An immunophenotype-coupled transcriptomic atlas of human hematopoietic progenitors.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.

Differential transpiration between pods and leaves during stress combination in soybean.

Climate change is causing an increase in the frequency and intensity of droughts, heat waves, and their combinations, diminishing agricultural productivity and destabilizing societies worldwide. We recently reported that during a combination of water deficit (WD) and heat stress (HS), stomata on leaves of soybean (Glycine max) plants are closed, while stomata on flowers are open. This unique stomatal response was accompanied by differential transpiration (higher in flowers, while lower in leaves) that cooled flowers during a combination of WD + HS. Here, we reveal that developing pods of soybean plants subjected to a combination of WD + HS use a similar acclimation strategy of differential transpiration to reduce internal pod temperature by approximately 4 °C. We further show that enhanced expression of transcripts involved in abscisic acid degradation accompanies this response and that preventing pod transpiration by sealing stomata causes a significant increase in internal pod temperature. Using an RNA-Seq analysis of pods developing on plants subjected to WD + HS, we also show that the response of pods to WD, HS, or WD + HS is distinct from that of leaves or flowers. Interestingly, we report that although the number of flowers, pods, and seeds per plant decreases under conditions of WD + HS, the seed mass of plants subjected to WD + HS increases compared to plants subjected to HS, and the number of seeds with suppressed/aborted development is lower in WD + HS compared to HS. Taken together, our findings reveal that differential transpiration occurs in pods of soybean plants subjected to WD + HS and that this process limits heat-induced damage to seed production.

Differential regulation of flower transpiration during abiotic stress in annual plants.

Heat waves occurring during droughts can have a devastating impact on yield, especially if they happen during the flowering and seed set stages of the crop cycle. Global warming and climate change are driving an alarming increase in the frequency and intensity of combined drought and heat stress episodes, critically threatening global food security. Because high temperature is detrimental to reproductive processes, essential for plant yield, we measured the inner temperature, transpiration, sepal stomatal aperture, hormone concentrations and transcriptomic response of closed soybean flowers developing on plants subjected to a combination of drought and heat stress. Here, we report that, during a combination of drought and heat stress, soybean plants prioritize transpiration through flowers over transpiration through leaves by opening their flower stomata, while keeping their leaf stomata closed. This acclimation strategy, termed 'differential transpiration', lowers flower inner temperature by about 2-3°C, protecting reproductive processes at the expense of vegetative tissues. Manipulating stomatal regulation, stomatal size and/or stomatal density of flowers could serve as a viable strategy to enhance the yield of different crops and mitigate some of the current and future impacts of global warming and climate change on agriculture.

12-Hydroxy-Jasmonoyl-l-Isoleucine Is an Active Jasmonate That Signals through CORONATINE INSENSITIVE 1 and Contributes to the Wound Response in Arabidopsis.

12-hydroxy-jasmonoyl-isoleucine (12OH-JA-Ile) is a metabolite in the catabolic pathway of the plant hormone jasmonate, and is synthesized by the cytochrome P450 subclade 94 enzymes. Contrary to the well-established function of jasmonoyl-isoleucine (JA-Ile) as the endogenous bioactive form of jasmonate, the function of 12OH-JA-Ile is unclear. Here, the potential role of 12OH-JA-Ile in jasmonate signaling and wound response was investigated. Exogenous application of 12OH-JA-Ile mimicked several JA-Ile effects including marker gene expression, anthocyanin accumulation and trichome induction in Arabidopsis thaliana. Genome-wide transcriptomics and untargeted metabolite analyses showed large overlaps between those affected by 12OH-JA-Ile and JA-Ile. 12OH-JA-Ile signaling was blocked by mutation in CORONATINE INSENSITIVE 1. Increased anthocyanin accumulation by 12OH-JA-Ile was additionally observed in tomato and sorghum, and was disrupted by the COI1 defect in tomato jai1 mutant. In silico ligand docking predicted that 12OH-JA-Ile can maintain many of the key interactions with COI1-JAZ1 residues identified earlier by crystal structure studies using JA-Ile as ligand. Genetic alternation of jasmonate metabolic pathways in Arabidopsis to deplete both JA-Ile and 12OH-JA-Ile displayed enhanced jasmonate deficient wound phenotypes and was more susceptible to insect herbivory than that depleted in only JA-Ile. Conversely, mutants overaccumulating 12OH-JA-Ile showed intensified wound responses compared with wild type with similar JA-Ile content. These data are indicative of 12OH-JA-Ile functioning as an active jasmonate signal and contributing to wound and defense response in higher plants.

Gene expression profiles of ion channels and receptors in mouse resistance arteries: Effects of cell type, vascular bed, and age.

OBJECTIVE: Receptors and ion channels of smooth muscle cells (SMCs) and endothelial cells (ECs) are integral to the regulation of vessel diameter and tissue blood flow. Physiological roles of ion channels and receptors in skeletal muscle and mesenteric arteries have been identified; however, their gene expression profiles are undefined. We tested the hypothesis that expression profiles for ion channels and receptors governing vascular reactivity vary with cell type, vascular bed, and age. METHODS: Mesenteric and superior epigastric arteries were dissected from Old (24-26 months) and Young (3-6 months) C57BL/6J mice. ECs and SMCs were collected for analysis with custom qRT-PCR arrays to determine expression profiles of 80 ion channel and receptor genes. Bioinformatics analyses were applied to gain insight into functional interactions. RESULTS: We identified 68 differences in gene expression with respect to cell type, vessel type, and age. Heat maps illustrate differential expression, and distance matrices predict patterns of coexpression. Gene networks based upon protein-protein interaction datasets and KEGG pathways illustrate biological processes affected by specific differences in gene expression. CONCLUSIONS: Differences in gene expression profiles are most pronounced between microvascular ECs and SMCs with subtle variations between vascular beds and age groups.

Multiomic profiling identifies predictors of survival in African American patients with acute myeloid leukemia.

Genomic profiles and prognostic biomarkers in patients with acute myeloid leukemia (AML) from ancestry-diverse populations are underexplored. We analyzed the exomes and transcriptomes of 100 patients with AML with genomically confirmed African ancestry (Black; Alliance) and compared their somatic mutation frequencies with those of 323 self-reported white patients with AML, 55% of whom had genomically confirmed European ancestry (white; BeatAML). Here we find that 73% of 162 gene mutations recurrent in Black patients, including a hitherto unreported PHIP alteration detected in 7% of patients, were found in one white patient or not detected. Black patients with myelodysplasia-related AML were younger than white patients suggesting intrinsic and/or extrinsic dysplasia-causing stressors. On multivariable analyses of Black patients, NPM1 and NRAS mutations were associated with inferior disease-free and IDH1 and IDH2 mutations with reduced overall survival. Inflammatory profiles, cell type distributions and transcriptional profiles differed between Black and white patients with NPM1 mutations. Incorporation of ancestry-specific risk markers into the 2022 European LeukemiaNet genetic risk stratification changed risk group assignment for one-third of Black patients and improved their outcome prediction.

Comparative Transcriptomics Reveal Metabolic Rather than Genetic Control of Divergent Antioxidant Metabolism in the Primary Root Elongation Zone of Water-Stressed Cotton and Maize.

Under water stress, the primary root elongation zones of cotton and maize exhibit both conserved and divergent metabolic responses, including variations in sulfur and antioxidant metabolism. To explore the relative importance of metabolic and genetic controls of these responses for each species, and the extent to which responses are mediated by similar gene expression networks within the framework of ortholog groups, comparative transcriptomics analyses were conducted under conditions of equivalent tissue water stress. Ortholog analysis revealed that 86% of the transcriptome response to water stress was phylogenetically unrelated between cotton and maize. Elevated transcript abundances for genes involved in abscisic acid (ABA) biosynthesis and signaling, as well as key enzymes that enable osmotic adjustment, were conserved between the species. In contrast, antioxidant responses, at least with regard to glutathione metabolism and anti-oxidative enzymes, did not exhibit such a transcript abundance adaptive signature. In particular, previously characterized differential responses of the glutathione and sulfur metabolic pathways between cotton and maize were not evident in the transcriptomic responses. The findings indicate that the antioxidant response in both species results from a metabolic acclimation to water stress, and thus represents an example of water stress-related metabolic plasticity.

Adventitial macrophage accumulation impairs perivascular nerve function in mesenteric arteries with inflammatory bowel disease.

INTRODUCTION: Inflammatory bowel disease (IBD) involves aberrant immune responses and is associated with both cardiovascular disease risk and altered intestinal blood flow. However, little is known about how IBD affects regulation of perivascular nerves that mediate blood flow. Previous work found perivascular nerve function is impaired in mesenteric arteries with IBD. The purpose of this study was to determine the mechanism of impaired perivascular nerve function. METHODS: RNA sequencing was performed on mesenteric arteries from IL10-/- mice treated with H.hepaticus to induce disease (IBD) or left non-gavaged (Control). For all other studies, Control and IBD mice received either saline or clodronate liposome injections to study the effect of macrophage depletion. Perivascular nerve function was assessed using pressure myography and electrical field stimulation. Fluorescent immunolabeling was used to label leukocyte populations and perivascular nerves. RESULTS: IBD was associated with increased in macrophage-associated gene expression, and immunolabeling showed accumulation of adventitial macrophages. Clodronate liposome injection eliminated adventitial macrophages, which reversed significant attenuation of sensory vasodilation, sympathetic vasoconstriction and sensory inhibition of sympathetic constriction in IBD. Acetylcholine-mediated dilation was impaired in IBD and restored after macrophage depletion, but sensory dilation remained nitric oxide independent regardless of disease and/or macrophage presence. CONCLUSION: Altered neuro-immune signaling between macrophages and perivascular nerves in the arterial adventitia contributes to impaired vasodilation, particularly via dilatory sensory nerves. Targeting the adventitial macrophage population may help preserve intestinal blood flow in IBD patients.

Adventitial macrophage accumulation impairs perivascular nerve function in mesenteric arteries with inflammatory bowel disease.

Introduction: Inflammatory bowel disease involves aberrant immune responses and is associated with both cardiovascular disease risk and altered intestinal blood flow. However, little is known about how inflammatory bowel disease affects regulation of perivascular nerves that mediate blood flow. Previous work found perivascular nerve function is impaired in mesenteric arteries with Inflammatory bowel disease. The purpose of this study was to determine the mechanism of impaired perivascular nerve function. Methods: RNA sequencing was performed on mesenteric arteries from IL10-/- mice treated with H. hepaticus to induce disease (inflammatory bowel disease) or left non-gavaged (Control). For all other studies, Control and Inflammatory bowel disease mice received either saline or clodronate liposome injections to study the effect of macrophage depletion. Perivascular nerve function was assessed using pressure myography and electrical field stimulation. Leukocyte populations, and perivascular nerves, and adventitial neurotransmitter receptors were labeled using fluorescent immunolabeling. Results: Inflammatory bowel disease was associated with increases in macrophage-associated gene expression, and immunolabeling showed accumulation of adventitial macrophages. Clodronate liposome injection eliminated adventitial macrophages, which reversed significant attenuation of sensory vasodilation, sympathetic vasoconstriction and sensory inhibition of sympathetic constriction in inflammatory bowel disease. Acetylcholine-mediated dilation was impaired in inflammatory bowel disease and restored after macrophage depletion, but sensory dilation remained nitric oxide independent regardless of disease and/or macrophage presence. Conclusion: Altered neuro-immune signaling between macrophages and perivascular nerves in the arterial adventitia contributes to impaired vasodilation, particularly via dilatory sensory nerves. Targeting the adventitial macrophage population may help preserve intestinal blood flow in Inflammatory bowel disease patients.

De novo transcriptome assembly from the nodal root growth zone of hydrated and water-deficit stressed maize inbred line FR697.

Certain cultivars of maize show increased tolerance to water deficit conditions by maintenance of root growth. To better understand the molecular mechanisms related to this adaptation, nodal root growth zone samples were collected from the reference inbred line B73 and inbred line FR697, which exhibits a relatively greater ability to maintain root elongation under water deficits. Plants were grown under various water stress levels in both field and controlled environment settings. FR697-specific RNA-Seq datasets were generated and used for a de novo transcriptome assembly to characterize any genotype-specific genetic features. The assembly was aided by an Iso-Seq library of transcripts generated from various FR697 plant tissue samples. The Necklace pipeline was used to combine a Trinity de novo assembly along with a reference guided assembly and the Viridiplantae proteome to generate an annotated consensus "SuperTranscriptome" assembly of 47,915 transcripts with a N50 of 3152 bp in length. The results were compared by Blastn to maize reference genes, a Benchmarking Universal Single-Copy Orthologs (BUSCO) genome completeness report and compared with three maize reference genomes. The resultant 'SuperTranscriptome' was demonstrated to be of high-quality and will serve as an important reference for analysis of the maize nodal root transcriptomic response to environmental perturbations.

Zinc is a master-regulator of sperm function associated with binding, motility, and metabolic modulation during porcine sperm capacitation.

Sperm capacitation is a post-testicular maturation step endowing spermatozoa with fertilizing capacity within the female reproductive tract, significant for fertility, reproductive health, and contraception. Recently discovered mammalian sperm zinc signatures and their changes during sperm in vitro capacitation (IVC) warranted a more in-depth study of zinc interacting proteins (further zincoproteins). Here, we identified 1752 zincoproteins, with 102 changing significantly in abundance (P < 0.05) after IVC. These are distributed across 8 molecular functions, 16 biological processes, and 22 protein classes representing 130 pathways. Two key, paradigm-shifting observations were made: i) during sperm capacitation, molecular functions of zincoproteins are both upregulated and downregulated within several molecular function categories; and ii) Huntington's and Parkinson's disease pathways were the two most represented, making spermatozoon a candidate model for studying neurodegenerative diseases. These findings highlight the importance of Zn2+ homeostasis in reproduction, offering new avenues in semen processing for human-assisted reproductive therapy, identification of somatic-reproductive comorbidities, and livestock breeding.

Transcriptomics Analysis Reveals New Insights into the Roles of Notch1 Signaling on Macrophage Polarization.

Naïve macrophages (Mφ) polarize in response to various environmental cues to a spectrum of cells that have distinct biological functions. The extreme ends of the spectrum are classified as M1 and M2 macrophages. Previously, we demonstrated that Notch1 deficiency promotes Tgf-ß2 dependent M2-polarization in a mouse model of abdominal aortic aneurysm. The present studies aimed to characterize the unique set of genes regulated by Notch1 signaling in macrophage polarization. Bone marrow derived macrophages isolated from WT or Notch1+/- mice (n = 12) were differentiated to Mφ, M1 or M2-phenotypes by 24 h exposure to vehicle, LPS/IFN-γ or IL4/IL13 respectively and total RNA was subjected to RNA-Sequencing (n = 3). Bioinformatics analyses demonstrated that Notch1 haploinsufficiency downregulated the expression of 262 genes at baseline level, 307 genes with LPS/IFN-γ and 254 genes with IL4/IL13 treatment. Among these, the most unique genes downregulated by Notch1 haploinsufficiency included fibromodulin (Fmod), caspase-4, Has1, Col1a1, Alpl and Igf. Pathway analysis demonstrated that extracellular matrix, macrophage polarization and osteogenesis were the major pathways affected by Notch1 haploinsufficiency. Gain and loss-of-function studies established a strong correlation between Notch1 haploinsufficiency and Fmod in regulating Tgf-ß signaling. Collectively, our studies suggest that Notch1 haploinsufficiency increases M2 polarization through these newly identified genes.

The DNA binding landscape of the maize AUXIN RESPONSE FACTOR family.

AUXIN RESPONSE FACTORS (ARFs) are plant-specific transcription factors (TFs) that couple perception of the hormone auxin to gene expression programs essential to all land plants. As with many large TF families, a key question is whether individual members determine developmental specificity by binding distinct target genes. We use DAP-seq to generate genome-wide in vitro TF:DNA interaction maps for fourteen maize ARFs from the evolutionarily conserved A and B clades. Comparative analysis reveal a high degree of binding site overlap for ARFs of the same clade, but largely distinct clade A and B binding. Many sites are however co-occupied by ARFs from both clades, suggesting transcriptional coordination for many genes. Among these, we investigate known QTLs and use machine learning to predict the impact of cis-regulatory variation. Overall, large-scale comparative analysis of ARF binding suggests that auxin response specificity may be determined by factors other than individual ARF binding site selection.

Phosphate Deficiency Negatively Affects Early Steps of the Symbiosis between Common Bean and Rhizobia.

Phosphate (Pi) deficiency reduces nodule formation and development in different legume species including common bean. Despite significant progress in the understanding of the genetic responses underlying the adaptation of nodules to Pi deficiency, it is still unclear whether this nutritional deficiency interferes with the molecular dialogue between legumes and rhizobia. If so, what part of the molecular dialogue is impaired? In this study, we provide evidence demonstrating that Pi deficiency negatively affects critical early molecular and physiological responses that are required for a successful symbiosis between common bean and rhizobia. We demonstrated that the infection thread formation and the expression of PvNSP2, PvNIN, and PvFLOT2, which are genes controlling the nodulation process were significantly reduced in Pi-deficient common bean seedlings. In addition, whole-genome transcriptional analysis revealed that the expression of hormones-related genes is compromised in Pi-deficient seedlings inoculated with rhizobia. Moreover, we showed that regardless of the presence or absence of rhizobia, the expression of PvRIC1 and PvRIC2, two genes participating in the autoregulation of nodule numbers, was higher in Pi-deficient seedlings compared to control seedlings. The data presented in this study provides a mechanistic model to better understand how Pi deficiency impacts the early steps of the symbiosis between common bean and rhizobia.