Dual targeting of histone deacetylases and MYC as potential treatment strategy for H3-K27M pediatric gliomas.

Diffuse midline gliomas (DMGs) are aggressive and fatal pediatric tumors of the central nervous system that are highly resistant to treatments. Lysine to methionine substitution of residue 27 on histone H3 (H3-K27M) is a driver mutation in DMGs, reshaping the epigenetic landscape of these cells to promote tumorigenesis. H3-K27M gliomas are characterized by deregulation of histone acetylation and methylation pathways, as well as the oncogenic MYC pathway. In search of effective treatment, we examined the therapeutic potential of dual targeting of histone deacetylases (HDACs) and MYC in these tumors. Treatment of H3-K27M patient-derived cells with Sulfopin, an inhibitor shown to block MYC-driven tumors in vivo, in combination with the HDAC inhibitor Vorinostat, resulted in substantial decrease in cell viability. Moreover, transcriptome and epigenome profiling revealed synergistic effect of this drug combination in downregulation of prominent oncogenic pathways such as mTOR. Finally, in vivo studies of patient-derived orthotopic xenograft models showed significant tumor growth reduction in mice treated with the drug combination. These results highlight the combined treatment with PIN1 and HDAC inhibitors as a promising therapeutic approach for these aggressive tumors.

Diffuse midline gliomas (DMGs) are among the most aggressive and fatal brain cancers in children. They are often associated with changes in histones, the proteins that control gene activity and give chromosomes their structure. Most children with DMGs, for example, share the same anomaly in their histone H3 protein (referred to as the H3-K27M mutation). This change affects how small chemical tags called methyl and acetyl groups can be added onto histone 3, which in turn alters the way the protein can switch genes on and off. As a result, tumours start to develop. One potential therapeutic strategy against DMGs is to use histone deacetylase inhibitors (HDACi), a promising type of drugs which inhibits the enzymes that remove acetyl groups from histones. Patients can develop resistance to HDACi, however, highlighting the need to explore other approaches. One possibility is to treat patients with several types of drugs, each usually targeting a distinct biological process that contributes to the emergence of cancer. This combined approach can have multiple benefits; the drugs potentially amplify each other's effect, for example, and it is also less likely for cells to become resistant to more than one compound at the time. In addition, each drug in the combination can be used in a lower dose to reduce side effects and benefit patients. DMG tumour cells often feature higher activity levels of a protein known as MYC, which can contribute to the growth of the tumour. Algranati, Oren et al. therefore set out to test whether combining an HDACi known as Vorinostat with a drug that blocks MYC activity (Sulfopin) can act as an effective treatment for this cancer. Tumour samples from eight DMG patients were treated with either Sulfopin alone, or Sulfopin in association with Vorinostat. Cells exposed to both drugs were less likely to survive, and additional genetic experiments showed that the combined treatment had resulted in pathways that promote tumour development being blocked. When both Sulfopin and Vorinostat were administered to mice made to grow human DMG tumors, the animals showed a greater reduction in tumor growth. Treatment options for DMG are usually limited, with chemotherapy often being ineffective and surgery impossible. The work by Algranati, Oren et al. suggests that combining HDACi and drugs targeting the MYC pathway is a strategy that should be examined further to determine whether clinical applications are possible.

Pediatric glioblastoma cells are sensitive to drugs that inhibit eIF2α dephosphorylation and its phosphomimetic S51D variant.

We found that pediatric glioblastoma (PED-GBM) cell lines from diffuse intrinsic pontine glioma (DIPG) carrying the H3K27M mutation or from diffuse hemispheric glioma expressing the H3G34R mutation are sensitive to the combination of vorinostat (a histone deacetylase inhibitor) and PARP-1 inhibitors. The combined treatment increased the phosphorylation of eIF2α (P-eIF2α) relative to each drug alone and enhanced the decrease in cell survival. To explore the role played by increased P-eIF2α in modulating PED-GBM survival and response to treatments, we employed brain-penetrating inhibitors of P-eIF2α dephosphorylation: salubrinal and raphin-1. These drugs increased P-eIF2α, DNA damage, and cell death, similarly affecting the sensitivity of DIPG cells and derived neurospheres to PARP-1 inhibitors. Interestingly, these drugs also decreased the level of eIF2Bϵ (the catalytic subunit of eIF2B) and increased its phosphorylation, thereby enhancing the effect of increased P-eIF2α. Transient transfection with the S51D phosphomimetic eIF2α variant recapitulated the effect of salubrinal and raphin-1 on PED-GBM survival and sensitivity to PARP-1 inhibitors. Importantly, either salubrinal or raphin-1 dramatically increased the sensitivity of DIPG cells to radiation, the main treatment modality of PED-GBM. Finally, PED-GBM was more sensitive than normal human astrocytes to salubrinal, raphin-1, and the treatment combinations described herein. Our results indicate that combinations of histone deacetylase inhibitors and PARP-1 inhibitors should be evaluated for their toxicity and efficacy in PED-GBM patients and point to drugs that increase P-eIF2α or modulate its downstream effectors as a novel means of treating PED-GBM.

Single-cell epigenetic analysis reveals principles of chromatin states in H3.3-K27M gliomas.

Cancer cells are highly heterogeneous at the transcriptional level and epigenetic state. Methods to study epigenetic heterogeneity are limited in throughput and information obtained per cell. Here, we adapted cytometry by time-of-flight (CyTOF) to analyze a wide panel of histone modifications in primary tumor-derived lines of diffused intrinsic pontine glioma (DIPG). DIPG is a lethal glioma, driven by a histone H3 lysine 27 mutation (H3-K27M). We identified two epigenetically distinct subpopulations in DIPG, reflecting inherent heterogeneity in expression of the mutant histone. These two subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity and expression of stem cell and differentiation markers. Moreover, we demonstrate the use of these high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity in cancer that could be applied to diverse biological systems.

Gut microbiota modulates weight gain in mice after discontinued smoke exposure.

Cigarette smoking constitutes a leading global cause of morbidity and preventable death1, and most active smokers report a desire or recent attempt to quit2. Smoking-cessation-induced weight gain (SCWG; 4.5 kg reported to be gained on average per 6-12 months, >10 kg year-1 in 13% of those who stopped smoking3) constitutes a major obstacle to smoking abstinence4, even under stable5,6 or restricted7 caloric intake. Here we use a mouse model to demonstrate that smoking and cessation induce a dysbiotic state that is driven by an intestinal influx of cigarette-smoke-related metabolites. Microbiome depletion induced by treatment with antibiotics prevents SCWG. Conversely, fecal microbiome transplantation from mice previously exposed to cigarette smoke into germ-free mice naive to smoke exposure induces excessive weight gain across diets and mouse strains. Metabolically, microbiome-induced SCWG involves a concerted host and microbiome shunting of dietary choline to dimethylglycine driving increased gut energy harvest, coupled with the depletion of a cross-regulated weight-lowering metabolite, N-acetylglycine, and possibly by the effects of other differentially abundant cigarette-smoke-related metabolites. Dimethylglycine and N-acetylglycine may also modulate weight and associated adipose-tissue immunity under non-smoking conditions. Preliminary observations in a small cross-sectional human cohort support these findings, which calls for larger human trials to establish the relevance of this mechanism in active smokers. Collectively, we uncover a microbiome-dependent orchestration of SCWG that may be exploitable to improve smoking-cessation success and to correct metabolic perturbations even in non-smoking settings.

Acute liver failure is regulated by MYC- and microbiome-dependent programs.

Acute liver failure (ALF) is a fulminant complication of multiple etiologies, characterized by rapid hepatic destruction, multi-organ failure and mortality. ALF treatment is mainly limited to supportive care and liver transplantation. Here we utilize the acetaminophen (APAP) and thioacetamide (TAA) ALF models in characterizing 56,527 single-cell transcriptomes to define the mouse ALF cellular atlas. We demonstrate that unique, previously uncharacterized stellate cell, endothelial cell, Kupffer cell, monocyte and neutrophil subsets, and their intricate intercellular crosstalk, drive ALF. We unravel a common MYC-dependent transcriptional program orchestrating stellate, endothelial and Kupffer cell activation during ALF, which is regulated by the gut microbiome through Toll-like receptor (TLR) signaling. Pharmacological inhibition of MYC, upstream TLR signaling checkpoints or microbiome depletion suppress this cell-specific, MYC-dependent program, thereby attenuating ALF. In humans, we demonstrate upregulated hepatic MYC expression in ALF transplant recipients compared to healthy donors. Collectively we demonstrate that detailed cellular/genetic decoding may enable pathway-specific ALF therapeutic intervention.

Diet Diurnally Regulates Small Intestinal Microbiome-Epithelial-Immune Homeostasis and Enteritis.

Throughout a 24-h period, the small intestine (SI) is exposed to diurnally varying food- and microbiome-derived antigenic burdens but maintains a strict immune homeostasis, which when perturbed in genetically susceptible individuals, may lead to Crohn disease. Herein, we demonstrate that dietary content and rhythmicity regulate the diurnally shifting SI epithelial cell (SIEC) transcriptional landscape through modulation of the SI microbiome. We exemplify this concept with SIEC major histocompatibility complex (MHC) class II, which is diurnally modulated by distinct mucosal-adherent SI commensals, while supporting downstream diurnal activity of intra-epithelial IL-10+ lymphocytes regulating the SI barrier function. Disruption of this diurnally regulated diet-microbiome-MHC class II-IL-10-epithelial barrier axis by circadian clock disarrangement, alterations in feeding time or content, or epithelial-specific MHC class II depletion leads to an extensive microbial product influx, driving Crohn-like enteritis. Collectively, we highlight nutritional features that modulate SI microbiome, immunity, and barrier function and identify dietary, epithelial, and immune checkpoints along this axis to be potentially exploitable in future Crohn disease interventions.

High-Throughput Screen Identifies Host and Microbiota Regulators of Intestinal Barrier Function.

BACKGROUND & AIMS: The intestinal barrier protects intestinal cells from microbes and antigens in the lumen-breaches can alter the composition of the intestinal microbiota, the enteric immune system, and metabolism. We performed a screen to identify molecules that disrupt and support the intestinal epithelial barrier and tested their effects in mice. METHODS: We performed an imaging-based, quantitative, high-throughput screen (using CaCo-2 and T84 cells incubated with lipopolysaccharide; tumor necrosis factor; histamine; receptor antagonists; and libraries of secreted proteins, microbial metabolites, and drugs) to identify molecules that altered epithelial tight junction (TJ) and focal adhesion morphology. We then tested the effects of TJ stabilizers on these changes. Molecules we found to disrupt or stabilize TJs were administered mice with dextran sodium sulfate-induced colitis or Citrobacter rodentium-induced intestinal inflammation. Colon tissues were collected and analyzed by histology, fluorescence microscopy, and RNA sequencing. RESULTS: The screen identified numerous compounds that disrupted or stabilized (after disruption) TJs and monolayers of epithelial cells. We associated distinct morphologic alterations with changes in barrier function, and identified a variety of cytokines, metabolites, and drugs (including inhibitors of actomyosin contractility) that prevent disruption of TJs and restore TJ integrity. One of these disruptors (putrescine) disrupted TJ integrity in ex vivo mouse colon tissues; administration to mice exacerbated colon inflammation, increased gut permeability, reduced colon transepithelial electrical resistance, increased pattern recognition receptor ligands in mesenteric lymph nodes, and decreased colon length and survival times. Putrescine also increased intestine levels and fecal shedding of viable C rodentium, increased bacterial attachment to the colonic epithelium, and increased levels of inflammatory cytokines in colon tissues. Colonic epithelial cells from mice given putrescine increased expression of genes that regulate metal binding, oxidative stress, and cytoskeletal organization and contractility. Co-administration of taurine with putrescine blocked disruption of TJs and the exacerbated inflammation. CONCLUSIONS: We identified molecules that disrupt and stabilize intestinal epithelial TJs and barrier function and affect development of colon inflammation in mice. These agents might be developed for treatment of barrier intestinal impairment-associated and inflammatory disorders in patients, or avoided to prevent inflammation.

IL-23-producing IL-10Rα-deficient gut macrophages elicit an IL-22-driven proinflammatory epithelial cell response.

Cytokines maintain intestinal health, but precise intercellular communication networks remain poorly understood. Macrophages are immune sentinels of the intestinal tissue and are critical for gut homeostasis. Here, we show that in a murine inflammatory bowel disease (IBD) model based on macrophage-restricted interleukin-10 (IL-10) receptor deficiency (Cx3cr1Cre:Il10rafl/fl mice), proinflammatory mutant gut macrophages cause severe spontaneous colitis resembling the condition observed in children carrying IL-10R mutations. We establish macrophage-derived IL-23 as the driving factor of this pathology. Specifically, we report that Cx3cr1Cre:Il10rafl/fl:Il23afl/fl mice harboring macrophages deficient for both IL-10R and IL-23 are protected from colitis. By analyzing the epithelial response to proinflammatory macrophages, we provide evidence that T cells of colitic animals produce IL-22, which induces epithelial chemokine expression and detrimental neutrophil recruitment. Collectively, we define macrophage-specific contributions to the induction and pathogenesis of colitis, as manifested in mice harboring IL-10R deficiencies and human IBDs.

Assessing Mucosal Inflammation in a DSS-Induced Colitis Mouse Model by MR Colonography.

Inflammatory bowel disease (IBD) is characterized by a chronic flaring inflammation of the gastrointestinal tract. To determine disease activity, the inflammatory state of the colon should be assessed. Endoscopy in patients with IBD aids visualization of mucosal inflammation. However, because the mucosa is fragile, there is a significant risk of perforation. In addition, the technique is based on grading of the entire colon, which is highly operator-dependent. An improved, noninvasive, objective magnetic resonance imaging (MRI) technique will effectively assess pathologies in the small intestinal mucosa, more specifically, along the colon, and the bowel wall and surrounding structures. Here, dextran sodium sulfate polymer induced acute colitis in mice that was subsequently characterized by multisection magnetic resonance colonography. This study aimed to develop a noninvasive, objective, quantitative MRI technique for detecting mucosal inflammation in a dextran sodium sulfate-induced colitis mouse model. MRI results were correlated with endoscopic and histopathological evaluations.

Induction of Nitric-Oxide Metabolism in Enterocytes Alleviates Colitis and Inflammation-Associated Colon Cancer.

Nitric oxide (NO) plays an established role in numerous physiological and pathological processes, but the specific cellular sources of NO in disease pathogenesis remain unclear, preventing the implementation of NO-related therapy. Argininosuccinate lyase (ASL) is the only enzyme able to produce arginine, the substrate for NO generation by nitric oxide synthase (NOS) isoforms. Here, we generated cell-specific conditional ASL knockout mice in combination with genetic and chemical colitis models. We demonstrate that NO derived from enterocytes alleviates colitis by decreasing macrophage infiltration and tissue damage, whereas immune cell-derived NO is associated with macrophage activation, resulting in increased severity of inflammation. We find that induction of endogenous NO production by enterocytes with supplements that upregulate ASL expression and complement its substrates results in improved epithelial integrity and alleviation of colitis and of inflammation-associated colon cancer.

ADAR1 deletion induces NFκB and interferon signaling dependent liver inflammation and fibrosis.

Adenosine deaminase acting on RNA (ADAR) 1 binds and edits double-stranded (ds) RNA secondary structures found mainly within untranslated regions of many transcripts. In the current research, our aim was to study the role of ADAR1 in liver homeostasis. As previous studies show a conserved immunoregulatory function for ADAR1 in mammalians, we focused on its role in preventing chronic hepatic inflammation and the associated activation of hepatic stellate cells to produce extracellular matrix and promote fibrosis. We show that hepatocytes specific ADAR1 knock out (KO) mice display massive liver damage with multifocal inflammation and fibrogenesis. The bioinformatics analysis of the microarray gene-expression datasets of ADAR1 KO livers reveled a type-I interferons signature and an enrichment for immune response genes compared to control littermate livers. Furthermore, we found that in vitro silencing of ADAR1 expression in HepG2 cells leads to enhanced transcription of NFκB target genes, foremost of the pro-inflammatory cytokines IL6 and IL8. We also discovered immune cell-independent paracrine signaling among ADAR1-depleted HepG2 cells and hepatic stellate cells, leading to the activation of the latter cell type to adopt a profibrogenic phenotype. This paracrine communication dependent mainly on the production and secretion of the cytokine IL6 induced by ADAR1 silencing in hepatocytes. Thus, our findings shed a new light on the vital regulatory role of ADAR1 in hepatic immune homeostasis, chiefly its inhibitory function on the crosstalk between the NFκB and type-I interferons signaling cascades, restraining the development of liver inflammation and fibrosis.

Genetic manipulation of iron biomineralization enhances MR relaxivity in a ferritin-M6A chimeric complex.

Ferritin has gained significant attention as a potential reporter gene for in vivo imaging by magnetic resonance imaging (MRI). However, due to the ferritin ferrihydrite core, the relaxivity and sensitivity for detection of native ferritin is relatively low. We report here on a novel chimeric magneto-ferritin reporter gene - ferritin-M6A - in which the magnetite binding peptide from the magnetotactic bacteria magnetosome-associated Mms6 protein was fused to the C-terminal of murine h-ferritin. Biophysical experiments showed that purified ferritin-M6A assembled into a stable protein cage with the M6A protruding into the cage core, enabling magnetite biomineralisation. Ferritin-M6A-expressing C6-glioma cells showed enhanced (per iron) r2 relaxivity. MRI in vivo studies of ferritin-M6A-expressing tumour xenografts showed enhanced R2 relaxation rate in the central hypoxic region of the tumours. Such enhanced relaxivity would increase the sensitivity of ferritin as a reporter gene for non-invasive in vivo MRI-monitoring of cell delivery and differentiation in cellular or gene-based therapies.

Chronic Akt1 deficiency attenuates adverse remodeling and enhances angiogenesis after myocardial infarction.

BACKGROUND: Akt1 is a key signaling molecule in multiple cell types, including endothelial cells. Accordingly, Akt1 was proposed as a therapeutic target for ischemic injury in the context of myocardial infarction (MI). The aim of this study was to use multimodal in vivo imaging to investigate the impact of systemic Akt1 deficiency on cardiac function and angiogenesis before and after MI. METHODS AND RESULTS: In vivo cardiac MRI was performed before and at days 1, 8, 15, and 29 to 30 after MI induction for wild-type, heterozygous, and Akt1-deficient mice. Noninfarcted hearts were imaged using ex vivo stereomicroscopy and microcomputed tomography. Histological examination was performed for noninfarcted hearts and for hearts at days 8 and 29 to 30 after MI. MRI revealed mildly decreased baseline cardiac function in Akt1 null mice, whereas ex vivo stereomicroscopy and microcomputed tomography revealed substantially reduced coronary macrovasculature. After MI, Akt1(-/-) mice demonstrated significantly attenuated ventricular remodeling and a smaller decrease in ejection fraction. At 8 days after MI, a larger functional capillary network at the remote and border zone, accompanied by reduced scar extension, preserved cardiac function, and enhanced border zone wall thickening, was observed in Akt1(-/-) mice when compared with littermate controls. CONCLUSIONS: Using multimodal imaging to probe the role of Akt1 in cardiac function and remodeling after MI, this study revealed reduced adverse remodeling in Akt1-deficient mice after MI. Augmented myocardial angiogenesis coupled with a more functional myocardial capillary network may facilitate revascularization and therefore be responsible for preservation of infarcted myocardium.

Zinc supplementation augments in vivo antitumor effect of chemotherapy by restoring p53 function.

Activated p53 is necessary for tumor suppression. Homeodomain-interacting protein kinase-2 (HIPK2) is a positive regulator of functional p53. HIPK2 modulates wild-type p53 activity toward proapoptotic transcription and tumor suppression by the phosphorylation of serine 46. Knock-down of HIPK2 interferes with tumor suppression and sensitivity to chemotherapy. Combined administration of adriamycin and zinc restores activity of misfolded p53 and enables the induction of its proapoptotic and tumor suppressor functions in vitro and in vivo. We therefore looked for a cancer model where HIPK2 expression is low. MMTV-neu transgenic mice overexpressing HER2/neu, develop mammary tumors at puberty with a long latency, showing very low expression of HIPK2. Here we show that whereas these tumors are resistant to adriamycin treatment, a combination of adriamycin and zinc suppresses tumor growth in vivo in these mice, an effect evidenced by the histological features of the mammary tumors. The combined treatment of adriamycin and zinc also restores wild-type p53 conformation and induces proapoptotic transcription activity. These findings may open up new possibilities for the treatment of human cancers via the combination of zinc with chemotherapeutic agents, for a selected group of patients expressing low levels of HIPK2, with an intact p53. In addition, HIPK2 may serve as a new biomarker for tumor aggressiveness.

In vivo characterization of tumor and tumor vascular network using multi-modal imaging approach.

We present a multi-modal optical diagnostic approach utilizing a combined use of Fluorescence Intravital Microscopy (FIM), Dynamic Light Scattering (DLS) and Spectrally Enhanced Microscopy (SEM) modalities for in vivo imaging of tumor vascular network and blood microcirculation. FIM is used for imaging of tumor surroundings and microenvironment, SEM provides information regarding blood vessels topography, whereas DLS is applied for functional imaging of vascular network and blood microcirculation. This complementary combination of the imaging approaches is extremely useful for functional in vivo imaging of blood vasculature and tumor microenvironment. The technique has also a great potential in vascular biology and can significantly expand the capabilities of tumor angiogenesis studies and notably contribute to the development of cancer treatment.

Survival and size are differentially regulated by placental and fetal PKBalpha/AKT1 in mice.

The importance of placental circulation is exemplified by the correlation of placental size and blood flow with fetal weight and survival during normal and compromised human pregnancies in such conditions as preeclampsia and intrauterine growth restriction (IUGR). Using noninvasive magnetic resonance imaging, we evaluated the role of PKBalpha/AKT1, a major mediator of angiogenesis, on placental vascular function. PKBalpha/AKT1 deficiency reduced maternal blood volume fraction without affecting the integrity of the fetomaternal blood barrier. In addition to angiogenesis, PKBalpha/AKT1 regulates additional processes related to survival and growth. In accordance with reports in adult mice, we demonstrated a role for PKBalpha/AKT1 in regulating chondrocyte organization in fetal long bones. Using tetraploid complementation experiments with PKBalpha/AKT1-expressing placentas, we found that although placental PKBalpha/AKT1 restored fetal survival, fetal PKBalpha/AKT1 regulated fetal size, because tetraploid complementation did not prevent intrauterine growth retardation. Histological examination of rescued fetuses showed reduced liver blood vessel and renal glomeruli capillary density in PKBalpha/Akt1 null fetuses, both of which were restored by tetraploid complementation. However, bone development was still impaired in tetraploid-rescued PKBalpha/Akt1 null fetuses. Although PKBalpha/AKT1-expressing placentas restored chondrocyte cell number in the hypertrophic layer of humeri, fetal PKBalpha/AKT1 was found to be necessary for chondrocyte columnar organization. Remarkably, a dose-dependent phenotype was exhibited for PKBalpha/AKT1 when examining PKBalpha/Akt1 heterozygous fetuses as well as those complemented by tetraploid placentas. The differential role of PKBalpha/AKT1 on mouse fetal survival and growth may shed light on its roles in human IUGR.

Functional phenotyping of the maternal albumin turnover in the mouse placenta by dynamic contrast-enhanced MRI.

PURPOSE: The purpose of this study was to develop a tool for functional phenotyping of the maternal circulation in the mouse placenta. PROCEDURES: In utero macromolecular dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed on embryonic day 10.5 (E10.5), E13.5, and E18.5. Fluorescence analysis was also used for validation of the results. RESULTS: The initial rate of contrast enhancement revealed an increased maternal blood volume fraction as the pregnancy progressed. Serial imaging of E10.5 and E13.5 placentas revealed a loss of contrast enhancement due to phagocytic uptake. A key application of macromolecular DCE-MRI would be to follow mouse pregnancies during fetal and placental manipulation including embryo transfer, tetraploid complementation, and fetal resorptions. We were able to resolve strain differences in ICR outbred mice carrying both ICR and C57Bl/6J embryos and to differentiate in utero resorptions from functional placentas. CONCLUSIONS: Our results highlight the importance of the functional in utero analysis of placental vascularization in physiological phenotyping of transgenic mice and suggest MRI, particularly macromolecular DCE-MRI, as a non-invasive tool for the analysis of the placenta.

Adenosine-to-inosine RNA editing shapes transcriptome diversity in primates.

Human and chimpanzee genomes are almost identical, yet humans express higher brain capabilities. Deciphering the basis for this superiority is a long sought-after challenge. Adenosine-to-inosine (A-to-I) RNA editing is a widespread modification of the transcriptome. The editing level in humans is significantly higher compared with nonprimates, due to exceptional editing within the primate-specific Alu sequences, but the global editing level of nonhuman primates has not been studied so far. Here we report the sequencing of transcribed Alu sequences in humans, chimpanzees, and rhesus monkeys. We found that, on average, the editing level in the transcripts analyzed is higher in human brain compared with nonhuman primates, even where the genomic Alu structure is unmodified. Correlated editing is observed for pairs and triplets of specific adenosines along the Alu sequences. Moreover, new editable species-specific Alu insertions, subsequent to the human-chimpanzee split, are significantly enriched in genes related to neuronal functions and neurological diseases. The enhanced editing level in the human brain and the association with neuronal functions both hint at the possible contribution of A-to-I editing to the development of higher brain function. We show here that combinatorial editing is the most significant contributor to the transcriptome repertoire and suggest that Alu editing adapted by natural selection may therefore serve as an alternate information mechanism based on the binary A/I code.

Bone vascularization and trabecular bone formation are mediated by PKB alpha/Akt1 in a gene-dosage-dependent manner: in vivo and ex vivo MRI.

PKBalpha/Akt1, a protein kinase, is a major mediator of angiogenic signaling. The purpose of this study was to determine the role of PKB alpha/Akt1 in bone vascularization and development. For that aim, macromolecular dynamic contrast enhanced MRI was applied to examine in vivo vascular changes in long bones of 40-day-old growing PKB alpha/Akt1-deficient, heterozygous, and wild-type mice. Ex vivo microMRI and microCT were applied to monitor the impact of PKB alpha/Akt1 gene dosage on trabecular bone formation during endochondral bone growth. PKB alpha/Akt1-deficient mice and, remarkably, also heterozygous mice showed significantly reduced blood volume fraction in the humerus compared to wild-type mice. Moreover, PKB alpha/Akt1-deficient mice showed a more severe vascular deficiency with reduced permeability. microCT and microMRI of trabeculae revealed impaired bone formation in both PKB alpha/Akt1-deficient and heterozygous mice, whereas cortical bone parameters were only reduced in PKB alpha/Akt1-deficient mice. Reduction of metaphyseal blood vessel invasion, concomitant with aberrant trabeculae and shorter long bones, demonstrates a gene-dose-dependent role for PKB alpha/Akt1 in regulation of overall size and endochondral bone growth. MRI proved to provide high sensitivity for in vivo detection of subtle gene dose effects leading to impaired bone vascularity and for uncovering changes in trabecular bone.

Ferritin as a reporter gene for MRI: chronic liver over expression of H-ferritin during dietary iron supplementation and aging.

The iron storage protein, ferritin, provides an important endogenous MRI contrast that can be used to determine the level of tissue iron. In recent years the impact of modulating ferritin expression on MRI contrast and relaxation rates was evaluated by several groups, using genetically modified cells, viral gene transfer and transgenic animals. This paper reports the follow-up of transgenic mice that chronically over-expressed the heavy chain of ferritin (h-ferritin) in liver hepatocytes (liver-hfer mice) over a period of 2 years, with the aim of investigating the long-term effects of elevated level of h-ferritin on MR signal and on the well-being of the mice. Analysis revealed that aging liver-hfer mice, exposed to chronic elevated expression of h-ferritin, have increased R(2) values compared to WT. As expected for ferritin, R(2) difference was strongly enhanced at high magnetic field. Histological analysis of these mice did not reveal liver changes with prolonged over expression of ferritin, and no differences could be detected in other organs. Furthermore, dietary iron supplementation significantly affected MRI contrast, without affecting animal wellbeing, for both wildtype and ferritin over expressing transgenic mice. These results suggest the safety of ferritin over-expression, and support the use of h-ferritin as a reporter gene for MRI.