed that L. elongatum chromosome 4E confers PSCR on wheat. The gene was mapped into a short distal region of L. elongatum arm 4ES and was tentatively named Pscr1. ILs harboring recombined chromosomes with 4ES segments, including Pscr1, incorporated into the distal part of the 4DS chromosome arm were identified. Based on the location, Pscr1 is not orthologous with the rice rhizome-development gene Rhz2 located on rice chromosome Os3, which is homoeologous with chromosome 4E, but it may correspond to the Teosinte branched1 (TB1) gene, which is located in the introgressed region in the L. elongatum and Ae. tauschii genomes. A hexaploid IL harboring a large portion of the E-genome but devoid of chromosome 4E also expressed PSCR, which provided evidence that perennial growth is controlled by genes on other L. elongatum chromosomes in addition to 4E.

Genome-wide introgression from a bread wheat × Lophopyrum elongatum amphiploid into wheat.

KEY MESSAGE: We introgressed wheatgrass germplasm from the octoploid amphiploid Triticum aestivum× Lophopyrum elongatum into wheat by manipulating the wheat Ph1 gene and discovered and characterized 130 introgression lines harboring single or, in various combinations, complete and recombined L. elongatum chromosomes. Diploid wheatgrass Lophopyrum elongatum (genomes EE) possesses valuable traits for wheat genetics and breeding. We evaluated several strategies for introgression of this germplasm into wheat. To detect it, we developed and validated multiplexed sets of Sequenom MassARRAY single nucleotide polymorphism (SNP) markers, which differentiated disomic and monosomic L. elongatum chromosomes from wheat chromosomes. We identified 130 introgression lines (ILs), which harbored 108 complete and 89 recombined L. elongatum chromosomes. Of the latter, 59 chromosomes were recombined by one or more crossovers and 30 were involved in centromeric (Robertsonian) translocations or were telocentric. To identify wheat chromosomes substituted for or recombined with L. elongatum chromosomes, we genotyped the ILs with the wheat 90-K Infinium SNP array. We found that most of the wheat 90-K probes correctly detected their targets in the L. elongatum genome and showed that some wheat SNPs are ancient and had originated prior to the divergence of the wheat and L. elongatum lineages. Of the 130 ILs, 52% were homozygous for Ph1 deletion and thus are staged to be recombined further. We failed to detect in the L. elongatum genome the 4/5 reciprocal translocation that has been reported in Thinopyrum bessarabicum and several other Triticeae genomes.

Structural variation and rates of genome evolution in the grass family seen through comparison of sequences of genomes greatly differing in size.

Homology was searched with genes annotated in the Aegilops tauschii pseudomolecules against genes annotated in the pseudomolecules of tetraploid wild emmer wheat, Brachypodium distachyon, sorghum and rice. Similar searches were performed with genes annotated in the rice pseudomolecules. Matrices of collinear genes and rearrangements in their order were constructed. Optical BioNano genome maps were constructed and used to validate rearrangements unique to the wild emmer and Ae. tauschii genomes. Most common rearrangements were short paracentric inversions and short intrachromosomal translocations. Intrachromosomal translocations outnumbered segmental intrachromosomal duplications. The densities of paracentric inversion lengths were approximated by exponential distributions in all six genomes. Densities of collinear genes along the Ae. tauschii chromosomes were highly correlated with meiotic recombination rates but those of rearrangements were not, suggesting different causes of the erosion of gene collinearity and evolution of major chromosome rearrangements. Frequent rearrangements sharing breakpoints suggested that chromosomes have been rearranged recurrently at some sites. The distal 4 Mb of the short arms of rice chromosomes Os11 and Os12 and corresponding regions in the sorghum, B. distachyon and Triticeae genomes contain clusters of interstitial translocations including from 1 to 7 collinear genes. The rates of acquisition of major rearrangements were greater in the large wild emmer wheat and Ae. tauschii genomes than in the lineage preceding their divergence or in the B. distachyon, rice and sorghum lineages. It is suggested that synergy between large quantities of dynamic transposable elements and annual growth habit have been the primary causes of the fast evolution of the Triticeae genomes.

Recombination between homoeologous chromosomes induced in durum wheat by the Aegilops speltoides Su1-Ph1 suppressor.

KEY MESSAGE: Su1-Ph1, which we previously introgressed into wheat from Aegilops speltoides, is a potent suppressor of Ph1 and a valuable tool for gene introgression in tetraploid wheat. We previously introgressed Su1-Ph1, a suppressor of the wheat Ph1 gene, from Aegilops speltoides into durum wheat cv Langdon (LDN). Here, we evaluated the utility of the introgressed suppressor for inducing introgression of alien germplasm into durum wheat. We built LDN plants heterozygous for Su1-Ph1 that simultaneously contained a single LDN chromosome 5B and a single Ae. searsii chromosome 5Sse, which targeted them for recombination. We genotyped 28 BC1F1 and 84 F2 progeny with the wheat 90-K Illumina single-nucleotide polymorphism assay and detected extensive recombination between the two chromosomes, which we confirmed by non-denaturing fluorescence in situ hybridization (ND-FISH). We constructed BC1F1 and F2 genetic maps that were 65.31 and 63.71 cM long, respectively. Recombination rates between the 5B and 5Sse chromosomes were double the expected rate computed from their meiotic pairing, which we attributed to selection against aneuploid gametes. Recombination rate between 5B and 5Sse was depressed compared to that between 5B chromosomes in the proximal region of the long arm. We integrated ND-FISH signals into the genetic map and constructed a physical map, which we used to map a 172,188,453-bp Ph1 region. Despite the location of the region in a low-recombination region of the 5B chromosome, we detected three crossovers in it. Our data show that Su1-Ph1 is a valuable tool for gene introgression and gene mapping based on recombination between homoeologous chromosomes in wheat.

Correction to: Recombination between homoeologous chromosomes induced in durum wheat by the Aegilops speltoides Su1-Ph1 suppressor.

Unfortunately, the 9th author name was incorrectly published in the original publication. The complete correct name is given below.

Reassessment of the evolution of wheat chromosomes 4A, 5A, and 7B.

KEY MESSAGE: Comparison of genome sequences of wild emmer wheat and Aegilops tauschii suggests a novel scenario of the evolution of rearranged wheat chromosomes 4A, 5A, and 7B. Past research suggested that wheat chromosome 4A was subjected to a reciprocal translocation T(4AL;5AL)1 that occurred in the diploid progenitor of the wheat A subgenome and to three major rearrangements that occurred in polyploid wheat: pericentric inversion Inv(4AS;4AL)1, paracentric inversion Inv(4AL;4AL)1, and reciprocal translocation T(4AL;7BS)1. Gene collinearity along the pseudomolecules of tetraploid wild emmer wheat (Triticum turgidum ssp. dicoccoides, subgenomes AABB) and diploid Aegilops tauschii (genomes DD) was employed to confirm these rearrangements and to analyze the breakpoints. The exchange of distal regions of chromosome arms 4AS and 4AL due to pericentric inversion Inv(4AS;4AL)1 was detected, and breakpoints were validated with an optical Bionano genome map. Both breakpoints contained satellite DNA. The breakpoints of reciprocal translocation T(4AL;7BS)1 were also found. However, the breakpoints that generated paracentric inversion Inv(4AL;4AL)1 appeared to be collocated with the 4AL breakpoints that had produced Inv(4AS;4AL)1 and T(4AL;7BS)1. Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 either originated sequentially, and Inv(4AL;4AL)1 was produced by recurrent chromosome breaks at the same breakpoints that generated Inv(4AS;4AL)1 and T(4AL;7BS)1, or Inv(4AS;4AL)1, Inv(4AL;4AL)1, and T(4AL;7BS)1 originated simultaneously. We prefer the latter hypothesis since it makes fewer assumptions about the sequence of events that produced these chromosome rearrangements.

A 4-gigabase physical map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the wheat D-genome progenitor.

The current limitations in genome sequencing technology require the construction of physical maps for high-quality draft sequences of large plant genomes, such as that of Aegilops tauschii, the wheat D-genome progenitor. To construct a physical map of the Ae. tauschii genome, we fingerprinted 461,706 bacterial artificial chromosome clones, assembled contigs, designed a 10K Ae. tauschii Infinium SNP array, constructed a 7,185-marker genetic map, and anchored on the map contigs totaling 4.03 Gb. Using whole genome shotgun reads, we extended the SNP marker sequences and found 17,093 genes and gene fragments. We showed that collinearity of the Ae. tauschii genes with Brachypodium distachyon, rice, and sorghum decreased with phylogenetic distance and that structural genome evolution rates have been high across all investigated lineages in subfamily Pooideae, including that of Brachypodieae. We obtained additional information about the evolution of the seven Triticeae chromosomes from 12 ancestral chromosomes and uncovered a pattern of centromere inactivation accompanying nested chromosome insertions in grasses. We showed that the density of noncollinear genes along the Ae. tauschii chromosomes positively correlates with recombination rates, suggested a cause, and showed that new genes, exemplified by disease resistance genes, are preferentially located in high-recombination chromosome regions.

Synteny analysis in Rosids with a walnut physical map reveals slow genome evolution in long-lived woody perennials.

BACKGROUND: Mutations often accompany DNA replication. Since there may be fewer cell cycles per year in the germlines of long-lived than short-lived angiosperms, the genomes of long-lived angiosperms may be diverging more slowly than those of short-lived angiosperms. Here we test this hypothesis. RESULTS: We first constructed a genetic map for walnut, a woody perennial. All linkage groups were short, and recombination rates were greatly reduced in the centromeric regions. We then used the genetic map to construct a walnut bacterial artificial chromosome (BAC) clone-based physical map, which contained 15,203 exonic BAC-end sequences, and quantified with it synteny between the walnut genome and genomes of three long-lived woody perennials, Vitis vinifera, Populus trichocarpa, and Malus domestica, and three short-lived herbs, Cucumis sativus, Medicago truncatula, and Fragaria vesca. Each measure of synteny we used showed that the genomes of woody perennials were less diverged from the walnut genome than those of herbs. We also estimated the nucleotide substitution rate at silent codon positions in the walnut lineage. It was one-fifth and one-sixth of published nucleotide substitution rates in the Medicago and Arabidopsis lineages, respectively. We uncovered a whole-genome duplication in the walnut lineage, dated it to the neighborhood of the Cretaceous-Tertiary boundary, and allocated the 16 walnut chromosomes into eight homoeologous pairs. We pointed out that during polyploidy-dysploidy cycles, the dominant tendency is to reduce the chromosome number. CONCLUSION: Slow rates of nucleotide substitution are accompanied by slow rates of synteny erosion during genome divergence in woody perennials.

Signaling Pathway Alterations Driven by BRCA1 and BRCA2 Germline Mutations are Sufficient to Initiate Breast Tumorigenesis by the PIK3CAH1047R Oncogene.

Single-cell transcriptomics studies have begun to identify breast epithelial cell and stromal cell specific transcriptome differences between BRCA1/2 mutation carriers and non-carriers. We generated a single-cell transcriptome atlas of breast tissues from BRCA1, BRCA2 mutation carriers and compared this single-cell atlas of mutation carriers with our previously described single-cell breast atlas of healthy non-carriers. We observed that BRCA1 but not BRCA2 mutations altered the ratio between basal (basal-myoepithelial), luminal progenitor (luminal adaptive secretory precursor, LASP), and mature luminal (luminal hormone sensing) cells in breast tissues. A unique subcluster of cells within LASP cells is underrepresented in case of BRCA1 and BRCA2 mutation carriers compared with non-carriers. Both BRCA1 and BRCA2 mutations specifically altered transcriptomes in epithelial cells which are an integral part of NFκB, LARP1, and MYC signaling. Signaling pathway alterations in epithelial cells unique to BRCA1 mutations included STAT3, BRD4, SMARCA4, HIF2A/EPAS1, and Inhibin A signaling. BRCA2 mutations were associated with upregulation of IL6, PDK1, FOXO3, and TNFSF11 signaling. These signaling pathway alterations are sufficient to alter sensitivity of BRCA1/BRCA2-mutant breast epithelial cells to transformation as epithelial cells from BRCA1 mutation carriers overexpressing hTERT + PIK3CAH1047R generated adenocarcinomas, whereas similarly modified mutant BRCA2 cells generated basal carcinomas in NSG mice. Thus, our studies provide a high-resolution transcriptome atlas of breast epithelial cells of BRCA1 and BRCA2 mutation carriers and reveal their susceptibility to PIK3CA mutation-driven transformation. SIGNIFICANCE: This study provides a single-cell atlas of breast tissues of BRCA1/2 mutation carriers and demonstrates that aberrant signaling due to BRCA1/2 mutations is sufficient to initiate breast cancer by mutant PIK3CA.

A genome sequence for the threatened whitebark pine.

Whitebark pine (WBP, Pinus albicaulis) is a white pine of subalpine regions in the Western contiguous United States and Canada. WBP has become critically threatened throughout a significant part of its natural range due to mortality from the introduced fungal pathogen white pine blister rust (WPBR, Cronartium ribicola) and additional threats from mountain pine beetle (Dendroctonus ponderosae), wildfire, and maladaptation due to changing climate. Vast acreages of WBP have suffered nearly complete mortality. Genomic technologies can contribute to a faster, more cost-effective approach to the traditional practices of identifying disease-resistant, climate-adapted seed sources for restoration. With deep-coverage Illumina short reads of haploid megagametophyte tissue and Oxford Nanopore long reads of diploid needle tissue, followed by a hybrid, multistep assembly approach, we produced a final assembly containing 27.6 Gb of sequence in 92,740 contigs (N50 537,007 bp) and 34,716 scaffolds (N50 2.0 Gb). Approximately 87.2% (24.0 Gb) of total sequence was placed on the 12 WBP chromosomes. Annotation yielded 25,362 protein-coding genes, and over 77% of the genome was characterized as repeats. WBP has demonstrated the greatest variation in resistance to WPBR among the North American white pines. Candidate genes for quantitative resistance include disease resistance genes known as nucleotide-binding leucine-rich repeat receptors (NLRs). A combination of protein domain alignments and direct genome scanning was employed to fully describe the 3 subclasses of NLRs. Our high-quality reference sequence and annotation provide a marked improvement in NLR identification compared to previous assessments that leveraged de novo-assembled transcriptomes.

Single-nucleus chromatin accessibility and transcriptomic map of breast tissues of women of diverse genetic ancestry.

Single-nucleus analysis allows robust cell-type classification and helps to establish relationships between chromatin accessibility and cell-type-specific gene expression. Here, using samples from 92 women of several genetic ancestries, we developed a comprehensive chromatin accessibility and gene expression atlas of the breast tissue. Integrated analysis revealed ten distinct cell types, including three major epithelial subtypes (luminal hormone sensing, luminal adaptive secretory precursor (LASP) and basal-myoepithelial), two endothelial and adipocyte subtypes, fibroblasts, T cells, and macrophages. In addition to the known cell identity genes FOXA1 (luminal hormone sensing), EHF and ELF5 (LASP), TP63 and KRT14 (basal-myoepithelial), epithelial subtypes displayed several uncharacterized markers and inferred gene regulatory networks. By integrating breast epithelial cell gene expression signatures with spatial transcriptomics, we identified gene expression and signaling differences between lobular and ductal epithelial cells and age-associated changes in signaling networks. LASP cells and fibroblasts showed genetic ancestry-dependent variability. An estrogen receptor-positive subpopulation of LASP cells with alveolar progenitor cell state was enriched in women of Indigenous American ancestry. Fibroblasts from breast tissues of women of African and European ancestry clustered differently, with accompanying gene expression differences. Collectively, these data provide a vital resource for further exploring genetic ancestry-dependent variability in healthy breast biology.

Integrated Single-Cell Multiomic Profiling of Caudate Nucleus Suggests Key Mechanisms in Alcohol Use Disorder.

Alcohol use disorder (AUD) is likely associated with complex transcriptional alterations in addiction-relevant brain regions. We characterize AUD-associated differences in cell type-specific gene expression and chromatin accessibility in the caudate nucleus by conducting a single-nucleus RNA-seq assay and a single-nucleus RNA-seq + ATAC-seq (multiome) assay on caudate tissue from 143 human postmortem brains (74 with AUD). We identified 17 cell types. AUD was associated with a higher proportion of microglia in an activated state and more astrocytes in a reactive state. There was widespread evidence for differentially expressed genes across cell types with the most identified in oligodendrocytes and astrocytes, including genes involved in immune response and synaptic regulation, many of which appeared to be regulated in part by JUND and OLIG2. Microglia-astrocyte communication via interleukin-1 beta, and microglia-astrocyte-oligodendrocyte interaction via transforming growth factor beta 1 were increased in individuals with AUD. Expression quantitative trait loci analysis revealed potential driver genes of AUD, including ADAL, that may protect against AUD in medium spiny neurons and interneurons. This work provides a thorough profile of the effects of AUD in the human brain and identifies several promising genes for further study.

Osteogenic Differentiation Potential of Mesenchymal Stem Cells Using Single Cell Multiomic Analysis.

Mesenchymal stem cells (MSC) are multipotent stem cells that can differentiate into multiple cell types, including osteoblasts, chondrocytes, and adipocytes. Osteoblast differentiation is reduced during osteoporosis development, resulting in reduced bone formation. Further, MSC isolated from different donors possess distinct osteogenic capacity. In this study, we used single-cell multiomic analysis to profile the transcriptome and epigenome of MSC from four healthy donors. Data were obtained from ~1300 to 1600 cells for each donor. These cells were clustered into four groups, indicating that MSC from different donors have distinct chromatin accessible regulatory elements for regulating gene expression. To investigate the mechanism by which MSC undergo osteogenic differentiation, we used the chromatin accessibility data from the single-cell multiome data to identify individual-specific enhancer-promoter pairs and evaluated the expression levels and activities of the transcriptional regulators. The MSC from four donors showed distinct differentiation potential into osteoblasts. MSC of donor 1 showed the largest average motif activities, indicating that MSC from donor 1 was most likely to differentiate into osteoblasts. The results of our validation experiments were consistent with the bioinformatics prediction. We also tested the enrichment of genome-wide association study (GWAS) signals of several musculoskeletal disease traits in the patient-specific chromatin accessible regions identified in the single-cell multiome data, including osteoporosis, osteopenia, and osteoarthritis. We found that osteoarthritis-associated variants were only enriched in the regions identified from donor 4. In contrast, osteoporosis and osteopenia variants were enriched in regions from donor 1 and least enriched in donor 4. Since osteoporosis and osteopenia are related to the density of bone cells, the enrichment of variants from these traits should be correlated with the osteogenic potential of MSC. In summary, this study provides large-scale data to link regulatory elements with their target genes to study the regulatory relationships during the differentiation of mesenchymal stem cells and provide a deeper insight into the gene regulatory mechanism.

A Genome Sequence for the Threatened Whitebark Pine.

Whitebark pine (WBP, Pinus albicaulis ) is a white pine of subalpine regions in western contiguous US and Canada. WBP has become critically threatened throughout a significant part of its natural range due to mortality from the introduced fungal pathogen white pine blister rust (WPBR, Cronartium ribicola ) and additional threats from mountain pine beetle ( Dendroctonus ponderosae ), wildfire, and maladaptation due to changing climate. Vast acreages of WBP have suffered nearly complete mortality. Genomic technologies can contribute to a faster, more cost-effective approach to the traditional practices of identifying disease-resistant, climate-adapted seed sources for restoration. With deep-coverage Illumina short-reads of haploid megametophyte tissue and Oxford Nanopore long-reads of diploid needle tissue, followed by a hybrid, multistep assembly approach, we produced a final assembly containing 27.6 Gbp of sequence in 92,740 contigs (N50 537,007 bp) and 34,716 scaffolds (N50 2.0 Gbp). Approximately 87.2% (24.0 Gbp) of total sequence was placed on the twelve WBP chromosomes. Annotation yielded 25,362 protein-coding genes, and over 77% of the genome was characterized as repeats. WBP has demonstrated the greatest variation in resistance to WPBR among the North American white pines. Candidate genes for quantitative resistance include disease resistance genes known as nucleotide-binding leucine-rich-repeat receptors (NLRs). A combination of protein domain alignments and direct genome scanning was employed to fully describe the three subclasses of NLRs (TNL, CNL, RNL). Our high-quality reference sequence and annotation provide a marked improvement in NLR identification compared to previous assessments that leveraged de novo assembled transcriptomes.

A Pilot Single Cell Analysis of the Zebrafish Embryo Cellular Responses to Uropathogenic Escherichia coli Infection.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) infections are common and when they disseminate can be of high morbidity. METHODS: We studied the effects of UPEC infection using single cell RNA sequencing (scRNAseq) in zebrafish. Bulk RNA sequencing has historically been used to evaluate gene expression patterns, but scRNAseq allows gene expression to be evaluated at the single cell level and is optimal for evaluating heterogeneity within cell types and rare cell types. Zebrafish cohorts were injected with either saline or UPEC, and scRNAseq and canonical pathway analyses were performed. RESULTS: Canonical pathway analysis of scRNAseq data provided key information regarding innate immune pathways in the cells determined to be thymus cells, ionocytes, macrophages/monocytes, and pronephros cells. Pathways activated in thymus cells included interleukin 6 (IL-6) signaling and production of reactive oxygen species. Fc receptor-mediated phagocytosis was a leading canonical pathway in the pronephros and macrophages. Genes that were downregulated in UPEC vs saline exposed embryos involved the cellular response to the Gram-negative endotoxin lipopolysaccharide (LPS) and included Forkhead Box O1a (Foxo1a), Tribbles Pseudokinase 3 (Trib3), Arginase 2 (Arg2) and Polo Like Kinase 3 (Plk3). CONCLUSIONS: Because 4-day post fertilization zebrafish embryos only have innate immune systems, the scRNAseq provides insights into pathways and genes that cell types utilize in the bacterial response. Based on our analysis, we have identified genes and pathways that might serve as genetic targets for treatment and further investigation in UPEC infections at the single cell level.

Nocturnal plant respiration is under strong non-temperature control.

Most biological rates depend on the rate of respiration. Temperature variation is typically considered the main driver of daily plant respiration rates, assuming a constant daily respiration rate at a set temperature. Here, we show empirical data from 31 species from temperate and tropical biomes to demonstrate that the rate of plant respiration at a constant temperature decreases monotonically with time through the night, on average by 25% after 8 h of darkness. Temperature controls less than half of the total nocturnal variation in respiration. A new universal formulation is developed to model and understand nocturnal plant respiration, combining the nocturnal decrease in the rate of plant respiration at constant temperature with the decrease in plant respiration according to the temperature sensitivity. Application of the new formulation shows a global reduction of 4.5 -6 % in plant respiration and an increase of 7-10% in net primary production for the present-day.