A chronic myelomonocytic leukemia (CMML)–like myelodysplastic/myeloproliferative disorder in -deficient mice

) Complete blood counts (white blood cell, lymphocyte, neutrophil, monocyte, red blood cell [RBC], hemoglobin, platelet) in wild-type mice at 2–5 months of age (n = 35), in mice at 2–5 months of age (n = 30), in wild-type mice more than 5 months old (n = 25), and in mice more than 5 months old with symptoms of CMML (n = 25). Means (and 95% confidence intervals) for cell concentrations are shown, and values were calculated using Student test. ) Wright–Giemsa staining of peripheral blood from an mouse with symptoms of CMML, showing teardrop poikilocytes (a, ), red cells with Howell–Jolly bodies (b, ), and nucleated red cells (c, ). Immature (d, ) and maturing (d, ) mononuclear cells were also observed, together with phagocytosis of RBC by a monocyte (e, ). Ten separate analyses were performed. = 5 μm. ) Survival of (n = 25) mice and wild-type (n = 25) mice. ) Reticulin staining of paraffin sections of bone marrow from a wild-type mouse and a sick mouse. The sample shows fibrous tissue stained with black color. Scale bars = 20 μm. ) Flow cytometric analysis of apoptotic cells in bone marrow from a wild-type and a sick mouse. The percentages of cells positive for annexin V are indicated. Five separate cytometric analyses were performed. ) Splenomegaly and ) hepatomegaly in a sick mouse. Hematoxylin and eosin–stained sections of ) spleen and ) liver from a wild-type mouse and a sick mouse. Extramedullary hematopoiesis was found in the spleen and liver, which were infiltrated with nucleated elements of blood cells. Ten separate analyses were performed. Scale bars = 20 μm. ) Flow cytometric analysis of cells from spleen in a wild-type mouse and an mouse stained with Ter119 surface antigen. The percentages of cells positive for the antigen are indicated. Twenty separate analyses were performed.<p><b>Copyright information:</b></p><p>Taken from "Identification of Chromatin Remodeling Genes and as Leukemia Suppressor Genes"</p><p></p><p>JNCI Journal of the National Cancer Institute 2008;100(17):1247-1259.</p><p>Published online 3 Sep 2008</p><p>PMCID:PMC2528019.</p><p></p

Gene expression analysis of bone marrow cells from wild-type, , and mice

) Reverse transcriptase–polymerase chain reaction was performed to analyze the genes indicated, with serving as the control gene. Three separate experiments were performed. ) Pathways by which and might regulate hematopoiesis through control of the and genes. In the scenario shown, controls erythropoiesis, possibly by positively regulating and genes. also controls the expression of , whose product blocks differentiation of hematopoietic stem cells and common myeloid progenitors. Control of B lymphopoiesis by may be achieved by increasing expression of . , together with , increases expression of , which acts on regulatory T (TR) cells to suppress conventional T (Tc) cells. also functions as a tumor suppressor gene. However, it is unclear whether suppresses leukemia malignancies.<p><b>Copyright information:</b></p><p>Taken from "Identification of Chromatin Remodeling Genes and as Leukemia Suppressor Genes"</p><p></p><p>JNCI Journal of the National Cancer Institute 2008;100(17):1247-1259.</p><p>Published online 3 Sep 2008</p><p>PMCID:PMC2528019.</p><p></p

Expression analysis of the paternally expressed gene <i>Ndn</i> in mice carrying the Δ4.8 mutation and/or the Δ<i>Ndn</i> mutation.

<p>(A) Genomic structure of the maternal wild-type <i>Ndn</i> allele and the paternal Δ<i>Ndn</i> allele in the m⁺p^Δ<i>Ndn</i> mice. The relative position of the primer pair used for RT-PCR and qRT-PCR is indicated (half-arrows). In the Δ<i>Ndn</i> mutation, the open reading frame of <i>Ndn</i> was deleted by a replacement of <i>β</i>-galactosidase (<i>β</i>-gal) and a neomycin-resistant cassette (Neo). (B, C) The <i>Ndn</i> transcripts were analyzed by RT-PCR (top) and quantitative RT-PCR (bottom). Total RNA was isolated from brains of wild-type mice (B and C, a, m⁺p⁺) (n = 8), mice with paternal inheritance of the Δ<i>Ndn</i> mutation (B, b, m⁺p^Δ<i>Ndn</i>) (n = 3), mice inheriting the Δ4.8 mutation maternally and the Δ<i>Ndn</i> mutation paternally (B, c, m^Δ4.8p^Δ<i>Ndn</i>) (n = 3), mice with paternal inheritance of the Δ4.8 mutation (C, d, m⁺p^Δ4.8) (n = 5), mice with maternal inheritance of the Δ4.8 mutation (C, e, m^Δ4.8p⁺) (n = 5), and mice with the Δ4.8 mutation from both the parents (C, f, m^Δ4.8p^Δ4.8) (n = 5). RT-PCR analyses were performed using 2.0 µg total RNA with (+) and without (−) reverse transcriptase (RT). For quantitative RT-PCR, the levels of gene expression from wild-type mice were set as 1. Transcripts of <i>Hprt</i> were amplified as an endogenous control. RT-PCR products: <i>Ndn</i>, 365 bp; <i>Hprt</i>, 266 bp. (D) Schematic representation of the mouse PWS/AS domain (top) and summary of <i>Ndn</i> expression in mice of the six different genotypes (bottom, a–f). The <i>Ndn</i> transcripts are marked as an arrow. The centromeric (cen) and the telomeric (tel) positions are indicated. Paternally expressed imprinted genes are marked as blue boxes. Δ<i>Ndn</i> indicates a deletion at <i>Ndn</i>. Δ4.8 indicates a 4.8-kb deletion at <i>Snrpn</i> exon 1. The level of the <i>Ndn</i> transcripts from wild-type mice was set as 100%. Mat, maternal chromosome; Pat, paternal chromosome.</p

Expression analysis of <i>Snrpn</i>, <i>Snrod116</i>, and <i>Snord115</i> in mice carrying the Δ4.8 mutation and/or the ΔS-U mutation.

<p>(A) Genomic structure of the maternal Δ4.8 allele and the paternal ΔS-U allele in the m^Δ4.8p^ΔS-U mice. The Δ4.8 mutation removes exon 1 of <i>Snrpn</i>. <i>Snrpn</i> is still able to transcribe from the upstream exons splicing to <i>Snrpn</i> exon 2. The relative positions of the primers specific for upstream exon 1 and exon 3 (u1-ex3) and for the downstream exon 7 (ex7) designed for RT-PCR and qRT-PCR are indicated (half-arrows). The ΔS-U mutation removes <i>Snrpn</i> from exon 2 to exon 10. (B–E) The <i>Snrpn</i> u1-ex3 (B), <i>Snrpn</i> exon 7 (C), <i>Snrod116</i> (D), and <i>Snord115</i> (E) transcripts were analyzed by RT-PCR (top) and quantitative RT-PCR (bottom). Total RNA was isolated from brains of wild-type mice (a, m⁺p⁺) (n = 5), mice inheriting the ΔS-U mutation maternally and the Δ4.8 mutation paternally (b, m^ΔS-Up^Δ4.8) (n = 5), mice inheriting the Δ4.8 mutation maternally and the ΔS-U mutation paternally (c, m^Δ4.8p^ΔS-U) (n = 5), mice with only the maternally inherited ΔS-U mutation (d, m^ΔS-Up⁺) (n = 5), and mice with only the paternally inherited ΔS-U mutation (e, m⁺p^ΔS-U) (n = 5). RT-PCR analyses were performed using 2.0 µg total RNA (top). For quantitative RT-PCR, the levels of gene expression from wild-type mice were set as 1 (bottom). Transcripts of <i>Hprt</i> were amplified as an endogenous control for the <i>Snrpn</i> u1-ex3 transcripts, since their sizes were similar. Transcripts of <i>Gapdh</i> were amplified as an endogenous control for the <i>Snrpn</i> exon 7, <i>Snrod116</i>, and <i>Snord115</i> transcripts. RT-PCR products: <i>Snrpn</i> u1-ex3, 295 bp; <i>Hprt</i>, 266 bp; <i>Snrpn</i> ex7, 171 bp; <i>Snrod116</i>, 98 bp; <i>Snrod115</i>, 79 bp; <i>Gapdh</i>, 97 bp. (F) Schematic representation of the mouse PWS/AS domain (top) and summary of gene expression in mice of the five different genotypes (bottom, a–e). The <i>Snrpn</i> sense/<i>Ube3a</i> antisense transcripts initiated from <i>Snrpn</i> exon 1 with the major promoter activity and from <i>Snrpn</i> upstream exons with weaker promoter activity are marked as bold and thin arrows, respectively. SnoRNAs are encoded within these large <i>Snrpn</i> sense/<i>Ube3a</i> antisense transcripts derived from both <i>Snrpn</i> major and upstream exon promoters. <i>Snord116</i> and <i>Snord115</i> are multiple copy gene clusters. The centromeric (cen) and the telomeric (tel) positions are indicated. Paternally and maternally expressed genes are marked as blue and red boxes, respectively. ΔS-U indicates a large deletion from <i>Snrpn</i> exon 2 to <i>Ube3a</i>. Δ4.8 indicates a 4.8-kb deletion at <i>Snrpn</i> exon 1. The levels of the <i>Snrpn</i> u1-ex3, <i>Snrpn</i> exon 7, <i>Snrod116</i>, and <i>Snord115</i> transcripts from wild-type mice were set as 100%. Mat, maternal chromosome; Pat, paternal chromosome.</p

ChIP-qPCR analyses for H3K4me3, H3Ac, and H3K4me3 at <i>Snrpn</i> and <i>Ndn</i>.

<p>(A) Schematic diagram of the <i>Snrpn</i> promoter. Gene structure is shown at the top, where the white box represents the <i>Snrpn</i> exon 1 with the +1 as the major transcriptional start site. The region deleted in the Δ4.8 mutation started from −2,702 is indicated as a gray line. A primer pair (half-arrows) was used for qPCR to amplify the <i>Snrpn</i> promoter from −2,919 to −2,705 right upstream of the Δ4.8 region (black box). This qPCR region includes the <i>Snrpn</i> peak 2 region shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034348#pone-0034348-g005" target="_blank">Figure 5B</a>. (B) Schematic diagram of <i>Ndn</i>. The gene structure is shown at the top, where the white box represents the <i>Ndn</i> exon with the +1 as the transcriptional start site. The region deleted in the Δ<i>Ndn</i> mutation started from +228 is indicated (gray line). A primer pair (half-arrows) was used for qPCR to amplify the region from +773 to +976 (black line). This qPCR region includes the <i>Ndn</i> peak 1 region shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034348#pone-0034348-g005" target="_blank">Figure 5C</a>. (C–E) Quantification of H3K4me3 at <i>Snrpn</i> (C) and <i>Ndn</i> (D, E) in the wild-type m⁺p⁺ mice (C–E), the m^Δ4.8p⁺ mice (C, D), the m⁺p^Δ4.8 mice(C, D), the m⁺p^Δ<i>Ndn</i> mice (E), and the m^Δ4.8p^Δ<i>Ndn</i> mice (E). (F–H) Quantification of H3Ac at <i>Snrpn</i> (F) and <i>Ndn</i> (G, H) in the wild-type m⁺p⁺ mice (F–H), the m^Δ4.8p⁺ mice (F, G), the m⁺p^Δ4.8 mice (F, G), the m⁺p^Δ<i>Ndn</i> mice (H), and the m^Δ4.8p^Δ<i>Ndn</i> mice (H). (I–H) Quantification of H3K9me3 at <i>Snrpn</i> (I) and <i>Ndn</i> (J, K) in the wild-type m⁺p⁺ mice (I–H), the m^Δ4.8p⁺ mice (I, J), the m⁺p^Δ4.8 mice(I, J), the m⁺p^Δ<i>Ndn</i> mice (K), and the m^Δ4.8p^Δ<i>Ndn</i> mice (K). The level of ChIP was normalized against the level of input in each sample. The normalized level of ChIP from the wild-type mouse was set as 1. m⁺p⁺, n = 6; m⁺p^Δ4.8, n = 4; m⁺p^Δ4.8, n = 4; m⁺p^Δ<i>Ndn</i>, n = 3; m^Δ4.8p^Δ<i>Ndn</i>, n = 3.</p

Schematic representation of genetic architecture at the PWS/AS domain.

<p>(A) Gene expression and DNA methylation associated with the Δ4.8 mutation, the AS-IC^an mutation, or the PWS-IC^Hs mutation were shown. On the paternal wild-type chromosome, the <i>Snrpn</i> and <i>Ndn</i> promoters are unmethylated and the paternally expressed imprinted genes are activated (a). When the PWS-IC Δ4.8 mutation deletes the CpG island at the <i>Snrpn</i> promoter on the paternal chromosome, the <i>Snrpn</i> sense/<i>Ube3a</i> antisense is transcribed only from the upstream exons, but is not transcribed from the major promoter <i>Snrpn</i> exon 1, resulting in partial activation of <i>Ube3a</i>. The <i>Ndn</i> promoter was partially methylated with decreased gene expression (b). On the maternal wild-type chromosome, silencing of <i>Ndn</i> and <i>Snrpn</i> is associated with DNA methylation at their promoters. <i>Ube3a</i> is activated (c). When the PWS-IC Δ4.8 mutation deletes the CpG island at the <i>Snrpn</i> promoter on the maternal chromosome, the <i>Snrpn</i> promoter at the upstream exons is activated and transcribes the <i>Snrpn</i> sense/<i>Ube3a</i> antisense, resulting in partial reduction of <i>Ube3a</i> expression. The <i>Ndn</i> promoter was partially activated with decreased DNA methylation (d). Maternal inheritance of an insertion/duplication mutation 13 kb upstream of <i>Snrpn</i> exon 1 (AS-IC^an) causes loss of <i>Snrpn</i> methylation, decreased <i>Ndn</i> methylation, activation of the maternally repressed genes, and silencing of <i>Ube3a</i> due to expression of the maternal copy of the <i>Snrpn</i> sense/<i>Ube3a</i> antisense (e). Maternal transmission of a targeted replacement of mouse PWS-IC with human PWS-IC (PWS-IC^Hs) expressed the <i>Snrpn</i> sense/<i>Ube3a</i> antisense transcripts from the inserted human <i>SNRPN</i> promoter, resulting in silencing of <i>Ube3a</i>. The PWS-IC^Hs does not affect any other paternally expressed imprinted transcripts on the maternal chromosome (f). (B) A model on how PWS-IC controls both paternal and maternal imprint at the PWS/AS domain. On the paternal chromosome, the PWS-IC functions as the major promoter for the <i>Snrpn</i> sense/<i>Ube3a</i> antisense transcripts. The paternal PWS-IC also acts at long distances to activate the <i>Snrpn</i> upstream exons and <i>Ndn</i> gene. The active <i>Snrpn</i> and <i>Ndn</i> promoters are unmethylated on the CpG islands and modified with H3K4me3 and H3Ac. On the other hand, the maternal PWS-IC acts <i>in cis</i> to silence the paternally expressed imprinted genes with the <i>Snrpn</i> and <i>Ndn</i> promoters methylated on the CpG islands and modified with H3K9me3, a mark of a repressive chromatin state. The maternally expressed imprinted genes <i>Ube3a</i> is expressed due to lack of the <i>Snrpn</i> sense/<i>Ube3a</i> antisense transcripts on the maternal chromosome.</p

DNA methylation analyses at <i>Ndn</i> and <i>Mkrn3</i>.

<p>(A) Schematic diagram of <i>Ndn</i>. Gene structure is shown at the top, where the white box represents the <i>Ndn</i> exon with the +1 as the transcriptional start site. A <i>Sac</i>II site at +750 for Southern blot analysis in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034348#pone.0034348.s001" target="_blank">Figure S1</a> is indicated. Locations of CpG dinucleotides are shown as vertical bars. The region deleted in the Δ<i>Ndn</i> mutation started from +228 is indicated (gray line). Two primer pairs (half-arrows) were used for nested PCR to amplify the bisulfite-treated DNA at the <i>Ndn</i> promoter from −69 to +470 (black line). A third primer pair (half-arrows) was used for the MeDIP-qPCR analysis to amplify the region from +237 to +470 (black line). (B) Sodium bisulfite sequencing analyses of methylation status of 42 CpG dinucleotides across the <i>Ndn</i> promoter (−69 to +470) in the wild-type m⁺p⁺ mice, the m^Δ4.8p⁺ mice, the m⁺p^Δ<i>Ndn</i> mice, and the m^Δ4.8p^Δ<i>Ndn</i> mice. Each line represents an individual clone with open and closed circles corresponding to unmethylated and methylated CpGs, respectively. (C) MeDIP-qPCR analyses of DNA methylation at the <i>Ndn</i> promoter (+237 to +470) in the wild-type m⁺p⁺ mice, the m^Δ4.8p⁺ mice, and the m⁺p^Δ4.8 mice (left), as well as in the m⁺p^Δ<i>Ndn</i> mice and the m^Δ4.8p^Δ<i>Ndn</i> mice (right). The level of MeDIP DNA was normalized against the level of input DNA in each sample. m⁺p⁺, n = 3; m⁺p^Δ4.8, n = 3; m⁺p^Δ4.8, n = 3; m⁺p^Δ<i>Ndn</i>, n = 3; m^Δ4.8p^Δ<i>Ndn</i>, n = 3. (D) Schematic diagram of the <i>Mkrn3</i> promoter. Gene structure is shown at the top, where the white box represents the partial <i>Mkrn3</i> exon with the +1 as the transcriptional start site. A <i>Not</i>I site at +139 is indicated. Locations of CpG dinucleotides are shown as vertical bars. Two primer pairs (half-arrows) were used for nested PCR to amplify the bisulfite-treated DNA at the <i>Mkrn3</i> promoter from −469 to +91 (black line). A third primer pair (half-arrows) was used for the MeDIP-qPCR analysis to amplify the region from +21 to +324 (black line). (E) Sodium bisulfite sequencing analyses of methylation status of 22 CpG dinucleotides across the <i>Mkrn3</i> promoter (−469 to +91) in the wild-type m⁺p⁺ mice and the m^Δ4.8p⁺ mice. Each line represents an individual clone with open and closed circles corresponding to unmethylated and methylated CpGs, respectively. (F) MeDIP-qPCR analyses of DNA methylation at the <i>Mkrn3</i> promoter (+21 to +324) in the wild-type m⁺p⁺ mice, the m^Δ4.8p⁺ mice, and the m⁺p^Δ4.8 mice. The level of MeDIP DNA was normalized against the level of input DNA in each sample. The normalized level of MeDIP DNA from the wild-type mouse was set as 1. m⁺p⁺, n = 3; m⁺p^Δ4.8, n = 3; m⁺p^Δ4.8, n = 3.</p

Rescue of growth retardation in the PWS mouse models by maternal inheritance of the Δ4.8 mutation.

<p>(A) Wild-type offspring were obtained from mating a wild-type female with a wild-type male. (B) Growth retardation was observed in the m⁺p^Δ4.8 offspring obtained from mating a wild-type female with a male carrying the Δ4.8 mutation. (C) Growth retardation was rescued in the m^Δ4.8p^Δ4.8 offspring inheriting the Δ4.8 mutation from both the parents. (D) Double heterozygous m^Δ4.8p^ΔS-U pups attained a normal body weight indistinguishable from the m^Δ4.8p⁺ littermates. All photographs were taken when groups of litters were 10 days of age. (E) The growth retardation was analyzed by weighting groups of mice with those five different genotypes up to 6 weeks of age. m⁺p⁺, n = 6; m⁺p^Δ4.8, n = 4; m^Δ4.8p^Δ4.8, n = 5; m^Δ4.8p^ΔS-U, n = 5; m^Δ4.8p⁺, n = 5.</p

Effects and safety of oral tolvaptan in patients with congestive heart failure: A systematic review and network meta-analysis

<div><p>Aims</p><p>Several studies reported treatment benefits of tolvaptan in patients with congestive heart failure (CHF). However, the optimal dosage remains unclear. We aimed to compare different dosage of tolvaptan to determine the optimal dosage in terms of the efficacy and safety.</p><p>Methods</p><p>We searched MEDLINE, PubMed, EMBASE, Cochrane CENTRAL and ClinicalTrials.gov through Aug 31, 2016. Randomized controlled trials (RCTs) comparing tolvaptan of different dosages or to placebo in patients with CHF were included. We used network meta-analysis to look for the optimal dosage in terms of effectiveness and safety. Urine output, body weight change and change in serum sodium were the main outcomes of efficacy. Adverse effects were the secondary outcomes. Quality was assessed by Cochrane risk-of-bias tool.</p><p>Results</p><p>Twelve RCTs reporting 14 articles with 5793 patients (mean age, 65.7 ± 11.9 years; 73.7% man) were included. Compared with placebo, the tolvaptan 30 mg had similar effects to tolvaptan 45–90 mg in terms of urine output (mean difference [MD] 2.03 liter; 95% confidence interval [CI] 1.3 to 2.71), body weight change (MD -1.12 kg; 95% CI -1.37 to -0.88) and change in serum sodium (MD 3.06 meq/L; 95% CI 2.43 to 3.68). Compared with placebo, tolvaptan of different dosage showed a non-significant higher risk of adverse effects.</p><p>Conclusions</p><p>These findings suggest that tolvaptan 30 mg and 45 mg may be the optimum dosage for CHF patients, because of its ability to provide favourable clinical results without greater adverse effects. However, tolvaptan is not beneficial for reducing all-cause mortality in CHF patients.</p></div

Results from network meta-analysis of primary outcomes.

<p>(A) Urine output. (B) Body weight change. (C) Change of serum sodium.</p