Inhibitor of DNA binding proteins revealed as orchestrators of steady state, stress and malignant hematopoiesis.

Adult mammalian hematopoiesis is a dynamic cellular process that provides a continuous supply of myeloid, lymphoid, erythroid/megakaryocyte cells for host survival. This process is sustained by regulating hematopoietic stem cells (HSCs) quiescence, proliferation and activation under homeostasis and stress, and regulating the proliferation and differentiation of downstream multipotent progenitor (MPP) and more committed progenitor cells. Inhibitor of DNA binding (ID) proteins are small helix-loop-helix (HLH) proteins that lack a basic (b) DNA binding domain present in other family members, and function as dominant-negative regulators of other bHLH proteins (E proteins) by inhibiting their transcriptional activity. ID proteins are required for normal T cell, B cell, NK and innate lymphoid cells, dendritic cell, and myeloid cell differentiation and development. However, recent evidence suggests that ID proteins are important regulators of normal and leukemic hematopoietic stem and progenitor cells (HSPCs). This chapter will review our current understanding of the function of ID proteins in HSPC development and highlight future areas of scientific investigation.

ID2 and HIF-1α collaborate to protect quiescent hematopoietic stem cells from activation, differentiation, and exhaustion.

Defining mechanism(s) that maintain tissue stem quiescence is important for improving tissue regeneration, cell therapies, aging, and cancer. We report here that genetic ablation of Id2 in adult hematopoietic stem cells (HSCs) promotes increased HSC activation and differentiation, which results in HSC exhaustion and bone marrow failure over time. Id2Δ/Δ HSCs showed increased cycling, ROS production, mitochondrial activation, ATP production, and DNA damage compared with Id2+/+ HSCs, supporting the conclusion that Id2Δ/Δ HSCs are less quiescent. Mechanistically, HIF-1α expression was decreased in Id2Δ/Δ HSCs, and stabilization of HIF-1α in Id2Δ/Δ HSCs restored HSC quiescence and rescued HSC exhaustion. Inhibitor of DNA binding 2 (ID2) promoted HIF-1α expression by binding to the von Hippel-Lindau (VHL) protein and interfering with proteasomal degradation of HIF-1α. HIF-1α promoted Id2 expression and enforced a positive feedback loop between ID2 and HIF-1α to maintain HSC quiescence. Thus, sustained ID2 expression could protect HSCs during stress and improve HSC expansion for gene editing and cell therapies.

BRCA2-DSS1 interaction is dispensable for RAD51 recruitment at replication-induced and meiotic DNA double strand breaks.

The interaction between tumor suppressor BRCA2 and DSS1 is essential for RAD51 recruitment and repair of DNA double stand breaks (DSBs) by homologous recombination (HR). We have generated mice with a leucine to proline substitution at position 2431 of BRCA2, which disrupts this interaction. Although a significant number of mutant mice die during embryogenesis, some homozygous and hemizygous mutant mice undergo normal postnatal development. Despite lack of radiation induced RAD51 foci formation and a severe HR defect in somatic cells, mutant mice are fertile and exhibit normal RAD51 recruitment during meiosis. We hypothesize that the presence of homologous chromosomes in close proximity during early prophase I may compensate for the defect in BRCA2-DSS1 interaction. We show the restoration of RAD51 foci in mutant cells when Topoisomerase I inhibitor-induced single strand breaks are converted into DSBs during DNA replication. We also partially rescue the HR defect by tethering the donor DNA to the site of DSBs using streptavidin-fused Cas9. Our findings demonstrate that the BRCA2-DSS1 complex is dispensable for RAD51 loading when the homologous DNA is close to the DSB.

SSSPTA is essential for serine palmitoyltransferase function during development and hematopoiesis.

Serine palmitoyltransferase complex (SPT) mediates the first and rate-limiting step in the de novo sphingolipid biosynthetic pathway. The larger subunits SPTLC1 and SPTLC2/SPTLC3 together form the catalytic core while a smaller third subunit either SSSPTA or SSSPTB has been shown to increase the catalytic efficiency and provide substrate specificity for the fatty acyl-CoA substrates. The in vivo biological significance of these smaller subunits in mammals is still unknown. Here, using two null mutants, a conditional null for ssSPTa and a null mutant for ssSPTb, we show that SSSPTA is essential for embryogenesis and mediates much of the known functions of the SPT complex in mammalian hematopoiesis. The ssSPTa null mutants are embryonic lethal at E6.5 much like the Sptlc1 and Sptlc2 null alleles. Mx1-Cre induced deletion of ssSPTa leads to lethality and myelopoietic defect. Chimeric and competitive bone marrow transplantation experiments show that the defect in myelopoiesis is accompanied by an expansion of the Lin-Sca1+c-Kit+ stem and progenitor compartment. Progenitor cells that fail to differentiate along the myeloid lineage display evidence of endoplasmic reticulum stress. On the other hand, ssSPTb null mice are homozygous viable, and analyses of the bone marrow cells show no significant difference in the proliferation and differentiation of the adult hematopoietic compartment. SPTLC1 is an obligatory subunit for the SPT function, and because Sptlc1-/- and ssSPTa-/- mice display similar defects during development and hematopoiesis, we conclude that an SPT complex that includes SSSPTA mediates much of its developmental and hematopoietic functions in a mammalian model.

Pogz deficiency leads to transcription dysregulation and impaired cerebellar activity underlying autism-like behavior in mice.

Several genes implicated in autism spectrum disorder (ASD) are chromatin regulators, including POGZ. The cellular and molecular mechanisms leading to ASD impaired social and cognitive behavior are unclear. Animal models are crucial for studying the effects of mutations on brain function and behavior as well as unveiling the underlying mechanisms. Here, we generate a brain specific conditional knockout mouse model deficient for Pogz, an ASD risk gene. We demonstrate that Pogz deficient mice show microcephaly, growth impairment, increased sociability, learning and motor deficits, mimicking several of the human symptoms. At the molecular level, luciferase reporter assay indicates that POGZ is a negative regulator of transcription. In accordance, in Pogz deficient mice we find a significant upregulation of gene expression, most notably in the cerebellum. Gene set enrichment analysis revealed that the transcriptional changes encompass genes and pathways disrupted in ASD, including neurogenesis and synaptic processes, underlying the observed behavioral phenotype in mice. Physiologically, Pogz deficiency is associated with a reduction in the firing frequency of simple and complex spikes and an increase in amplitude of the inhibitory synaptic input in cerebellar Purkinje cells. Our findings support a mechanism linking heterochromatin dysregulation to cerebellar circuit dysfunction and behavioral abnormalities in ASD.

Lsh/HELLS is required for B lymphocyte development and immunoglobulin class switch recombination.

Mutation of HELLS (Helicase, Lymphoid-Specific)/Lsh in human DNA causes a severe immunodeficiency syndrome, but the nature of the defect remains unknown. We assessed here the role of Lsh in hematopoiesis using conditional Lsh knockout mice with expression of Mx1 or Vav Cre-recombinase. Bone marrow transplantation studies revealed that Lsh depletion in hematopoietic stem cells severely reduced B cell numbers and impaired B cell development in a hematopoietic cell-autonomous manner. Lsh-deficient mice without bone marrow transplantation exhibited lower Ig levels in vivo compared to controls despite normal peripheral B cell numbers. Purified B lymphocytes proliferated normally but produced less immunoglobulins in response to in vitro stimulation, indicating a reduced capacity to undergo class switch recombination (CSR). Analysis of germline transcripts, examination of double-stranded breaks using biotin-labeling DNA break assay, and End-seq analysis indicated that the initiation of the recombination process was unscathed. In contrast, digestion-circularization PCR analysis and high-throughput sequencing analyses of CSR junctions and a chromosomal break repair assay indicated an impaired ability of the canonical end-joining pathway in Lsh-deficient B cells. Our data suggest a hematopoietic cell-intrinsic role of Lsh in B cell development and in CSR providing a potential target for immunodeficiency therapy.

Protection of hematopoietic stem cells from stress-induced exhaustion and aging.

PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) are defined by their ability to self-renew and differentiate to replenish all blood lineages throughout adult life. Under homeostasis, the majority of HSCs are quiescent, and few stem cells are cycling to sustain hematopoiesis. However, HSCs can be induced to proliferate and differentiate in response to stress signals produced during infection, inflammation, chemotherapy, radiation, bone marrow transplantation, and aging. Recent evidence suggests that acute and chronic stress impact the number and function of HSCs including their ability to repopulate and produce mature cells. This review will focus on how chronic stress affects HSC biology and methods to mitigate HSC loss during chronic hematopoietic stress. RECENT FINDINGS: Quiescent HSCs exit dormancy, divide, and differentiate to maintain steady-state hematopoiesis. Under conditions of acute stress including infection or blood loss some HSCs are pushed into division by cytokines and proinflammatory stimuli to differentiate and provide needed myeloid and erythroid cells to protect and reconstitute the host; after which, hematopoiesis returns to steady-state with minimal loss of HSC function. However, under conditions of chronic stress including serial bone marrow transplantation (BMT), chronic inflammation, and genotoxic stress (chemotherapy) and aging, HSCs are continuously induced to proliferate and undergo accelerated exhaustion. Recent evidence demonstrates that ablation of inhibitor of DNA binding 1 (Id1) gene can protect HSCs from exhaustion during chronic proliferative stress by promoting HSC quiescence. SUMMARY: Increasing our understanding of the molecular processes that protect HSCs from chronic proliferative stress could lead to therapeutic opportunities to prevent accelerated HSC exhaustion during physiological stress, genotoxic stress, BMT, and aging.

Placental defects lead to embryonic lethality in mice lacking the Formin and PCP proteins Daam1 and Daam2.

The actin cytoskeleton plays a central role in establishing cell polarity and shape during embryonic morphogenesis. Daam1, a member of the Formin family of actin cytoskeleton regulators, is a Dvl2-binding protein that functions in the Wnt/Planar Cell Polarity (PCP) pathway. To examine the role of the Daam proteins in mammalian development, we generated Daam-deficient mice by gene targeting and found that Daam1, but not Daam2, is necessary for fetal survival. Embryonic development of Daam1 mutants was delayed most likely due to functional defects in the labyrinthine layer of the placenta. Examination of Daam2 and Daam1/2 double mutants revealed that Daam1 and Daam2 are functionally redundant during placental development. Of note, neural tube closure defects (NTD), which are observed in several mammalian PCP mutants, are not observed in Wnt5a or Daam1 single mutants, but arise in Daam1;Wnt5a double mutants. These findings demonstrate a unique function for Daam genes in placental development and are consistent with a role for Daam1 in the Wnt/PCP pathway in mammals.

Id1 and Id3 Maintain Steady-State Hematopoiesis by Promoting Sinusoidal Endothelial Cell Survival and Regeneration.

Investigating mechanisms that regulate endothelial cell (EC) growth and survival is important for understanding EC homeostasis and how ECs maintain stem cell niches. We report here that targeted loss of Id genes in adult ECs results in dilated, leaky sinusoids and a pro-inflammatory state that increases in severity over time. Disruption in sinusoidal integrity leads to increased hematopoietic stem cell (HSC) proliferation, differentiation, migration, and exhaustion. Mechanistically, sinusoidal ECs (SECs) show increased apoptosis because of reduced Bcl2-family gene expression following Id gene ablation. Furthermore, Id1-/-Id3-/- SECs and upstream type H vessels show increased expression of cyclin-dependent kinase inhibitors p21 and p27 and impaired ability to proliferate, which is rescued by reducing E2-2 expression. Id1-/-Id3-/- mice do not survive sublethal irradiation because of impaired vessel regeneration and hematopoietic failure. Thus, Id genes are required for the survival and regeneration of BM SECs during homeostasis and stress to maintain HSC development.

Crisis Leadership Education for Critical Care Fellows. A Longitudinal Curriculum Using Simulation.

Background: Leadership and teamwork are critical to the performance of a multidisciplinary team responding to emergencies in the intensive care unit; yet, these skills are variably taught to pulmonary and critical care trainees. Currently, there is no standardized leadership curriculum in critical care training. Objective: We developed a longitudinal crisis leadership curriculum for first-year pulmonary and critical care fellows using high-fidelity simulation as a medium to practice and solidify skills. The goal was to improve leadership skills and trainee confidence when leading a team during life-threatening emergencies. Methods: Guided by a needs assessment of current and recently graduated fellows, we developed a leadership curriculum from a review of the available literature and local expert opinion. Four sessions were conducted over the academic years of 2016 to 2017 and 2017 to 2018, each including small-group teaching on effective leadership behaviors, followed by simulation with postsession leadership debriefing to review performance. Fellows were surveyed regarding their experiences with the curriculum. Results: Over two academic years, 100% of targeted fellows (N = 13) completed every session. Participants reported improved understanding of key elements of effective leadership, greater confidence in leading a multidisciplinary team, and increased preparedness to lead during a crisis. Simulation with debriefing was viewed as an effective medium for learning leadership skills, and fellows provided positive feedback regarding the experience. Conclusion: Implementation of a longitudinal crisis leadership curriculum within the first year of pulmonary and critical care fellowship was feasible and highly valued by learners. More research is needed to determine effective methods for teaching and assessing leadership skills.

Differential Expression of the Transcription Factor GATA3 Specifies Lineage and Functions of Innate Lymphoid Cells.

Lymphoid tissue inducer (LTi) cells are regarded as a subset of innate lymphoid cells (ILCs). However, these cells are not derived from the ILC common progenitor, which generates other ILC subsets and is defined by the expression of the transcription factor PLZF. Here, we examined transcription factor(s) determining the fate of LTi progenitors versus non-LTi ILC progenitors. Conditional deletion of Gata3 resulted in the loss of PLZF+ non-LTi progenitors but not the LTi progenitors that expressed the transcription factor RORγt. Consistently, PLZF+ non-LTi progenitors expressed high amounts of GATA3, whereas GATA3 expression was low in RORγt+ LTi progenitors. The generation of both progenitors required the transcriptional regulator Id2, which defines the common helper-like innate lymphoid progenitor (ChILP), but not cytokine signaling. Nevertheless, low GATA3 expression was necessary for the generation of functionally mature LTi cells. Thus, differential expression of GATA3 determines the fates and functions of distinct ILC progenitors.

Sptlc1 is essential for myeloid differentiation and hematopoietic homeostasis.

Serine palmitoyltransferase (SPT) long-chain base subunit 1 (SPTLC1) is 1 of the 2 main catalytic subunits of the SPT complex, which catalyzes the first and rate-limiting step of sphingolipid biosynthesis. Here, we show that Sptlc1 deletion in adult bone marrow (BM) cells results in defective myeloid differentiation. In chimeric mice from noncompetitive BM transplant assays, there was an expansion of the Lin- c-Kit+ Sca-1+ compartment due to increased multipotent progenitor production, but myeloid differentiation was severely compromised. We also show that defective biogenesis of sphingolipids in the endoplasmic reticulum (ER) leads to ER stress that affects myeloid differentiation. Furthermore, we demonstrate that transient accumulation of fatty acid, a substrate for sphingolipid biosynthesis, could be partially responsible for the ER stress. Independently, we find that ER stress in general, such as that induced by the chemical thapsigargin or the fatty acid palmitic acid, compromises myeloid differentiation in culture. These results identify perturbed sphingolipid metabolism as a source of ER stress, which may produce diverse pathological effects related to differential cell-type sensitivity.

A Whole-Body Mathematical Model of Sepsis Progression and Treatment Designed in the BioGears Physiology Engine.

Sepsis is a debilitating condition associated with a high mortality rate that greatly strains hospital resources. Though advances have been made in improving sepsis diagnosis and treatment, our understanding of the disease is far from complete. Mathematical modeling of sepsis has the potential to explore underlying biological mechanisms and patient phenotypes that contribute to variability in septic patient outcomes. We developed a comprehensive, whole-body mathematical model of sepsis pathophysiology using the BioGears Engine, a robust open-source virtual human modeling project. We describe the development of a sepsis model and the physiologic response within the BioGears framework. We then define and simulate scenarios that compare sepsis treatment regimens. As such, we demonstrate the utility of this model as a tool to augment sepsis research and as a training platform to educate medical staff.

Mechanical Ventilation Training During Graduate Medical Education: Perspectives and Review of the Literature.

BACKGROUND: Management of mechanical ventilation (MV) is an important and complex aspect of caring for critically ill patients. Management strategies and technical operation of the ventilator are key skills for physicians in training, as lack of expertise can lead to substantial patient harm. OBJECTIVE: We performed a narrative review of the literature describing MV education in graduate medical education (GME) and identified best practices for training and assessment methods. METHODS: We searched MEDLINE, PubMed, and Google Scholar for English-language, peer-reviewed articles describing MV education and assessment. We included articles from 2000 through July 2018 pertaining to MV education or training in GME. RESULTS: Fifteen articles met inclusion criteria. Studies related to MV training in anesthesiology, emergency medicine, general surgery, and internal medicine residency programs, as well as subspecialty training in critical care medicine, pediatric critical care medicine, and pulmonary and critical care medicine. Nearly half of trainees assessed were dissatisfied with their MV education. Six studies evaluated educational interventions, all employing simulation as an educational strategy, although there was considerable heterogeneity in content. Most outcomes were assessed with multiple-choice knowledge testing; only 2 studies evaluated the care of actual patients after an educational intervention. CONCLUSIONS: There is a paucity of information describing MV education in GME. The available literature demonstrates that trainees are generally dissatisfied with MV training. Best practices include establishing MV-specific learning objectives and incorporating simulation. Next research steps include developing competency standards and validity evidence for assessment tools that can be utilized across MV educational curricula.

ICU Emergencies Simulation Curriculum for Critical Care Fellows: Neurologic Emergencies.

Introduction: The management of neurologic emergencies is an important component of critical care fellowship training. Additional training in neurocritical care has been demonstrated to improve clinical outcomes, though exposure to these emergencies during training can be limited. Methods: Three simulation cases are presented as part of a comprehensive neurologic emergencies curriculum for critical care trainees. The cases represent neurologic catastrophes encountered in the intensive care unit consisting of symptomatic hyponatremia, severe alcohol withdrawal syndrome, and brain herniation syndrome. The case descriptions are complete with learning objectives, critical actions checklists, and debriefing material for facilitators, as well as all necessary personnel briefs and required equipment. Results: The scenarios were completed over the course of the 2016-2017 academic year by first-year critical care fellows. Following curriculum implementation, there was an improvement in self-perceived confidence of fellows in neurologic emergency management skills. Discussion: The cases were felt to be realistic and beneficial and led to perceived improvement in management of neurologic emergencies and leadership during clinical crises.

Courtship behavior and coloration influence conspicuousness of wolf spiders (Schizocosa ocreata (Hentz)) to avian predators.

Signalers must balance the benefits of detection by intended receivers with the costs of detection by eavesdroppers. This trade-off is exemplified by sexual signaling systems, in which signalers experience sexual selection for conspicuousness to mates as well as natural selection for crypsis to predators. In this study, we examined how courtship behavior and body coloration influenced the conspicuousness of males to avian predators in the well-studied brush-legged wolf spider system (Schizocosa ocreata (Hentz)). We focused on three behaviors (courtship, walking, and freezing) and two coloration schemes (natural coloration and idealized background-matching coloration). We presented captive blue jays (Cyanocitta cristata) with video playbacks of male spiders in a presence-absence detection task and characterized conspicuousness by measuring response latency and detectability. We found that any type of motion significantly increased detectability, and that body coloration and behavior interacted to determine detectability while the spiders were in motion. Among spiders in motion, courting spiders were detected faster than walking spiders. Stationary (frozen) spiders, in contrast, were rarely detected. These results illustrate that male S. ocreata can be both highly conspicuous and highly cryptic to avian predators. Thus, while we find that courtship is conspicuous to avian predators in this system, we suggest that behavioral plasticity may mitigate some of the predation costs of the sexual signal.