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Genetic diseases can be diagnosed early during pregnancy, but many monogenic disorders continue to cause considerable neonatal and pediatric morbidity and mortality. Early intervention through intrauterine gene editing, however, could correct the genetic defect, potentially allowing for normal organ development, functional disease improvement, or cure. Here we demonstrate safe intravenous and intra-amniotic administration of polymeric nanoparticles to fetal mouse tissues at selected gestational ages with no effect on survival or postnatal growth. In utero introduction of nanoparticles containing peptide nucleic acids (PNAs) and donor DNAs corrects a disease-causing mutation in the β-globin gene in a mouse model of human β-thalassemia, yielding sustained postnatal elevation of blood hemoglobin levels into the normal range, reduced reticulocyte counts, reversal of splenomegaly, and improved survival, with no detected off-target mutations in partially homologous loci. This work may provide the basis for a safe and versatile method of fetal gene editing for human monogenic disorders., The correction of genetic defects in utero could allow for improved outcomes of gene therapy. Here, the authors demonstrate safe delivery of nanoparticles to fetal mouse tissues, and show that nanoparticles containing peptide nucleic acids to edit the beta-globin gene are effective in a mouse model of beta-thalassemia.

Objective— The calcium composition of atherosclerotic plaque is thought to be associated with increased risk for cardiovascular events, but whether plaque calcium itself is predictive of worsening clinical outcomes remains highly controversial. Inflammation is likely a key mediator of vascular calcification, but immune signaling mechanisms that promote this process are minimally understood. Approach and Results— Here, we identify Rac2 as a major inflammatory regulator of signaling that directs plaque osteogenesis. In experimental atherogenesis, Rac2 prevented progressive calcification through its suppression of Rac1-dependent macrophage interleukin-1β (IL-1β) expression, which in turn is a key driver of vascular smooth muscle cell calcium deposition by its ability to promote osteogenic transcriptional programs. Calcified coronary arteries from patients revealed decreased Rac2 expression but increased IL-1β expression, and high coronary calcium burden in patients with coronary artery disease was associated with significantly increased serum IL-1β levels. Moreover, we found that elevated IL-1β was an independent predictor of cardiovascular death in those subjects with high coronary calcium burden. Conclusions— Overall, these studies identify a novel Rac2-mediated regulation of macrophage IL-1β expression, which has the potential to serve as a powerful biomarker and therapeutic target for atherosclerosis.

Nature Communications 8: Article number: 14433 (2017) Published online 14 February 2017; Updated 20 April 2018 In this Article, a subset of the H3K27ac ChIP-seq data (15 benign meningiomas and 2 dura samples (Sample IDs: MN-297, MN-288, MN-292, MN-163, MN-1037, MN-105, MN-201, MN-249, MN-191, MN-1066, MN-169, MN-291, MN-24, MN-79, MN-1044, CONTROL1, CONTROL2) was reported previously in a publication by the corresponding author1.

The fat mass and obesity associated (FTO) gene has previously been associated with a variety of diseases and conditions, notably obesity, acute coronary syndrome and metabolic syndrome. Reports describing mutations in FTO as well as in FTO animal models have further demonstrated a role for FTO in the development of the brain and other organs. Here, we describe a patient born of consanguineous union who presented with microcephaly, developmental delay, behavioral abnormalities, dysmorphic facial features, hypotonia and other various phenotypic abnormalities. Whole-exome sequencing revealed a novel homozygous missense mutation in FTO and a nonsense mutation in the cholesteryl ester transfer protein (CETP). Exome copy number variation analysis revealed no disease-causing large duplications or deletions within coding regions. Patient's, her parents' and non-related control' fibroblasts were analyzed for morphologic defects, abnormal proliferation, apoptosis and transcriptome profile. We have shown that FTO is located in the nucleus of cells from each tested sample. Western blot analysis demonstrated no changes in patient FTO. Quantitative (qPCR) analysis revealed slightly decreased levels of FTO expression in patient cells compared with controls. No morphological or proliferation differences between the patient and control fibroblasts were observed. There is still much to be learned about the molecular mechanisms by which mutations in FTO contribute to such severe phenotypes.

The calcium composition of atherosclerotic plaque has predictive value for increased risk of cardiovascular events. Inflammation is associated with atherosclerotic calcification, but the immune signaling that regulates calcium mineralization in plaque is minimally understood. The hematopoietic Rac family member, Rac2, modulates the activation of immune cells and has potential to influence plaque osteogenesis. Both aortic plaque from ApoE -/- mice fed a high fat diet and coronary plaque from patients revealed increased Rac1:Rac2 expression ratios, driven by dynamic Rac2 expression, to be associated with calcified plaque. On high fat diet, Rac2 -/- ApoE -/- mice demonstrated comparable serum cholesterol and plaque burden relative to ApoE -/- mice, but histology identified differences in plaque structure and cellularity. MicroCT and calcium-targeted imaging identified increased atherosclerotic calcification, which was associated with elevated expression of osteogenic transcription factors and was dependent on the hematopoietic compartment. Calcified plaque expressed higher IL-1β mRNA levels, and serum revealed increased IL-1β protein concentrations. Rac2 -/- ApoE -/- macrophages demonstrated increased activation of Rac1 and consequent Rac1-dependent IL-1β secretion. Downstream of Rac1, NF-κB and reactive oxygen species (ROS) signaling drove IL-1β production by increasing IL-1β mRNA expression and caspase1 activation. Cultured mouse aorta smooth muscle cells mineralized calcium in an IL-1β dose-dependent manner, and the enhanced atherosclerotic calcification in vivo was inhibited by IL-1 receptor antagonist, confirming a cause-and-effect relationship. In patients with stable coronary artery disease, high coronary calcium burden was associated with increased serum IL-1β, and patients with combined elevations in calcium and IL-1β had more events driven by higher mortality, reinforcing the relevance of this inflammatory calcification signaling axis to human disease. Therapeutic targeting of IL-1β expression through the balance of Rac activation has potential to impact patient care by modulating atherosclerotic calcification and consequent cardiovascular events.

Gliomas represent approximately 30\% of all central nervous system tumors and 80\% of malignant brain tumors(1). To understand the molecular mechanisms underlying the malignant progression of low-grade gliomas with mutations in IDH1 (encoding isocitrate dehydrogenase 1), we studied paired tumor samples from 41 patients, comparing higher-grade, progressed samples to their lower-grade counterparts. Integrated genomic analyses, including whole-exome sequencing and copy number, gene expression and DNA methylation profiling, demonstrated nonlinear clonal expansion of the original tumors and identified oncogenic pathways driving progression. These include activation of the MYC and RTK-RAS-PI3K pathways and upregulation of the FOXM1- and E2F2-mediated cell cycle transitions, as well as epigenetic silencing of developmental transcription factor genes bound by Polycomb repressive complex 2 in human embryonic stem cells. Our results not only provide mechanistic insight into the genetic and epigenetic mechanisms driving glioma progression but also identify inhibition of the bromodomain and extraterminal (BET) family as a potential therapeutic approach.

Objective— The regeneration of the hematopoietic system in bone marrow after chemotherapy depends on a balance between the quiescence and proliferation of lineage-specific progenitor cells. Even though the vascular network in bone is damaged by cytoablation, the transcriptional control of quiescence in endothelial cells is not well known. In this study, we investigated the role of the transcription factor E74-like factor (ELF4) in the proliferation of endothelial cells in bone marrow. Methods and Results— Loss-of-function models were used to study the role of ELF4 in human and murine endothelial cells. ELF4 promotes cell cycle entry by activating cyclin-dependent kinase-4 in human umbilical vein endothelial cells. Elf4 -null mice exhibited enhanced recovery of bone marrow CD45 − CD31 + endothelial cells and sinusoidal blood vessels following administration of 5-fluorouracil. Conclusion— Loss of ELF4 leads to increased quiescence in bone marrow endothelial cells by the deregulation of cyclin-dependent kinase-4 expression and to enhanced regeneration of sinusoidal blood vessels.

Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53(515C) (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2(-/-) mice. Mdm2(-/-) p53(515C/515C) mice, however, die by postnatal day 13 resulting from hematopoietic failure. Hematopoietic stem cells and progenitors of Mdm2(-/-) p53(515C/515C) mice were normal in fetal livers but were depleted in postnatal bone marrows. After birth, these mice had elevated reactive oxygen species (ROS) thus activating p53R172P. In the absence of Mdm2, stable p53R172P induced ROS and cell cycle arrest, senescence, and cell death in the hematopoietic compartment. This phenotype was partially rescued with antioxidant treatment and upon culturing of hematopoietic cells in methycellulose at 3% oxygen. p16 was also stabilized because of ROS, and its loss increased cell cycling and partially rescued hematopoiesis and survival. Thus, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis.

WOS: 000364783100007, PubMed: 25740784, Background. Malignant high-grade gliomas (HGGs), including the most aggressive form, glioblastoma multiforme, show significant clinical and genomic heterogeneity. Despite recent advances, the overall survival of HGGs and their response to treatment remain poor. In order to gain further insight into disease pathophysiology by correlating genomic landscape with clinical behavior, thereby identifying distinct HGG molecular subgroups associated with improved prognosis, we performed a comprehensive genomic analysis. Methods. We analyzed and compared 720 exome-sequenced gliomas (136 from Yale, 584 from The Cancer Genome Atlas) based on their genomic, histological, and clinical features. Results. We identified a subgroup of HGGs (6 total, 4 adults and 2 children) that harbored a statistically significantly increased number of somatic mutations (mean = 9257.3 vs 76.2, P = .002). All of these "ultramutated" tumors harbored somatic mutations in the exonuclease domain of the polymerase epsilon gene (POLE), displaying a distinctive genetic profile, characterized by genomic stability and increased C-to-A transversions. Histologically, they all harbored multinucleated giant or bizarre cells, some with predominant infiltrating immune cells. One adult and both pediatric patients carried homozygous germline mutations in the mutS homolog 6 (MSH6) gene. In adults, POLE mutations were observed in patients younger than 40 years and were associated with a longer progression-free survival. Conclusions. We identified a genomically, histologically, and clinically distinct subgroup of HGGs that harbored somatic POLE mutations and carried an improved prognosis. Identification of distinctive molecular and pathological HGG phenotypes has implications not only for improved classification but also for potential targeted treatments., Gregory M. Kiez and Mehmet Kutman Foundation, This study was supported by the Gregory M. Kiez and Mehmet Kutman Foundation.

Hematopoietic stem cells (HSCs) have been used therapeutically for decades, yet their widespread clinical use is hampered by the inability to expand HSCs successfully in vitro. In culture, HSCs rapidly differentiate and lose their ability to self-renew. We hypothesize that by mimicking aspects of the bone marrow microenvironment in vitro we can better control the expansion and differentiation of these cells. In this work, derivatives of poly(ethylene glycol) diacrylate hydrogels were used as a culture substrate for hematopoietic stem and progenitor cell (HSPC) populations. Key HSC cytokines, stem cell factor (SCF) and interferon-γ (IFNγ), as well as the cell adhesion ligands RGDS and connecting segment 1 were covalently immobilized onto the surface of the hydrogels. With the use of SCF and IFNγ, we observed significant expansion of HSPCs, ∼97 and ∼104 fold respectively, while maintaining c-kit(+) lin(-) and c-kit(+) Sca1(+) lin(-) (KSL) populations and the ability to form multilineage colonies after 14 days. HSPCs were also encapsulated within degradable poly(ethylene glycol) hydrogels for three-dimensional culture. After expansion in hydrogels, ∼60% of cells were c-kit(+), demonstrating no loss in the proportion of these cells over the 14 day culture period, and ∼50% of colonies formed were multilineage, indicating that the cells retained their differentiation potential. The ability to tailor and use this system to support HSC growth could have implications on the future use of HSCs and other blood cell types in a clinical setting.

Hematopoietic stem cells (HSC) are multi-potent cells that have ability to self-renew and differentiate to all blood cell lineages. To function properly, HSC are regulated within unique microenvironments, or so called HSC niches , which are composed of specialized supporting cells and extracellular matrix. During mammalian embryogenesis, sites of hematopoiesis change over the course of gestation, as does the HSC niche composition: from extraembryonic yolk sac and placenta, to embryonic aorta-gonad-mesonephros region, fetal liver, and finally fetal bone marrow where HSC predominantly reside postnatally. From their first emergence to their final destination in bone marrow, HSC reside in close proximity to, and interact with vascular cells. There are three main vascular niche types; sinusoidal, arteriolar and perivascular. The interactions between HSC and vascular niche cells have been of great research interest; however, our understanding of the cellular phenotypes within vascular niches, and the molecular basis of vascular niche-HSC interactions, is still rudimentary. In this chapter, we will discuss the developmental relationships between HSC and vascular endothelial cells during HSC ontogeny, and the role of postnatal vascular niches in the regulation of HSC maintenance and function.

Hematopoietic stem cells (HSCs) reside within a specialized niche where interactions with vasculature, osteoblasts, and stromal components regulate their self-renewal and differentiation. Little is known about bone marrow niche formation or the role of its cellular components in HSC development; therefore, we established the timing of murine fetal long bone vascularization and ossification relative to the onset of HSC activity. Adult-repopulating HSCs emerged at embryonic day 16.5 (E16.5), coincident with marrow vascularization, and were contained within the c-Kit(+)Sca-1(+)Lin(-) (KSL) population. We used Osterix-null (Osx(-/-)) mice that form vascularized marrow but lack osteolineage cells to dissect the role(s) of these cellular components in HSC development. Osx(-/-) fetal bone marrow cells formed multilineage colonies in vitro but were hyperproliferative and failed to home to and/or engraft transplant recipients. Thus, in developing bone marrow, the vasculature can sustain multilineage progenitors, but interactions with osteolineage cells are needed to regulate long-term HSC proliferation and potential.

Objective: Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Pulmonary are among the most common causes of mortality in AML. This single-center retrospective study aimed to evaluate the relationship between radiological findings of pulmonary at presentation and post chemotherapy on prognosis and clinical outcome in a group of AML patients. Material and Methods: The study included 278 AML patients. Clinical and radiological findings, laboratory findings, and microbiological culture results were evaluated. Pulmonary complications at presentation and post chemotherapy were compared. Results: Pulmonary complications were observed in 53 of the patients (19%). Mean age of the patients with and without pulmonary complications was 43.1 ± 15.2 years and 38.8 ± 16.3 years, respectively (P < 0.001). Pulmonary complications were not correlated with gender, AML subtype, or the serum lactate dehydrogenase (LDH) level. The most common cause of pulmonary complications was infection. Pulmonary complications were observed in 29% and 71% of the patients at presentation and post chemotherapy, respectively. Conclusion: Pulmonary complications were observed more frequently at presentation in neutropenic AML patients of advanced age. The mortality rate was higher among the AML patients that had pulmonary complications at presentation.

Hematopoietic stem cells (HSC) are multi-potent cells that function to generate a lifelong supply of all blood cell types. During mammalian embryogenesis, sites of hematopoiesis change over the course of gestation: from extraembryonic yolk sac and placenta, to embryonic aorta-gonad-mesonephros region, fetal liver, and finally fetal bond marrow where HSC reside postnatally. These tissues provide microenviroments for de novo HSC formation, as well as HSC maturation and expansion. Within adult bone marrow, HSC self-renewal and differentiation are thought to be regulated by two major cellular components within their so-called niche: osteoblasts and vascular endothelial cells. This review focuses on HSC generation within, and migration to, different tissues during development, and also provides a summary of major regulatory factors provided by osteoblasts and vascular endothelial cells within the adult bone marrow niche.

Poly(3-hydroxybutyrate) (P3HB), its co-polymers with 3-hydroxyvalerate (HV) (PHBV8 and PHBV22), and their hydroxyapatite (HAp) containing composites (5 and 15%, w/w) were prepared by injection molding. PHBV bone plates with low valerate contents and 15% (w/w) HAp appear to have better mechanical properties than the others. Flexural strengths of 15% (w/w) HAp-loaded P3HB, PHBV8 and PHBV22 were 78.28, 63.45 and 39.38 MPa, respectively. Tensile strengths of 15% (w/w) HAp-loaded P3HB, PHBV8 and PHBV22 were 18.99, 15.44 and 11.02 MPa, respectively. For the ageing test, bone plates were incubated in phosphate-buffered saline PBS (0.1 M, pH 7.4) at 37 degrees C and at pre-determined time points they were removed and subjected to a three-point bending test. Incubation in PBS caused a sharp decrease in the mechanical properties within the first 24 h, followed either by a gradual decrease or no change for a period of about 1 month. SEM results showed that there was no significant material erosion in the 4-week incubation period. P3HB loaded with 15% HAp appeared to yield the most suitable bone plate, insofar as mechanical properties are concerned with potential for further testing in vivo.

SummaryDelineating the mechanism or mechanisms that regulate the specification of hemogenic endothelial cells from primordial endothelium is critical for optimizing their derivation from human stem cells for clinical therapies. We previously determined that retinoic acid (RA) is required for hemogenic specification, as well as cell-cycle control, of endothelium during embryogenesis. Herein, we define the molecular signals downstream of RA that regulate hemogenic endothelial cell development and demonstrate that cell-cycle control is required for this process. We found that re-expression of c-Kit in RA-deficient (Raldh2−/−) primordial endothelium induced Notch signaling and p27 expression, which restored cell-cycle control and rescued hemogenic endothelial cell specification and function. Re-expression of p27 in RA-deficient and Notch-inactivated primordial endothelial cells was sufficient to correct their defects in cell-cycle regulation and hemogenic endothelial cell development. Thus, RA regulation of hemogenic endothelial cell specification requires c-Kit, notch signaling, and p27-mediated cell-cycle control.