The inducible tissue-specific expression of the human IL-3/GM-CSF locus is controlled by a complex array of developmentally regulated enhancers.

The closely linked human IL-3 and GM-CSF genes are tightly regulated and are expressed in activated T cells and mast cells. In this study, we used transgenic mice to study the developmental regulation of this locus and to identify DNA elements required for its correct activity in vivo. Because these two genes are separated by a CTCF-dependent insulator, and the GM-CSF gene is regulated primarily by its own upstream enhancer, the main objective in this study was to identify regions of the locus required for correct IL-3 gene expression. We initially found that the previously identified proximal upstream IL-3 enhancers were insufficient to account for the in vivo activity of the IL-3 gene. However, an extended analysis of DNase I-hypersensitive sites (DHSs) spanning the entire upstream IL-3 intergenic region revealed the existence of a complex cluster of both constitutive and inducible DHSs spanning the -34- to -40-kb region. The tissue specificity of these DHSs mirrored the activity of the IL-3 gene, and included a highly inducible cyclosporin A-sensitive enhancer at -37 kb that increased IL-3 promoter activity 40-fold. Significantly, inclusion of this region enabled correct in vivo regulation of IL-3 gene expression in T cells, mast cells, and myeloid progenitor cells.

Whole genome structural predictions reveal hidden diversity in putative oxidative enzymes of the lignocellulose-degrading ascomycete Parascedosporium putredinis NO1.

IMPORTANCE: An annotated reference genome has revealed P. putredinis NO1 as a useful resource for the identification of new lignocellulose-degrading enzymes for biorefining of woody plant biomass. Utilizing a "structure-omics"-based searching strategy, we identified new potentially lignocellulose-active sequences that would have been missed by traditional sequence searching methods. These new identifications, alongside the discovery of novel enzymatic functions from this underexplored lineage with the recent discovery of a new phenol oxidase that cleaves the main structural ß-O-4 linkage in lignin from P. putredinis NO1, highlight the underexplored and poorly represented family Microascaceae as a particularly interesting candidate worthy of further exploration toward the valorization of high value biorenewable products.

The human IL-3/granulocyte-macrophage colony-stimulating factor locus is epigenetically silent in immature thymocytes and is progressively activated during T cell development.

The closely linked IL-3 and GM-CSF genes are located within a cluster of cytokine genes co-expressed in activated T cells. Their activation in response to TCR signaling pathways is controlled by specific, inducible upstream enhancers. To study the developmental regulation of this locus in T lineage cells, we created a transgenic mouse model encompassing the human IL-3 and GM-CSF genes plus the known enhancers. We demonstrated that the IL-3/GM-CSF locus undergoes progressive stages of activation, with stepwise increases in active modifications and the proportion of cytokine-expressing cells, throughout the course of T cell differentiation. Looking first at immature cells, we found that the IL-3/GM-CSF locus was epigenetically silent in CD4/CD8 double positive thymocytes, thereby minimizing the potential for inappropriate activation during the course of TCR selection. Furthermore, we demonstrated that the locus did not reach its maximal transcriptional potential until after T cells had undergone blast cell transformation to become fully activated proliferating T cells. Inducible locus activation in mature T cells was accompanied by noncoding transcription initiating within the enhancer elements. Significantly, we also found that memory CD4 positive T cells, but not naive T cells, maintain a remodeled chromatin structure resembling that seen in T blast cells.

Integrated miRNA/cytokine/chemokine profiling reveals severity-associated step changes and principal correlates of fatality in COVID-19.

Inflammatory cytokines and chemokines (CC) drive COVID-19 pathology. Yet, patients with similar circulating CC levels present with different disease severity. Here, we determined 171 microRNAomes from 58 hospitalized COVID-19 patients (Cohort 1) and levels of 25 cytokines and chemokines (CC) in the same samples. Combining microRNA (miRNA) and CC measurements allowed for discrimination of severe cases with greater accuracy than using miRNA or CC levels alone. Severity group-specific associations between miRNAs and COVID-19-associated CC (e.g., IL6, CCL20) or clinical hallmarks of COVID-19 (e.g., neutrophilia, hypoalbuminemia) separated patients with similar CC levels but different disease severity. Analysis of an independent cohort of 108 patients from a different center (Cohort 2) demonstrated feasibility of CC/miRNA profiling in leftover hospital blood samples with similar severe disease CC and miRNA profiles, and revealed CCL20, IL6, IL10, and miR-451a as key correlates of fatal COVID-19. These findings highlight that systemic miRNA/CC networks underpin severe COVID-19.

A novel mechanism of sodium iodide symporter repression in differentiated thyroid cancer.

Differentiated thyroid cancers and their metastases frequently exhibit reduced iodide uptake, impacting on the efficacy of radioiodine ablation therapy. PTTG binding factor (PBF) is a proto-oncogene implicated in the pathogenesis of thyroid cancer. We recently reported that PBF inhibits iodide uptake, and have now elucidated a mechanism by which PBF directly modulates sodium iodide symporter (NIS) activity in vitro. In subcellular localisation studies, PBF overexpression resulted in the redistribution of NIS from the plasma membrane into intracellular vesicles, where it colocalised with the tetraspanin CD63. Cell-surface biotinylation assays confirmed a reduction in plasma membrane NIS expression following PBF transfection compared with vector-only treatment. Coimmunoprecipitation and GST-pull-down experiments demonstrated a direct interaction between NIS and PBF, the functional consequence of which was assessed using iodide-uptake studies in rat thyroid FRTL-5 cells. PBF repressed iodide uptake, whereas three deletion mutants, which did not localise within intracellular vesicles, lost the ability to inhibit NIS activity. In summary, we present an entirely novel mechanism by which the proto-oncogene PBF binds NIS and alters its subcellular localisation, thereby regulating its ability to uptake iodide. Given that PBF is overexpressed in thyroid cancer, these findings have profound implications for thyroid cancer ablation using radioiodine.

Subtype-specific regulatory network rewiring in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease caused by a variety of alterations in transcription factors, epigenetic regulators and signaling molecules. To determine how different mutant regulators establish AML subtype-specific transcriptional networks, we performed a comprehensive global analysis of cis-regulatory element activity and interaction, transcription factor occupancy and gene expression patterns in purified leukemic blast cells. Here, we focused on specific subgroups of subjects carrying mutations in genes encoding transcription factors (RUNX1, CEBPα), signaling molecules (FTL3-ITD, RAS) and the nuclear protein NPM1). Integrated analysis of these data demonstrates that each mutant regulator establishes a specific transcriptional and signaling network unrelated to that seen in normal cells, sustaining the expression of unique sets of genes required for AML growth and maintenance.

Microexon gene transcriptional profiles and evolution provide insights into blood processing by the Schistosoma japonicum esophagus.

BACKGROUND: Adult schistosomes have a well-developed alimentary tract comprising an oral sucker around the mouth, a short esophagus and a blind ending gut. The esophagus is not simply a muscular tube for conducting blood from the mouth to gut but is divided into compartments, surrounded by anterior and posterior glands, where processing of ingested blood is initiated. Self-cure of rhesus macaques from a Schistosoma japonicum infection appears to operate by blocking the secretory functions of these glands so that the worms cease feeding and slowly starve to death. Here we use subtractive RNASeq to characterise the genes encoding the principal secretory products of S. japonicum esophageal glands, preparatory to evaluating their relevance as targets of the self-cure process. METHODOLOGY/PRINCIPAL FINDINGS: The heads and a small portion of the rear end of male and female S. japonicum worms were separately enriched by microdissection, for mRNA isolation and library construction. The sequence reads were then assembled de novo using Trinity and those genes enriched more than eightfold in the head preparation were subjected to detailed bioinformatics analysis. Of the 62 genes selected from the male heads, more than one third comprised MEGs encoding secreted or membrane-anchored proteins. Database searching using conserved motifs revealed that the MEG-4 and MEG-8/9 families had counterparts in the bird schistosome Trichobilharzia regenti, indicating an ancient association with blood processing. A second group of MEGs, including a MEG-26 family, encoded short peptides with amphipathic properties that most likely interact with ingested host cell membranes to destabilise them. A number of lysosomal hydrolases, two protease inhibitors, a secreted VAL and a putative natterin complete the line-up. There was surprisingly little difference between expression patterns in males and females despite the latter processing much more blood. SIGNIFICANCE/CONCLUSIONS: The mixture of approximately 40 proteins specifically secreted by the esophageal glands is responsible for initiating blood processing in the adult worm esophagus. They comprise the potential targets for the self-cure process in the rhesus macaque, and thus represent a completely new cohort of secreted proteins that can be investigated as vaccine candidates.

How does glucose insulin potassium improve hemodynamic performance? Evidence for altered expression of beta-adrenoreceptor and calcium handling genes.

BACKGROUND: Glucose insulin potassium (GIK) improves hemodynamic performance after coronary artery surgery (CABG). We investigated whether this is associated with changes in gene expression of beta1-adrenergic receptor (ADRB1) or other calcium handling proteins. METHODS AND RESULTS: During a randomized double-blind placebo-controlled trial, 48 patients undergoing on-pump CABG, allocated to receive pre-ischemic placebo (5% dextrose) or GIK (40% dextrose, K+ 100 mmol.L(-1), insulin 70 u.L(-1); 0.75 mL.kg(-1).h(-1)) continued for 6 hours after the removal of the aortic cross-clamp (AXC), underwent left ventricular biopsy for analysis of specific mRNAs immediately before AXC, before release of AXC, and 10 minutes after reperfusion (placebo n=24, GIK n=24). GIK or placebo was infused for a mean of 79+/-21 minutes or 79+/-18 minutes pre-ischemia respectively. Serial hemodynamic measurements were performed. Biopsy samples were snap-frozen and stored at -80 degrees C, mRNA was extracted and TaqMan real-time polymerase chain reaction was performed to investigate expression of ADRB1, sarcoplasmic reticulum Ca-ATPase (SERCA2a), and phospholamban (PLB). GIK significantly increased cardiac index versus placebo (P=0.037). TaqMan reverse-transcriptase polymerase chain reaction showed significantly greater ADRB1 mRNA expression at all time points (4.9-fold, 7.4-fold, and 15.6-fold increase, respectively; P<0.001), significantly greater SERCA2a mRNA expression after reperfusion (13.2-fold; P<0.001), and increased PLB mRNA expression at pre-ischemia and reperfusion (P<0.001 for both time-points) in GIK groups versus placebo. CONCLUSIONS: The beneficial hemodynamic effects of GIK therapy are associated with increased ADRB1 and SERCA2a mRNA expression. Further work is therefore warranted to investigate these mRNA effects at the protein level.

Placental transport of thyroid hormone.

Thyroid hormones are vital for fetal development and can act directly on placental tissues to modify their metabolism, differentiation and development. There is evidence that maternal thyroid hormones can cross the human placenta and act to modulate fetal development before the onset of the fetus's own thyroid hormone production. Plasma membrane transport of thyroid hormones has now been shown to require specific transporter proteins. Several proteins have recently been identified as specific thyroid hormone transporters. However, as yet few data are available to define the functionally important transporter proteins in the human placenta. To date, members of the organic anion-transporting polypeptide, L-type amino acid, and of the monocarboxylate transporter families, have been identified as thyroid hormone transporters that are active in a variety of placental cell types. However, further research is necessary to determine the role of these and other proteins in placental transport of thyroid hormone, and to investigate how modulations of their function could affect fetal pathologies such as intrauterine growth restriction.

Chronic FLT3-ITD Signaling in Acute Myeloid Leukemia Is Connected to a Specific Chromatin Signature.

Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect the epigenetic regulatory machinery and signaling molecules, leading to a block in hematopoietic differentiation. Constitutive signaling from mutated growth factor receptors is a major driver of leukemic growth, but how aberrant signaling affects the epigenome in AML is less understood. Furthermore, AML cells undergo extensive clonal evolution, and the mutations in signaling genes are often secondary events. To elucidate how chronic growth factor signaling alters the transcriptional network in AML, we performed a system-wide multi-omics study of primary cells from patients suffering from AML with internal tandem duplications in the FLT3 transmembrane domain (FLT3-ITD). This strategy revealed cooperation between the MAP kinase (MAPK) inducible transcription factor AP-1 and RUNX1 as a major driver of a common, FLT3-ITD-specific gene expression and chromatin signature, demonstrating a major impact of MAPK signaling pathways in shaping the epigenome of FLT3-ITD AML.

Identification of a dynamic core transcriptional network in t(8;21) AML that regulates differentiation block and self-renewal.

Oncogenic transcription factors such as RUNX1/ETO, which is generated by the chromosomal translocation t(8;21), subvert normal blood cell development by impairing differentiation and driving malignant self-renewal. Here, we use digital footprinting and chromatin immunoprecipitation sequencing (ChIP-seq) to identify the core RUNX1/ETO-responsive transcriptional network of t(8;21) cells. We show that the transcriptional program underlying leukemic propagation is regulated by a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes, which bind to identical DNA sites in a mutually exclusive fashion. Perturbation of this equilibrium in t(8;21) cells by RUNX1/ETO depletion leads to a global redistribution of transcription factor complexes within preexisting open chromatin, resulting in the formation of a transcriptional network that drives myeloid differentiation. Our work demonstrates on a genome-wide level that the extent of impaired myeloid differentiation in t(8;21) is controlled by the dynamic balance between RUNX1/ETO and RUNX1 activities through the repression of transcription factors that drive differentiation.

Induction of differentiation and apoptosis in leukaemic cell lines by the novel benzamide family histone deacetylase 2 and 3 inhibitor MI-192.

Histone deacetylase inhibitors (HDACIs) are in advanced clinical development as cancer therapeutic agents. However, first generation HDACIs such as butyrate and valproate are simple short chain aliphatic compounds with moieties resembling acetyl groups, and have a broad spectrum of activity against HDACs. More complex second generation HDACIs undergoing clinical trials, such as the benzamide group compounds MS-275 and MGCD0103, are specific primarily for HDAC1 and HDAC2. To expand the repertoire of available HDACIs and HDAC specificities we created a novel benzamide-based compound named MI-192. When tested against purified recombinant HDACs, MI-192 had marked selectivity for the class I enzymes, HDAC2 and HDAC3. Screening in the NCI60 screen demonstrated that MI-192 had greatly enhanced efficacy against cells of leukaemic origin. When tested in culture against the acute myeloid leukaemic cell lines U937, HL60 and Kasumi-1, MI-192 induced differentiation and was cytotoxic through promotion of apoptosis. MI-192 therefore justifies further investigation and development as a potential therapeutic agent for use in leukaemia.

The effects of acute triiodothyronine therapy on myocardial gene expression in brain stem dead cardiac donors.

CONTEXT: After brain stem death (BSD), a low T(3) state is common, and T(3) supplementation has been advocated to improve heart function and yield for transplantation. OBJECTIVES: The aim of the study was to assess the effects of T(3) on expression of mRNAs encoding T(3)-responsive genes in the post-BSD human heart. DESIGN: Within a prospective double-blind trial, potential BSD cardiac donors undergoing hemodynamic optimization were randomized to T(3) (0.8 microg . kg(-1) bolus; infusion 0.113 microg . kg(-1) . h(-1)) or placebo (5% dextrose) for up to 6 h. Left ventricular biopsies were obtained at end-assessment from 30 donors (T(3); n=16). TaqMan real-time PCR was performed to investigate mRNA expression of the voltage-gated potassium channel Kv1.5, beta-1 adrenergic receptor (ADRB1), sarcoplasmic reticulum calcium ATPase type 2a (SERCA2a), and phospholamban (PLB). RESULTS: Time between diagnosis of BSD and donor management was 13.2 h (range, 9.7-16.8 h). T(3) donors were managed for 7.6 (6.9-8.3) h. Median serum free T(3) (fT3) at baseline was 2.9 (2.3-3.8) pmol . liter(-1) (reference range, 3.3-7.5 pmol . liter(-1)). At baseline, 19 of 30 (56.7%) had low serum fT3, and T(3) treatment increased fT3 to supraphysiological levels (P < 0.001). Expression of mRNAs encoding Kv1.5 and SERCA2a was increased 1.99-fold and 1.51-fold (P = 0.015 and 0.043). There was no significant change in the expression of mRNAs encoding ADRB1 and PLB. Treatment with T(3) did not improve hemodynamic function compared with placebo. CONCLUSIONS: Acute administration of T(3) in the BSD cardiac donor reverses the low T(3) state and increases expression of the mRNAs encoding Kv1.5 and SERCA2a, but not ADRB1 or PLB and is not associated with any improvement in hemodynamic performance.

Transcriptional and epigenetic regulation of the GM-CSF promoter by RUNX1.

The RUNX1 gene, which is essential for normal hematopoiesis, is frequently rearranged by the t(8;21) chromosomal translocation in acute myeloid leukemia. The resulting RUNX1-ETO fusion protein contributes to leukemic progression by directing aberrant association of transcriptional cofactors and epigenetic modifiers to RUNX1 target genes. For example, the GM-CSF gene is activated by RUNX1, but is repressed by RUNX1-ETO. Here we show that RUNX1 normally cooperates with the histone acetyltransferase, CBP, to regulate GM-CSF expression at two levels. Firstly, it directs the establishment of a competent chromatin environment at the GM-CSF promoter prior to gene activation. It then participates in the transcriptional activation of the promoter in response to immune stimuli. In contrast, RUNX1-ETO, which cannot associate with CBP, is unable to transactivate the GM-CSF promoter and is associated with the generation of a repressive chromatin environment at the promoter.

N-terminal pro-brain-type natriuretic peptide: a biochemical surrogate of cardiac function in the potential heart donor.

OBJECTIVES: N-terminal pro-B-type natriuretic peptide (NT-proBNP) is elevated in subarachnoid haemorrhage, brainstem death (BSD) and heart failure. We examined the relationship between NT-proBNP and cardiac functional status after BSD and left ventricular (LV) BNP precursor gene expression. METHODS: We assayed NT-proBNP in the serum of potential heart donors investigated with pulmonary artery flotation catheters, transthoracic echocardiography and cardiac troponin (cTn) I and T. After 6.9 h of optimisation, haemodynamic studies were repeated to determine haemodynamic functional suitability for transplantation. Median (interquartile range (IQR)) NT-proBNP levels are reported according to initially measured dichotomised pulmonary capillary wedge pressure (PCWP), cardiac index (CI), indexed cardiac power output (CPOi), left ventricular ejection fraction (LVEF), wall motion score (WMS), extravascular lung water index (EVLWI), cTnT and cTnI and end-management functional suitability. LV biopsies were snap-frozen, mRNA extracted and reverse-transcribed, allowing performance of Taqman real-time polymerase chain reaction assays of mRNA-BNP precursor. RESULTS: There were 79 subjects. Median NT-proBNP was 121 pg ml(-1) (range 5-4139) and levels correlated with time from coning (p<0.01, r=-0.379). Higher NT-proBNP was found in donors with PCWP >14 mmHg; 504 (120-1544) versus 101 (38-285); p=0.01; CI <2.4 l min(-1) m(-2) 410 (123-1511) versus 95 (37-264); p=0.001; CPOi <0.5 Wm(-2) 256 (78-694) versus 105 (37-315); p=0.02; LVEF <50% 231 (75-499) versus 72 (36-177); p=0.04; WMS >2; 343 (80-673) versus 99 (37-236); p=0.01; cTnT >0.1 microg ml(-1) 499 (127-967) versus 80 (36-173); p<0.001 and cTnI >1 mg ml(-1) 410 (97-684) versus 88 (36-190); p<0.01 and in hearts functionally unsuitable at end-optimisation; 189 (74-522) versus 85 (39-243); p=0.02. Hearts functionally suitable for transplantation expressed significantly less mRNA encoding for BNP precursor (0.19-fold; p=0.01). CONCLUSION: During or after BSD, NT-proBNP is released and the heart is a likely source. Higher NT-proBNP levels are associated with donor heart dysfunction and a failure to achieve haemodynamic functional suitability criteria. This supports the hypothesis that biomarkers, including NT-proBNP, may be useful in donor heart assessment.

Modified cell cycle status in a mouse model of altered neuronal vulnerability (slow Wallerian degeneration; Wlds).

BACKGROUND: Altered neuronal vulnerability underlies many diseases of the human nervous system, resulting in degeneration and loss of neurons. The neuroprotective slow Wallerian degeneration (Wlds) mutation delays degeneration in axonal and synaptic compartments of neurons following a wide range of traumatic and disease-inducing stimuli, providing a powerful experimental tool with which to investigate modulation of neuronal vulnerability. Although the mechanisms through which Wlds confers neuroprotection remain unclear, a diverse range of downstream modifications, incorporating several genes/pathways, have been implicated. These include the following: elevated nicotinamide adenine dinucleotide (NAD) levels associated with nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1; a part of the chimeric Wlds gene); altered mRNA expression levels of genes such as pituitary tumor transforming gene 1 (Pttg1); changes in the location/activity of the ubiquitin-proteasome machinery via binding to valosin-containing protein (VCP/p97); and modified synaptic expression of proteins such as ubiquitin-activating enzyme E1 (Ube1). RESULTS: Wlds expression in mouse cerebellum and HEK293 cells induced robust increases in a broad spectrum of cell cycle-related genes. Both NAD-dependent and Pttg1-dependent pathways were responsible for mediating different subsets of these alterations, also incorporating changes in VCP/p97 localization and Ube1 expression. Cell proliferation rates were not modified by Wlds, suggesting that later mitotic phases of the cell cycle remained unaltered. We also demonstrate that Wlds concurrently altered endogenous cell stress pathways. CONCLUSION: We report a novel cellular phenotype in cells with altered neuronal vulnerability. We show that previous reports of diverse changes occurring downstream from Wlds expression converge upon modifications in cell cycle status. These data suggest a strong correlation between modified cell cycle pathways and altered vulnerability of axonal and synaptic compartments in postmitotic, terminally differentiated neurons.