Mature Population Research Articles

Cpne7 deficiency induces cellular senescence and premature aging of dental pulp.

Once tooth development is complete, odontoblasts and their progenitor cells in the dental pulp play a major role in protecting tooth vitality from external stresses. Hence, understanding the homeostasis of the mature pulp populations is just as crucial as understanding that of the young, developing ones for managing age-related dentinal damage. Here, it is shown that loss of Cpne7 accelerates cellular senescence in odontoblasts due to oxidative stress and DNA damage accumulation. Thus, in Cpne7-null dental pulp, odontoblast survival is impaired, and aberrant dentin is extensively formed. Intraperitoneal or topical application of CPNE7-derived functional peptide, however, alleviates the DNA damage accumulation and rescues the pathologic dentin phenotype. Notably, a healthy dentin-pulp complex lined with metabolically active odontoblasts is observed in 23-month-old Cpne7-overexpressing transgenic mice. Furthermore, physiologic dentin was regenerated in artificial dentinal defects of Cpne7-overexpressing transgenic mice. Taken together, Cpne7 is indispensable for the maintenance and homeostasis of odontoblasts, while promoting odontoblastic differentiation of the progenitor cells. This research thereby introduces its potential in oral disease-targeted applications, especially age-related dental diseases involving dentinal loss.

Exercise to transform tumours from cold to hot and improve immunotherapy responsiveness.

Exercise provides significant health benefits to patients diagnosed with cancer including improved survival outcomes, quality of life and reduced cancer recurrence. Across multiple murine cancer models, aerobic exercise and resistance training has exhibited anti-tumour properties illustrated by inhibited tumour growth, reduced metastatic potential and modulation of the tumour microenvironment to allow the recognition and destruction of cancer cells. Clinical studies have demonstrated the rapid mobilisation and circulatory release of mature lymphoid populations, myokines and cytokines that occurs with exercise along with tumour vasculature normalisation. Tumour microenvironments enriched with immune cells with anti-cancer potential, such as CD8+ T cells, are termed 'hot', whilst those favouring an immunosuppressive environment and lacking in effector immune cells are classed as 'cold'. Pre-clinical evidence suggests exercise training has the potential to reprogramme cold tumours to become hot, although this requires validation in clinical studies. This hot environment could potentiate immunotherapy responsiveness, improving survival outcomes of patients undergoing cancer immunotherapy and allow those with typically cold tumours to benefit from immunotherapy. This review discusses the complex interactions between exercise and cancer, including exercise-induced alterations within the tumour microenvironment and systemic immunity. The potential role exercise may play in improving cancer immunotherapy responsiveness is explored. This review also highlights the need for translational studies exploring the role of exercise in patients with cancer with the potential to widen the spectrum of tumours that derive significant benefit from immunotherapy.

Perioperative Characteristics and Outcomes of Fontan Versus Non-Fontan Patients Undergoing Combined Heart-Liver Transplantation: A Retrospective Cohort Study

Combined heart-liver transplantation (CHLT) is becoming increasingly frequent as a maturing population of patients with Fontan-palliated congenital heart disease develop advanced liver fibrosis or cirrhosis. The authors present their experience with CHLT for congenital and noncongenital indications, and identify characteristics associated with poor outcomes that may guide intervention in high-risk patients. This was a single-center retrospective cohort study. This study was conducted at Vanderbilt University Medical Center in Nashville, Tennessee. The study included 16 consecutive adult recipients of CHLT at the authors' institution between April 2017 and February 2022. Eleven patients underwent transplantation for Fontan indications, and 5 were transplanted for non-Fontan indications. Compared with non-Fontan patients, Fontan recipients had longer cardiopulmonary bypass duration (199 v 119 minutes, p =m0.002), operative times (786 v 599 minutes, p = 0.01), and larger blood product transfusions (15.4 v 6.3 L, p = 0.18). Six of 16 patients required extracorporeal membrane oxygenation (ECMO), of whom 4 were Fontan patients who subsequently died. Patients who required ECMO had lower 5-hour lactate clearance (0.0 v 3.5 mmol/L, p = 0.001), higher number of vasoactive infusions, lower pulmonary artery pulsatility indices (0.58 v 1.77, p = 0.03), and higher peak inspiratory pressures (28.0 v 18.5 mmHg, p = 0.01) after liver reperfusion. Combined heart-liver transplantation in patients with Fontan-associated end-organ disease is particularly challenging and associated with higher recipient morbidity compared with non-Fontan-related CHLT. Early hemodynamic intervention for signs of ventricular dysfunction may improve outcomes in this growing high-risk population.

Srcap Plays an Essential Role in Normal Hematopoiesis

The incorporation of variant histones into chromatin has been revealed to be an important mechanism of epigenetic regulation. Deposition of H2A.Z, in particular, is known to mediate several essential cellular processes including chromatin organization, DNA damage response, and transcriptional regulation. Nonsense and frameshift mutations in the H2A.Z exchanger, SRCAP, have been recurrently identified in both clonal hematopoiesis and AML, suggesting the importance of this gene in both non-malignant hematopoiesis and leukemogenesis. However, the precise role of SRCAP and the impact of its dysregulation on hematopoiesis remain unknown. To investigate the role of Srcap in hematopoiesis, we generated a conditional Srcap loss of function murine model with loxP sites flanking exons 11-16. Cre-induced deletion of these exons results in a loss of the catalytic ATPase domain along with a frameshift within the remaining protein sequence. When Vav1-iCre +/-Srcap fl/+ and Srcap fl/fl mice are crossed, no Vav1-iCre +/-Srcap fl/fl pups are born, suggesting that hematopoietic-specific-loss of Srcap is embryonic lethal. At E14.5/15.5, Vav1-iCre +/-Srcap fl/fl embryos are paler than other genotypes ( Figure 1) with a reduced number of Ter119 + fetal liver cells. Interestingly, the decrease in Ter119 + cells upon Srcap loss occurs primarily in later stage erythroid progenitors, whereas the number of stage 1 erythroid progenitors is relatively well preserved, suggesting that loss of Srcap adversely impacts fetal erythroid maturation. Furthermore, Srcap loss impacts fetal liver myeloid maturation. While the number of granulocytes is unchanged in Srcap deficient embryos, they have a near complete absence of monocytes, monocyte-dendritic cell progenitors, and monocyte progenitors. In contrast to the changes observed within mature hematopoietic populations, phenotypic hematopoietic stem cells (HSCs) are maintained in Vav1-iCre +/-Srcap fl/fl fetal livers. However, their function is impaired as fetal liver HSCs fail to repopulate upon transplantation ( Figure 2). Importantly, adult HSCs isolated from Mx1-Cre +/-Srcap fl/flmice show a very similar phenotype in that they fail to maintain hematopoiesis when transplanted and treated with poly (I:C). In total, this data shows that Srcap is essential for the maintenance and proper function of both mature hematopoietic populations and hematopoietic progenitors with different lineages and developmental stages displaying varied sensitivities to Srcap loss. To investigate the cellular consequences of SRCAP loss, we knocked down SRCAP expression in hematopoietic cell lines using an inducible shRNA approach. In agreement with the results obtained from our murine model, SRCAP deficient MOLM13 cells show a baseline competitive growth disadvantage that becomes apparent within one week of culture. This is associated with increased γ-H2AX activity, suggesting enhanced DNA damage and consistent with the previously identified role of SRCAP in the cellular DNA damage response. Interestingly, murine fetal liver HSCs with loss of Srcap also exhibit increased γ-H2AX activity, and Srcap null fetal liver stage 1 erythroid progenitors display marked transcriptional upregulation of multiple p53 downstream genes. Our data demonstrate that, in hematopoietic populations, Srcap is an essential gene, with its complete loss resulting in enhanced cellular stress potentially due to increased DNA damage. In humans, nonsense and frameshift mutations in SRCAP appear to be heterozygous in nature, suggesting that the impact of SRCAP deficiency is dose dependent. Indeed, murine HSCs with heterozygous loss of Srcap maintain normal repopulating activity in both primary and secondary transplantation assays ( Figure 2). It remains to be determined how the intermediate deficiencies in SRCAP levels found in patients impact hematopoiesis and lead to clonal expansion and whether the impact of SRCAP loss on the cellular response to stress, including DNA damage, is mediated by its canonical function as a histone variant H2A.Z exchanger. These studies promise to reveal novel insights on the nature of H2A.Z-mediated epigenetic regulation in HSCs and on how its dysregulation may lead to leukemogenesis.

Isocitrate Dehydrogenase 2 Mutation Allows Myeloid Differentiation but Impairs Bone Marrow Macrophage Polarization and Function Via Metabolic Dysregulation

Isocitrate dehydrogenase 2 (IDH2) mutations, while less common than other mutations associated with myelodysplastic syndromes (MDS), are also found in clonal hematopoiesis and are associated with increased risk of transformation to leukemia. IDH2 mutations lead to metabolic dysfunction and DNA hypermethylation due to the accumulation of D-2-hydroxyglutarate (2HG), an oncometabolite derived from alpha-ketoglutarate (aKG), which disrupts hematopoiesis. However, the mechanisms that induce clonal progression and transformation remain incompletely understood. Furthermore, the intrinsic and extrinsic impact of IDH mutations on mature myeloid cell functions and the bone marrow microenvironment remains to be elucidated. Previous literature suggested that hypermethylation via this mutant leads to incomplete differentiation and failed mature myeloid cell generation. Our lab previously demonstrated that efferocytic defects in bone marrow macrophages can skew hematopoietic stem and progenitor cells via increased inflammatory mediators. Based on these data, we hypothesized that IDH2 mutations prevent complete polarization down pro-efferocytic phenotypes in macrophages due to blocking of metabolic pathways required for mature macrophage function. Congenic CD45.1+ mice were transplanted with retrovirally infected hematopoietic stem cells containing human WT or R172K IDH2 mutant with an mCherry reporter and showed macrocytic anemia, thrombocytopenia, and elevated reticulocytes by 16 weeks post-transplant. In contrast to prior literature, IDH2 mutants gave rise to mature reporter positive monocytes, macrophages, and neutrophils in the bone marrow and peripheral blood. As expected, we observed myeloid skewed hematopoiesis, a trademark of MDS. When cultured ex vivo, there was loss of efferocytic capacity in IDH2 mutant bone marrow macrophages when fed apoptotic neutrophils. We then generated a genetic murine model using the Vav1 Cre IDH2 R140Q floxed mutant. The genetic model allowed us to study disease progression in the absence of radiation induced stress, and to observe mature myeloid populations while clinically defined MDS was absent. We performed ATACseq and bulk RNAseq analysis on bone marrow macrophages isolated from 3-month-old and 6-month-old mice, which demonstrated a significant decrease in global chromosomal accessibility; however, this had limited impact on the transcriptional program. Based on existing literature, the majority of macrophages in the bone marrow are in a poised state and have not yet activated genes associated with pro-efferocytic/pro-resolution or pro-inflammatory phenotypes. Since aKG is accumulated in macrophages during polarization toward reparative and pro-efferocytic phenotypes and is necessary for metabolic reprogramming for oxidative phosphorylation, we hypothesized that, in the presence of IDH2 mutations, which drain aKG from the system, pro-efferocytic polarized macrophages would be metabolically stunned. To test this theory, we isolated bone marrow macrophages from IDH2 mutants and cultured them to generate pro-efferocytic polarized macrophages. We then tested their efferocytic potential and metabolic capacity by seahorse assay. Continual efferocytosis, a hallmark of pro-efferocytic macrophages, was impaired in IDH2 mutant macrophages as early as 2-3 months of age. Consistent with this finding, mito stress testing by seahorse demonstrated that IDH2 mutant macrophages had severely impaired maximal respiratory capacity compared to WT macrophages. Treatment with global hypomethylating agent azacytidine or a cell permeable aKG analog were unable to restore respiratory capacity or efferocytic activity in the IDH2 mutant. These data strongly suggest that the efferocytic defect in IDH2 mutant macrophages is due to metabolic rather than epigenetic reprogramming. In summary, we identify defects in efferocytic function and oxidative phosphorylation in IDH2 mutant macrophages. This represents a novel mechanism that could alter the bone marrow microenvironment and impact progression of MDS and leukemic transformation. Furthermore, our data suggest that the metabolic dysregulation induced by IDH mutations may play a greater role than epigenetic impairment in IDH2 mutant mature myeloid cells, which could explain why approaches targeting hypermethylation have shown limited efficacy in clinical settings.

Genome-Wide Mapping of R-Loops in Single Cells Reveals Cell-Type Specific Roles for R-Loops in Hematopoietic Cell Identity

R-loops are three-stranded structures composed of an RNA-DNA hybrid and a single strand of DNA. R-loops play critical roles in gene regulation and pathologic roles in DNA damage response. Current methods to map R-loops require at least 500,000 cells. Consequently, R-loop characterization in primary cells is limited. Furthermore, there are no genomic tools to identify R-loops in single cells. We therefore developed here methods to map genome-wide R-loops at single cell resolution. R-loop mapping in primary stem and mature hematopoietic cell populations, and individual single cells revealed striking cell-type specific R-loops. Current methods to map R-loops utilize immunoprecipitation with a monoclonal antibody (clone “S9.6”) or catalytically inactive, but R-loop binding competent, RNAseH1. Motivated by success mapping chromatin modifications in single cells through fusion of Tn5 transposase to a nanobody as well as insertion of T7 promoters to amplify DNA at sites of transposition, we combined both techniques to identify R-loops in limited cell numbers, down to the single cell level. We used a nanobody-Tn5 fusion to identify S9.6 and developed an orthogonal recombinant fusion protein of enzymatic dead RNAseH1 fused to Tn5 (Figure A). We first applied our nanobody-Tn5 method to identify R-loops bound by S9.6 in mouse lineage-negative c-Kit + bone marrow cells in biological duplicates of increasingly reduced cell numbers (64,000, 32,000, 16,000, and 8,000 cells). This revealed remarkable reproducibility of R-loop detection and characteristics. R-loops identified were sensitive to RNAseH1 and enriched at sites with G/C skew and mitochondrial DNA (a site with abundant physiologic R-loops), suggesting authentic, bona fide R-loops mapped in as few 8,000 cells. We next evaluated R-loops across FACS-isolated hematopoietic stem and mature cells from mouse and human bone marrow. Mapping of R-loops in 10,000 cells in duplicate revealed reproducible R-loops which were characteristic of each cell population. Hierarchical clustering of R-loop profiles delineated mature from stem and progenitor cells and discovered increased abundance of genic R-loops in hematopoietic stem and progenitors relative to mature cells (Figure B). Application of our enzymatic dead RNaseH1/Tn5 fusion protein to CD34 + cells revealed a similar genome-wide distribution of R-loops as with the S9.6/nanobody-Tn5 method. There was high correlation of long- and short-term HSC-specific R-loops with sites characteristic of active transcription (based on H3K4me1/2/3 as well as H3K27Ac). Transcription factor motifs activated in unique hematopoietic cell types were enriched at R-loops within the same cell types. For example, GATA motif usage was enriched at R-loops in CMP, MEP, and erythroid precursors. We also detected massive increases in R-loop abundance at the immunoglobulin heavy chain locus in primary B-cells stimulated to undergo class switch recombination. This provides further proof of our low input method to identify R-loops, as physiologic R-loops are known to occur at this locus to promote class switch recombination. Despite recent advances in single cell genomics there are no single cell maps of R-loops. We therefore profiled R-loops using our nanobody-Tn5, low-input S9.6 CUT&Tag approach in individual human CD34 + and mouse c-Kit + cells. We obtained 1,579 single-cell profiles from human and 1,716 from mouse. Uniform manifold approximation and projection (UMAP) identified 12 clusters in human and 9 mouse hematopoietic clusters. Aggregated single-cell R-loop profiles closely resembled bulk profiles indicating that single-cell data captured features of multi-cell maps. Pseudotime analyses of R-loops inferred LT-HSCs as the shared root of hematopoiesis in both mouse and human and identified seven terminal states without prior knowledge of the number of branches in the hematopoietic development. These data provide the first maps of R-loops in primary normal hematopoietic cells and identify scheduled, physiologic cell-type specific R-loops which have not previously been uncovered. The atlas of R-loops across hematopoiesis generated here will motivate future studies to understand the roles of R-loops in hematopoietic differentiation. Moreover, the methods presented map R-loop abundance at nucleotide resolution in primary and limited cell types which can be used for wide-reaching applications.

Arid1b Loss Impairs Hematopoietic Stem Cell Function in Normal Hematopoiesis

Switch Sugar Non- Fermenting (SWI/SNF) nucleosome remodeling complexes are multi subunits complexes that utilize the energy from ATP to remodel chromatin, affecting cellular processes on chromatin including that of gene transcription. SWI/SNF complexes play important roles in normal development and differentiation as well as in oncogenesis. ARID1 proteins, ARID1A and ARID1B, are unique and mutually exclusive subunits of the canonical SWI/SNF complex BRG-1/BRM Associated Factor (BAF) complex. Arid1a is the most frequently mutated SWI/SNF subunit in cancers and plays a vital role in hematopoiesis. Arid1b is also frequently mutated in cancers, and its loss promotes Acute Myeloid Leukemia leukemogenesis. However, its role in normal hematopoiesis is not known. In this study we used hematopoietic specific Cre strains, VavCre and Mx1Cre, to delete Arid1b in a mouse genetic model to study its role in normal hematopoiesis. Analysis of steady state hematopoiesis in the VavCre model showed no significant differences in complete blood count (CBC) between Arid1b fl/flVavCre+ and VavCre+ mice. Further, bone marrow (BM) cellularity was not significantly different between control and experimental mice. Flow cytometry analysis of the percentage of mature myeloid, B cell, or T cell populations in the peripheral blood (PB), BM, or spleen (SPL) did not show significant differences between Arid1b fl/flVavCre+ and VavCre+ control mice. Further, we did not observe any significant differences in percentages of hematopoietic stem and progenitor cells (HSPCs), including HSCs, common myeloid progenitors (CMPs), granulocyte macrophage progenitors (GMPs), megakaryocyte/erythrocyte progenitors (MEPs), and common lymphoid progenitors (CLPs) in the BM of Arid1b fl/flVavCre+ mice versus VavCre+ controls. Analysis of Mx1Cre model showed similar results. Taken together, these results suggest that Arid1b is dispensable for steady state hematopoiesis. To assess the effect of loss Arid1b in a regenerative setting, we conducted non-competitive and competitive transplantation experiments in VavCre and Mx1Cre models. We transplanted BM cells from Arid1b fl/flCre+ or Cre+ mice (CD45.2) into lethally irradiated recipient CD45.1 mice with (CD45.1 at 1:1 ratio) or without competitor cells for competitive or non-competitive transplantations respectively. In Mx1Cre model, deletion of Arid1b was induced 4 weeks after transplantation. We monitored donor chimerism in PB over time as well as in the BM and SPL at end point. In both VavCre and Mx1Cre non-competitive transplants, we did not observe significant differences in CBC or donor chimerism in mature cells between Arid1b fl/flCre+ and control Cre+ mice in the PB over time. Further, no significant differences were observed between knockout and control mice in donor chimerism in mature cell populations in the BM or SPL, nor HSPC populations in the BM at end point (24-28 weeks). Deletion of Arid1b in the BM was confirmed. These results indicate that Arid1b loss does not affect regenerative potential of HSPCs in a non-competitive setting. In our competitive transplants, we observed significant decreases in PB donor chimerism for both myeloid and B cell populations over time in Arid1b fl/flVavCre+ compared to VavCre+ controls. Further, we observed significant decreases in donor chimerism of mature myeloid cells and B cells in the BM and SPL, as well as HSCs, CMPs, GMPs, and MEPs in the BM of Arid1b fl/flVavCre+ mice compared to VavCre+ controls at endpoint. However, there seemed to be incomplete penetrance as the observed phenotype varied in cohorts of different donor mice and was not significant in all cohorts. A similar phenotype was observed in the Mx1Cre model with the exception of a trend, albeit insignificant, towards a decrease in donor derived mature B cell populations in Arid1b fl/flMx1Cre+ compared to Mx1Cre+ mice in PB, SPL, and BM and no significant difference in HSCs in the BM at end point (20 weeks). Deletion of Arid1b in the BM was confirmed in both models. Overall, our results suggest that Arid1b deficient HSCs can regenerate the hematopoietic system but have defects in competitive transplantations with incomplete penetrance. Further work is needed to address the observed incomplete penetrance phenotype in competitive transplantations, and to understand the mechanism behind the requirement of Arid1b in normal hematopoiesis.

Dynamic Recruitment of Inhibitory Complexes Controls Oncogenic Signaling in B-Cell Malignancies

Background and Significance: Initiation of B-cell receptor (BCR) signaling, and subsequent antigen-encounter in germinal centers represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL) and lymphoma also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells, the significance of its expression on B-cells was unclear. High expression levels on the surface of B-ALL cells are associated with poor clinical outcomes in pediatric (COG P9906) and adult (ECOG E2993) B-ALL cohorts. In DLBCL, aggressive disease is associated with CD25-inactivation by shedding and high serum levels of soluble CD25. Results: Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKCδ, immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors and inhibitory phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKCδ and phosphatases, conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early stages of B-cell development (e.g. B-ALL) CD25-loss induced hyperactivation of oncogenic signaling followed by cell death. In contrast, genetic deletion of CD25 in models for mature B-cell lymphoma accelerated proliferation of malignant cells. Biochemical and interactome studies revealed a critical role of CD25 in feedback regulation of oncogenic signaling in B-cell malignancies: Signaling from BCR-ABL1 and RAS-oncogenes in B-ALL as well as oncogenic BCR-signaling in DLBCL induced PKCδ-mediated phosphorylation of CD25 on its cytoplasmic tail (S 268). Importantly, CD25-phosphorylation stabilized its expression at the cell membrane and opposed both inactivation by CD25-shedding and internalization. Moreover CD25-S 268 tail-phosphorylation enabled CD25 to recruit inhibitory receptors, including CD22, that bear ITIM-motifs for the activation of inhibitory phosphatases (e.g. SHP1, Figure 1). Thereby, the positively charged cytoplasmic tail of CD25 engages negatively charged residues in the cytoplasmic tails of the BCR signaling chain CD79B and the inhibitory CD22-ITIM. Molecular dynamics simulations revealed that the ternary complex between CD25, CD79B and CD22 enables recruitment and activation of the phosphatase SHP1 within reach of its substrate, the BCR signaling chain CD79B. Thereby, the SH2-domain of SHP1 engages the CD22-ITIM motif, while its phosphatase domain makes contact with the CD79B-ITAM to dephosphorylate and terminate BCR-signaling. This complex, while engaging and activating SHP1, is held together by CD25 ( Figure 1). To test functional consequences of destabilization of this complex, we generated a knockin mouse model carrying a single point mutation (S268A) in the cytoplasmic tail of CD25. The CD25 S268A knockin mutation abolished recruitment and activation of SHP1. In addition, the S268A-mutant failed to assemble CD22 and CD79B in complex with SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function and failure to terminate oncogenic signaling resulted in constitutive activation of ERK and NF-κB, autonomous Ca 2+-oscillations. Hyperactivation of oncogenic signaling resulted in exhaustion and cell death in B-ALL cells, as opposed to excessive proliferation and acceleration of disease in mature B-cell lymphoma. Conclusions: While CD25 has an established function in IL2 signal transduction in T- and NK-cells, these findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and provides an explanation for the common feature of CD25 shedding and high serum levels of functionally inactive soluble CD25 in aggressive B-cell lymphomas.

Roles of Ribosomal S6 Kinases in Acute Leukemia and Normal Hematopoiesis

Ribosomal S6 kinases (RSKs) are serine-threonine kinases that are activated by the Ras/mitogen activated protein kinase signaling pathways and regulate cell proliferation and survival. There are 4 isoforms of RSK (RSK1, 2, 3, 4), and there is evidence that the isoforms mediate tissue-specific cellular functions. We hypothesized that RSK isoforms have distinct roles in hematopoiesisand evaluated RSK as a target for the treatment of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). To investigate isoform expression in hematopoiesis, we sorted cord blood mononuclear cells into hematopoietic progenitor stages and quantified RSK isoform mRNA by RT-qPCR. RSK1 expression remained consistent throughout hematopoiesis, whereas RSK2 expression decreased in the more differentiated populations. RSK3 was elevated in the lymphoid-primed multipotential progenitor and common myeloid progenitor stages and was greatly reduced in the more mature megakaryocyte-erythroid progenitor population. These results suggest that RSK proteins have isoform-specific roles in hematopoiesis. To analyze the importance of each isoform on hematopoiesis, we transduced cord blood CD34+ cells with lentiviral constructs expressing isoform-specific or control shRNAs. After liquid culture for 5 days, the cells were plated in methylcellulose media containing SCF, IL-3, GM-CSF, and EPO to determine the colony-forming potential. RSK1 and RSK2 knockdown decreased the colony-forming unit - erythroid colonies and increased the granulocyte/macrophage colony numbers. RSK3 knockdown increased the burst-forming unit - erythroid colonies and total colony numbers. After liquid culture days 8, 11, and 14, the erythroid and myeloid populations were analyzed by flow cytometry. RSK1 knockdown increased the CD11b+ and CD14+ myeloid populations and decreased the CD71+ erythroid population. RSK3 knockdown increased the erythroid population and decreased the myeloid populations. These data demonstrate that RSK1 and RSK3 have opposing effects on hematopoietic lineage determination. Overexpression of RSK proteins leads to aberrant cell survival and proliferation in many cancers including leukemia. We have previously reported that RSK and phosphorylated RSK are elevated in pediatric AML and are associated with a worse prognosis. There are only two orally bioavailable RSK inhibitors that have moved to phase I clinical trials: TAS0612 and PMD-026. TAS0612 is a multi-kinase inhibitor of RSK, AKT, and S6K. PMD-026 has high specificity to all four RSK isoforms. We evaluated the effects of both compounds in AML and ALL. To determine the effects of the RSK inhibitors on AML, we treated AML cell lines and patient samples with a range of doses of PMD-026, TAS0612, or 0.1% DMSO. After 3 days, we performed CellTiter Glo assays to determine the relative viable cell number and calculate the concentration of drug that inhibits cell growth by 50% (IC50). TAS0612 had an IC50 of 0.096-0.64 µM in AML cell lines Molm13, U937, AML193, and MV4;11. To test the cytotoxicity of TAS0612 on normal hematopoiesis, we plated healthy bone marrow mononuclear cells with TAS0612 in methylcellulose media. Colony formation was not adversely affected by TAS0612 up to 1 µM. PMD-026 was effective in Molm13, Kasumi-1, and MV4;11 with an IC50 of 0.34-0.55 µM and in AML patient samples 3123 and 4192 with an IC50 of 1.5-2.3 µM. PMD-026 did not affect healthy bone marrow colony formation up to 5 µM for a 2.5- to 10-fold therapeutic window. Furthermore, we tested the effects of RSK inhibitors in ALL. While PMD-026 was not effective, we found that B-ALL cell lines (REH, Nalm-6) and T-ALL cell lines (Loucy, Jurkat) were sensitive to RSK inhibition by TAS0612 with an IC50 of 0.056-0.19 µM. Western blot analysis of RSK protein revealed that RSK1, RSK2, and phospho-RSK proteins are elevated in ALL cell lines compared to healthy peripheral blood mononuclear cells. We are investigating the role of RSK and RSK isoforms in ALL. We found that RSK isoform expression is modified during hematopoiesis. Isoform expression influences early-fate decisions to differentiate into erythroid and myeloid lineages. RSK inhibitors PMD-026 and TAS0612 are effective at targeting AML and ALL cells with limited cytotoxicity to healthy cells, making them promising candidates for translation into clinical trials for AML and ALL.

Integrative Single-Cell RNA-Seq and Single-Cell ATAC-Seq Analysis of Human Plasma Cell Differentiation

Plasma cells (PC) are highly specialized cells representing the end stage of B cell differentiation. They play an important role in humoral immunity by synthesizing and secreting antibodies protecting the host against infections. We have shown that PC differentiation (PCD) can be reproduced in vitrofrom memory B cells (MBC). MBC differentiate into CD20 low/-CD38 - pre-plasmablasts (prePB), CD20 -CD38 +CD138 - plasmablasts (PB) and CD20 -CD38 +CD138 + PC. The molecular changes occurring during PCD are recapitulated in this in vitrodifferentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drives the early stages of PCD. We combined single-cell (sc) RNA-seq and scATAC-seq to decipher the trajectories involved in PCD. scRNA-seq experiments on the four populations (MBC, prePB, PB and PC) generated during normal PCD revealed a highly specific transcriptomic profile for MBC and PC, and a strong heterogeneity of the prePB and PB. The prePB stage presented the most differentially expressed genes (DEG) with almost 2000 DEG showing that the most important changes take place during this stage. Epigenetic analysis using scATAC-seq technology showed that prePB, PB and PC stages were clearly separated from the MBC highlighting chromatin remodeling induced by B cell activation and differentiation. As observed on the transcriptomic level, the number of differentially accessible peaks was higher in prePB (4660) than in other stages (MBC: 641; PB: 44; PC: 105). Among genes that were commonly identified using scATAC-seq and RNA-seq, we identified several TF such as BATF and BATF3 in prePB. Interestingly, differentially accessible peaks characterizing the prePB stage were enriched in motifs of BATF3, FOS and BATF belonging to the TF AP-1 family and among these differentially accessible peaks, 38% of peaks presented BATF3 motif. Since BATF3 TF was previously identified operating in short impulse manner at prePB stage, BATF3 target genes may represent a key transcriptional node involved in PCD. The integration of the chromatin accessibility and transcriptomic data revealed a more mature population of prePB cells characterized by open chromatin in PC genes without significant expression suggesting that a population of prePB were already committed to become antibody-secreting cells. To focus on the understanding of the processes occurring during the transition from prePB to PB which remains largely unknown, we computationally clustered and ordered cells. Pseudotemporal analyses underlined maturation trajectories in prePB with early prePB characterized by downregulation of B cell markers ( CD19, CD22, CD83, CCR7, CCL17 and CCL22) and B cell TF (SPIB, PAX5, STAT5A, RUNX3 and BATF3) together with upregulation of PC markers, adhesion molecules and growth factor receptors ( CD27, CD38, SLAMF7, BCMA, ITGA4, IL-6R, IL-6ST and INSR). The transition from early prePB to more mature prePB is associated with downregulation of AICDA and PRC2 complex ( EZH2, EED). In the transitional prePB, a first wave of UPR activation is observed, associated with mTORC1 pathway activation. In these prePB, HSAP5, also called binding immunoglobulin protein (BiP) is overexpressed together with ERN1, leading to the activation of the Ire1 pathway, known to splice XBP-1 (sXBP-1) producing a highly active TF. Then, prePB overexpress EGR1, FOS, CXCR4 and TFRC. Mature prePB overexpress KLF2 that participate in bone marrow PC homing. Then, PB overexpress gene involved in conventional UPR and protein secretion associated to immunoglobulin gene expression. Altogether, these data underlined that prePB present already UPR priming through mTORC1 pathway activation to prepare for PC function. XBP1 driven UPR activation will be coordinated in PB related to antibody synthesis increase. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of prePB already undergone epigenetic remodeling related to PC profile together with UPR activation and are committed to differentiate in PC. These results and the supporting data generated with our PCD model provide a resource for the identification of molecular circuits that are crucial for early and mature PC biological function and survival. These data thus provide critical insights into epigenetic- and transcriptional-mediated reprogramming events that sustain PCD.

Human cytomegalovirus induces significant structural and functional changes in terminally differentiated human cortical neurons.

Human cytomegalovirus (HCMV) is a highly prevalent viral pathogen that can cause serious neurological deficits in infants experiencing an in utero infection. Also, as a life-long infection, HCMV has been associated with several diseases in the adult brain. HCMV is known to infect early neural progenitor cells, but whether it also infects terminally differentiated neurons is still debated. Here, we differentiated human-induced pluripotent stem cells into neurons for 84-120 days to test the ability of HCMV to infect terminally differentiated neurons and assess the downstream functional consequences. We discovered that mature human neurons are highly permissive to HCMV infection, exhibited late replication hallmarks, and produced infectious virus. Moreover, infection in terminally differentiated neurons essentially eliminated neuron function. These results demonstrate that terminally differentiated human neurons are permissive to HCMV infection, which can significantly alter both structural and functional features of this mature neuron population.

CSF1R Inhibition Promotes Neuroinflammation and Behavioural Deficits during Graft-Versus-Host Disease in Mice

Chronic graft-versus-host disease (cGVHD) remains a significant complication of allogeneic hematopoietic stem cell transplantation. Central nervous system (CNS) involvement is becoming increasingly recognised, where brain-infiltrating donor MHC class II + bone marrow-derived macrophages (BMDM) are implicated mediators of pathology. Colony-stimulating factor 1 receptor (CSF1R)-dependent BMDM are established mediators of cutaneous and pulmonary cGVHD, and clinical trials assessing the efficacy of CSF1R antibody blockade to deplete macrophages are promising. We hypothesized that CSF1R antibody blockade may also be a useful strategy to prevent/treat CNS cGVHD. We leveraged studies in acute GVHD (aGVHD) to determine the feasibility of an antibody-based approach for depleting brain microglia and macrophages. Moreover, we queried whether CSF1R blockade may be a viable therapy for CNS aGVHD, which has been shown to be mediated by host microglia. Increased blood-brain barrier permeability during aGVHD facilitated CNS antibody access. However, CSF1R blockade early post-transplant induced region-specific microglia depletion and was associated with unexpected exacerbation of aGVHD neuroinflammation, including increased T cell and inflammatory monocyte infiltration, and whole brain cytokine levels. In established cGVHD, vascular changes and anti-CSF1R efficacy was more limited. Anti-CSF1R-treated mice retained donor BMDM, activated microglia, CD8 + and CD4 + T cells, and local cytokine expression in the brain. These findings were recapitulated in GVHD recipients where CSF1R was conditionally depleted in donor CX3CR1 + BMDM. Notably, inhibition of CSF1R signalling post-transplant failed to reverse either acute or chronic GVHD-induced behavioral changes. Taken together, these data suggest that ongoing clinical trials of CSF1R inhibition should assess neurological adverse events in patients. In contrast to the lack of efficacy with CSF1R inhibition, transfer of Ifngr-/- grafts reduced MHC class II + BMDM infiltration, resulting in improved behaviour. Furthermore, single nuclei RNA sequencing of brains from mice receiving wild-type or Ifngr-/- allografts revealed a disease-associated interferon-inducible mature oligodendrocyte population in the cGVHD brain. This was markedly reduced in recipients of Ifngr-/- grafts, indicating that targeting this axis can promote brain repair beyond the myeloid compartment during cGVHD. In summary, our findings reveal unexpected neurological immune toxicity during CSF1R blockade and highlight the IFNGR pathway as an alternative target for the treatment of cGVHD within the CNS.

C/EBPα Acts As an RNA Binding Protein Essential for, and Promotes End-Stage Macrophage Differentiation

The transcription factor C/EBPα is a well characterized DNA binding protein with essential functions in controlling and regulating myeloid differentiation and lipid metabolism. Herein, we describe C/EBPα's separate and distinct RNA binding characteristics. Using Chromatin RNA Immunoprecipitation, we identified that C/EBPα interacts primarily with RNA introns in both HL-60 and THP-1 cells, with a preference for a palindromic GC-rich binding motif. Structural prediction in conjunction with RNA electrophoretic mobility shift assays show that C/EBPα interacts with RNA through two previously undescribed domains located towards the proteins N terminus. These domains are distinct from C/EBPα's DNA binding b-ZIP domain which is instead located on the proteins C terminus. Mouse bone marrow transplantation and in vitro cytokine assays reveal that C/EBPα RNA binding appears to be essential for macrophage maturation but not neutrophil differentiation. To better understand these phenotypic differences, we expanded our observations into the transcriptomic space by utilizing single-cell CITE-Seq. In line with biochemical data, notable mature cell populations were absent when the C/EBPα's RNA binding domains are excluded from the protein. Intriguingly, differential gene expression revealed strong, selective upregulation of Lipoprotein Lipase (Lpl) in monocyte, but not neutrophil populations (Panel 1). It has been previously reported that dysregulation of Lpl affects bone marrow monocyte progenitor differentiation and results in dysregulated cellular mobilization from the bone marrow, a phenotype that is commonly seen in cancers such as AML. The increased abundance of monocyte and neutrophil populations in the C/EBPα RNA deletion samples is therefore suggestive that C/EBPα RNA binding is critical in regulating terminal differentiation and mobilization circuits which may become dysregulated or perturbed in diseases such as AML. On-going analyses are investigating the RNA velocity trajectory of captured cell populations in an attempt to better understand at which stage C/EBPα RNA binding becomes essential for terminal maturation and differentiation of select cell populations. Taken together, we demonstrate that C/EBPα is also a bona fida RNA binding protein with unique functions distinct from its DNA binding activity that are essential for monocyte maturation, differentiation and mobilization. Panel 1 - Identified Cell Populations Following scCITE-Seq with C/EBPα Full-Length (WT) and C/EBPα RNA Binding Deletion (DD). Each circle represents a single cell presented in UMAP space. The left panel shows populations identified with the full-length C/EBPα construct. The right panel shows the cell populations identified with the RNA domain deletion construct. Notable differences in cell populations and numbers are evident. Overlayed onto the cells is the expression of Lipoprotein Lipase (Lpl). Expression is shown as a gradient from black up to green, representing a log2(4) expression. Lpl expression is shown to be expressed uniquely in the monocyte population of cells almost exclusively in the RNA deletion sample.

Deficiency of the Heterogeneous Nuclear Ribonucleoprotein U locus leads to delayed hindbrain neurogenesis.

Genetic variants affecting Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU) have been identified in several neurodevelopmental disorders (NDDs). HNRNPU is widely expressed in the human brain and shows the highest postnatal expression in the cerebellum. Recent studies have investigated the role of HNRNPU in cerebral cortical development, but the effects of HNRNPU deficiency on cerebellar development remain unknown. Here, we describe the molecular and cellular outcomes of HNRNPU locus deficiency during in vitro neural differentiation of patient-derived and isogenic neuroepithelial stem cells with a hindbrain profile. We demonstrate that HNRNPU deficiency leads to chromatin remodeling of A/B compartments, and transcriptional rewiring, partly by impacting exon inclusion during mRNA processing. Genomic regions affected by the chromatin restructuring and host genes of exon usage differences show a strong enrichment for genes implicated in epilepsies, intellectual disability, and autism. Lastly, we show that at the cellular level HNRNPU downregulation leads to an increased fraction of neural progenitors in the maturing neuronal population. We conclude that the HNRNPU locus is involved in delayed commitment of neural progenitors to differentiate in cell types with hindbrain profile.

Financial development in Africa: Do expenditure and population matter? Evidence through pooled mean group and generalized method of moments estimators

AbstractThis article assesses the impact of government expenditure and population structure on financial development among selected African economies for a panel of 37 selected African economies from years 1980 to 2018. The pooled mean group regression estimator was used, which suitably isolates short run from long run impacts while allowing for heterogeneities and homogeneities for each run respectively. Additionally, generalized method of moments technique was applied for robustness check and allows for dynamism in the estimations. Findings show that financial development is sensitive to both productive and non‐productive government expenditures in the long run. They affirm positive impacts convergences on financial development. Financial development responds only to non‐productive expenditures in the short run. Young and elderly populations do not benefit financial development but mature population does due to its active role in the private sector. Further, it helps support the negative impact of inflation and provides positive impacts of economy openness and individual's economic output. Also, results support short‐run adjustment towards long‐run equilibrium. These results are significant both statistically and economically. Unlike former studies, this work comprehensively disaggregated government expenditure into productive and unproductive types. It isolated population structure age‐wise. In order to stimulate financial development, governments in Africa need to favour policies that stabilize government expenditures, having both short‐run and long‐run foci, encourage public–private partnership and mutually beneficial linkages, devise population policies based on respective age‐structures, and promote economy openness, and growth per capita, and control inflation.

Optimizing industrial growth through alternative forest biomass resources: A mathematical model using DDE

Abstract This paper focuses on the classification of forest biomass into two categories: premature and mature forest biomass. The third variable considered is industrialization. The growth of the wood-based industry is believed to be closely tied to the population of mature forest biomass. Any scarcity of the mature population could have a negative impact on industrialization. So, pre-mature forest biomass is provided as an alternative for industrial growth. The industrialization growth is assumed to be based on a modified Leslie-Gower equation. The positivity and boundedness of the system are calculated using the comparison theorem. Stability analysis is done about nonzero equilibrium points with the help of the Routh-Hurwitz theorem. When there is no delay in the system, the system is stable. At τ< 1.8, the system shows asymptotic stability, but at τ ≥ 1.8, system shows Hopf-bifurcation and periods oscillations occur. Furthermore, sensitivity analysis is examined about different parameters of the systems. MATLAB is used to draw the numerical simulation.

Plants and age-structured pest dynamics with natural enemy as control strategy: A fractional differential equations model

In recent years, plant pests have drawn much attention from researchers. Biological control proved an efficient replacement for chemical and other pest control techniques. The present study proposes the cognition of an immature pest population unaffected by the natural enemy and grows exponentially by the harvesting effort of the mature pest population to plants. The fractional calculus environment proposes A four-compartment: plant, immature and mature pest, and natural enemy model. Equilibrium points and the system’s stability are studied theoretically and numerically. Simulation study for different values of fractional order generates the optimum solution. The suggested method is much more benignant than the existing method of ordinary differential equations as we can figure out the infrequent changes in the variable using a fractional derivative. Creating the memory effect in the system using fractional derivatives makes it more realistic.

The invasive ant Solenopsis invicta is established in Europe

The red imported fire ant (Solenopsis invicta) is classified as one of the worst invasive alien species1 and as the fifth costliest worldwide2, impacting ecosystems, agriculture and human health3. We report the establishment of S. invicta in Europe for the first time, documenting a mature population in Sicily. We use genetic analyses to assess its putative origin, as well as wind tracking and species distribution modelling to predict its potential range on the continent. We show that half of the urban areas in Europe are already suitable and that climate warming expected under current trends will favor the expansion of this invasive ant.

Population size as a major determinant of mating system and population genetic differentiation in a narrow endemic chasmophyte

BackgroundMating system is one of the major determinants of intra- and interspecific genetic structure, but may vary within and between plant populations. Our study model included all known populations of Moehringia tommasinii (Caryophyllaceae), a narrow endemic plant inhabiting rock crevices in the northwestern Adriatic, and some populations of co-occurring and widespread M. muscosa, an ecologically divergent relative with an overlapping flowering period. We performed reciprocal crosses within and between taxa and used molecular markers to assess the extent of gene flow within and between populations and taxa. Using coefficient of inbreeding, population size, seed weight, pollen-to-ovule ratio, and flower display size, we also looked for evidence of a selfing syndrome.ResultsA surprisingly high variation in mating systems was observed among populations of M. tommasinii. These populations exhibited genetic structuring, with their size positively correlated with both seed weight and pollen production. Although a selfing syndrome could not be confirmed as the majority of selfing resulted from allogamous treatments, the occurrence of selfing was notable. In the presence of M. muscosa, at a site where both species coexist closely, a distinct pattern of fruit production was observed in M. tommasinii following various pollination treatments. Molecular and morphometric data provided evidence of hybridization followed by local extinction at this site.ConclusionsPopulation size proved to be the most important factor affecting the mating system in genetically structured populations of M. tommasinii. Lighter seeds and lower pollen production observed in populations with pronounced selfing do not provide enough evidence for the selfing syndrome. Detected gene flow between M. tommasinii and the sympatric M. muscosa suggested weak reproductive barriers between the taxa, which could pose a conservation problems for the former species. Hybridization leading to local extinction may also resulted in floral polymorphism and disruption of mating patterns of M. tommasinii.

Government expenditure impacts on financial development: Do population age structures moderations matter?

PurposeThis study examined the roles of public spending and population moderating characteristic structure of selected African economies on bank-based financial development through credit to private sector.Design/methodology/approachThe study sampled 37 selected African economies for the years 1991–2018, and it applied a pooled mean group (PMG) estimator to account for short-run and long-run causal effects, and confirmed short-run adjustments towards the long-run convergences between the variables. Specific suitable tests were also applied.FindingsEvidence confirms positive impacts of both capital formation and final consumption expenditures on financial development in the short run and long run. The moderation of population structures on expenditure structures help to speed up convergences.Originality/valueThis work attests its innovation by accounting for the separate effects of the expenditure types, the moderation effects of young and mature populations for capital and final consumption expenditure on financial development among selected economies in Africa.