B-MYB Is Essential for Normal Cell Cycle Progression and Chromosomal Stability of Embryonic Stem Cells

Background: The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1. Methodology/Principal Findings: In the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death. Conclusions/Significance: Appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death

Proteomic Landscape and Deduced Functions of the Cardiac 14-3-3 Protein Interactome

Rationale: The 14-3-3 protein family is known to interact with many proteins in non-cardiac cell types to regulate multiple signaling pathways, particularly those relating to energy and protein homeostasis; and the 14-3-3 network is a therapeutic target of critical metabolic and proteostatic signaling in cancer and neurological diseases. Although the heart is critically sensitive to nutrient and energy alterations, and multiple signaling pathways coordinate to maintain the cardiac cell homeostasis, neither the structure of cardiac 14-3-3 protein interactome, nor potential functional roles of 14-3-3 protein–protein interactions (PPIs) in heart has been explored. Objective: To establish the comprehensive landscape and characterize the functional role of cardiac 14-3-3 PPIs. Methods and Results: We evaluated both RNA expression and protein abundance of 14-3-3 isoforms in mouse heart, followed by co-immunoprecipitation of 14-3-3 proteins and mass spectrometry in left ventricle. We identified 52 proteins comprising the cardiac 14-3-3 interactome. Multiple bioinformatic analyses indicated that more than half of the proteins bound to 14-3-3 are related to mitochondria; and the deduced functions of the mitochondrial 14-3-3 network are to regulate cardiac ATP production via interactions with mitochondrial inner membrane proteins, especially those in mitochondrial complex I. Binding to ribosomal proteins, 14-3-3 proteins likely coordinate protein synthesis and protein quality control. Localizations of 14-3-3 proteins to mitochondria and ribosome were validated via immunofluorescence assays. The deduced function of cardiac 14-3-3 PPIs is to regulate cardiac metabolic homeostasis and proteostasis. Conclusions: Thus, the cardiac 14-3-3 interactome may be a potential therapeutic target in cardiovascular metabolic and proteostatic disease states, as it already is in cancer therapy

Side effects of statins in patient with compensated hypothyroidism and SLCO1B1 *5 (c.521T>C) polymorphism

Aim: to assess the influence of compensated hypothyroidism and SLCO1B1 *5 (c.521TC) gene polymorphism on the clinical and laboratory signs of the muscle damage during statin therapy. Methods: assessment of symptoms and markers of the muscle damage and SLCO1B1 *5 (c.521TC) genotyping were performed in 33 patients with primary hypothyroidism taking statins, in 31 patients taking statins without hypothyroidism and in 33 patients with primary hypothyroidism without statins taking. Results: muscle pain was observed more often in the group of the patients with compensated hypothyroidism on the background of statins taking compared with other groups (45,5, 16,1 and 30,3 %, respectively, p=0,048). Only in this group the pain was associated with increased levels of creatine- kinase (171,0108,12 and 110,043,81U/L, in the presence and absence of the pain, p=0,049), LDH (369,566,22 and 305,641,98 U/L, р=0,007), myoglobin titer (90,7109,89 and 41,128,56, р=0,005), and more frequent occurrence of TC and CC genotypes of SLCO1B1*5 (c.521TC) (68,4 и 28,6%, р=0,0027). Conclusions: the patients with compensated hypothyroidism have a higher risk of statin-induced myopathy increasing if the TC heterozygotes or CC homozygotes of SLCO1B1 *5 (c.521TC) gene are present, which requires thorough monitoring of clinical and biochemical muscle damage signs in case of its detection

Temperatura do ar: dados climáticos relativos ao mês de março de 1989

Os dados foram recolhidos e registados pelo técnico da ESACB João Nunes sob a supervisão da Prof.ª Maria do Carmo Horta Monteiro.Dados climáticos relativos ao ano de 1989, recolhidos e registados no Posto Meteorológico da Escola Superior Agrária do Instituto Politécnico de Castelo Branco

Examination of known somatic cell B-Myb target gene expression patterns following knockdown of B-Myb in ESCs.

<p>A) Western blot and graphic presentation of Cyclin B1 (Ccnb1) protein expression following knockdown of B-MYB by shRNAs 1, 2 and 5. B) RNA (left) and protein (right) analysis of the B-MYB target gene Polo-like kinase 1 (Plk1) following Bγ-Myb KD with shRNA1 and shRNA5. The effects of B-MYB KD on Plk1 RNA were transient; however, the loss of RNA led to a significant and sustained decrease in its protein abundance. C) Western blot and bar graph showing the abundance of selected proteins following knockdown of B-MYB and CCNB1 using shRNAs and microRNAs, respectively. D) Reduced expression of CCNB1 could not mimic the cell cycle related defects associated with B-MYB deficiency, such as monopolar or multipolar centrosomes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042350#pone-0042350-g001" target="_blank">Figure 1</a>. Ctl – control; KD – Knockdown; NS- non-silencing. *, p<0.05. Data are expressed as mean ± standard deviation (SD).</p

Biological processes and pathways associated with B-MYB deficiency.

<p>A total 18,097 genes were ranked by fold-change of gene expression between control and B-MYB knockdown in mouse ESCs based on microarray experiments (see Methods). Ranked genes were analyzed using GSEA based on GO biological processes. The biological processes in the table represents enriched ones with P value of 0 for under-expressed genes by B-MYB knockdown.</p

Pathway enrichment analysis.

<p>A) Hierarchical clustering of differentially expressed genes (fold-change ≥1.5) in control and B-MYB deficient cells. Red and green colors denote increased and decreased expression, respectively. Each row represents a unique experiment (n = 4, ctl and n = 4, KD) B) KEGG pathway enrichment among the B-MYB knockdown-repressed genes. The enrichment profiles for cell cycle and insulin signaling pathways for genes with reduced expression in B-MYB deficient cells are shown. C) Heatmaps showing gene expression in cell cycle and insulin signaling pathways, indicating that the majority of transcripts are decreased (blue) in abundance in the absence of B-MYB. D) Confirmation of shRNA1 specificity and qPCR analysis of selected cell cycle products and epigenetic regulators. Most of the transcripts analyzed by qPCR showed consistent trends in expression after nucleofection with shRNA1, shRNA2 or shRNA5. Exceptions included Cip1 and Dbf4, both of which had unexpectedly low transcript levels with shRNA5 relative to shRNAs 1 and 2. Almost all of the transcripts with decreased expression 48 hours after nucleofection had modest to significant increases in mRNA expression 24 hours later.</p