MTAP-related increased erythroblast proliferation as a mechanism of polycythaemia vera

Polycythaemia vera (PV) is a haematological disorder caused by an overproduction of erythroid cells. To date, the molecular mechanisms involved in the disease pathogenesis are still ambiguous. This study aims to identify aberrantly expressed proteins in erythroblasts of PV patients by utilizing mass spectrometry-based proteomic analysis. Haematopoietic stem cells (HSCs) were isolated from newly-diagnosed PV patients, PV patients who have received cytoreductive therapy, and healthy subjects. In vitro erythroblast expansion confirmed that the isolated HSCs recapitulated the disease phenotype as the number of erythroblasts from newly-diagnosed PV patients was significantly higher than those from the other groups. Proteomic comparison revealed 17 proteins that were differentially expressed in the erythroblasts from the newly-diagnosed PV patients compared to those from healthy subjects, but which were restored to normal levels in the patients who had received cytoreductive therapy. One of these proteins was S-methyl-5′-thioadenosine phosphorylase (MTAP), which had reduced expression in PV patients’ erythroblasts. Furthermore, MTAP knockdown in normal erythroblasts was shown to enhance their proliferative capacity. Together, this study identifies differentially expressed proteins in erythroblasts of healthy subjects and those of PV patients, indicating that an alteration of protein expression in erythroblasts may be crucial to the pathology of PV

Optimization of an erythroid culture system to reduce the cost of in vitro production of red blood cells

In vitro generation of red blood cells has become a goal for scientists globally. Directly, in vitro-generated red blood cells (RBCs) may close the gap between blood supply obtained through blood donation and high demand for therapeutic uses. In addition, the cells obtained can be used as a model for haematologic disorders to allow the study of their pathophysiology and novel treatment discovery. For those reasons, a number of RBC culture systems have been established and shown to be successful; however, the cost of each millilitre of packed RBC is still extremely high. In order to reduce the cost, we aim to see if we can reduce the number of factors used in the existing culture system. In this study, we examined how well haematopoietic stem cells proliferate and differentiate into mature red blood cells with modified culture system. • Absence of extra heparin or insulin or both from the erythroid differentiation media did not affect haematopoietic stem cell proliferation and differentiation.Therefore, we show that the cost and complexity of erythroid culture can be reduced, which may improve the feasibility of in vitro generation of red blood cells. Method name: A 3-stage erythroid culture system, Keywords: In vitro erythropoiesis, Insulin, Heparin, Erythroid culture syste

<i>ERECTA</i> Corresponds to the Petal Shape Quantitative Trait Locus (QTL) <i>CL-S1</i> on Chromosome II.

<p>(<b>A</b>) Chromosome II diagram showing introgression lines and mutants used for QTL fine mapping. The Stepped Aligned Inbred Recombinant Strain (STAIRS) introgression lines are numbered and their L<i>er</i> -0 introgressions are drawn to scale in black and the otherwise entirely Columbia (Col-4) background is shown in white. The location of the <i>CL-S1</i> 2-LOD interval is drawn to scale with the boundaries marked as black horizontal lines, and the <i>er</i> mutants used, such as <i>er-105</i>, have an <i>er</i> mutation in an otherwise entirely Columbia (Col-0) background (apart from L<i>er</i>-0 which is an <i>er</i> mutant in a Landsberg background). (<b>B</b>) Histogram showing shape measurements of stage 13 petals using STAIRS, natural isolates, and <i>er</i> mutants. Error bars indicate 95% confidence intervals. For each line a minimum of 20 petals were scored. (<b>C</b>) Stage 13 representative petal images. Scale bar = 1 mm.</p

Quantitative Trait Loci for Natural Variation in Petal Shape and Size.

<p>The first two letters of the QTL name designate the parental lines from which the recombinant inbred lines were derived, C for Columbia, E for Estland-1, L for Landsberg <i>erecta</i>. Chr, the chromosome to which the QTl was mapped. LOD, likelihood of difference score. PVE, percentage of the variance explained (R2). CI, Confidence Interval. All petal trait measurements are normalized to comparable sepal data apart from petal shape, which is calculated as the ratio of petal length/petal width.</p

Chromosomal Locations of Quantitative Trait Loci (QTL) for Natural Variation in Petal Form in <i>Arabidopsis thaliana</i>.

<p>The chromosomal location of identified QTLs is shown on the five <i>Arabidopsis</i> chromosomes. The location of several known floral or growth regulatory genes are indicated and the centromeres marked as open circles. QTLs identified using Columbia (Col-4)×Landsberg <i>erecta</i> (L<i>er</i>) recombinant inbred lines are shown in black and named <i>CL</i>, and those identified from Columbia (Col-0)×Estland (Est) recombinant inbred lines are shown in white and named <i>CE</i>. Each QTL is indicated by a shape, with the extent of the shape indicating the 1-LOD support interval: triangle (area), rectangle (length), diamond (width), or oval (shape) and named as A, L, W, or S, respectively. The location of each QTL peak is marked with a black horizontal line and the 2-LOD support interval boundary is indicated by arrows. Where the 1-LOD and 2-LOD boundaries are the same for a QTL, no arrows are marked. Adjacent to each QTL the Percentage of Variance Explained (PVE) is noted.</p

Natural Variation Identifies Multiple Loci Controlling Petal Shape and Size in <em>Arabidopsis thaliana</em>

<div><p>Natural variation in organ morphologies can have adaptive significance and contribute to speciation. However, the underlying allelic differences responsible for variation in organ size and shape remain poorly understood. We have utilized natural phenotypic variation in three <em>Arabidopsis thaliana</em> ecotypes to examine the genetic basis for quantitative variation in petal length, width, area, and shape. We identified 23 loci responsible for such variation, many of which appear to correspond to genes not previously implicated in controlling organ morphology. These analyses also demonstrated that allelic differences at distinct loci can independently affect petal length, width, area or shape, suggesting that these traits behave as independent modules. We also showed that <em>ERECTA</em> (<em>ER</em>), encoding a leucine-rich repeat (LRR) receptor-like serine-threonine kinase, is a major effect locus determining petal shape. Allelic variation at the <em>ER</em> locus was associated with differences in petal cell proliferation and concomitant effects on petal shape. <em>ER</em> has been previously shown to be required for regulating cell division and expansion in other contexts; the <em>ER</em> receptor-like kinase functioning to also control organ-specific proliferation patterns suggests that allelic variation in common signaling components may nonetheless have been a key factor in morphological diversification.</p> </div

miR-34 activity is modulated through 5'-end phosphorylation in response to DNA damage.

MicroRNA (miRNA) expression is tightly regulated by several mechanisms, including transcription and cleavage of the miRNA precursor RNAs, to generate a mature miRNA, which is thought to be directly correlated with activity. MiR-34 is a tumour-suppressor miRNA important in cell survival, that is transcriptionally upregulated by p53 in response to DNA damage. Here, we show for the first time that there is a pool of mature miR-34 in cells that lacks a 5'-phosphate and is inactive. Following exposure to a DNA-damaging stimulus, the inactive pool of miR-34 is rapidly activated through 5'-end phosphorylation in an ATM- and Clp1-dependent manner, enabling loading into Ago2. Importantly, this mechanism of miR-34 activation occurs faster than, and independently of, de novo p53-mediated transcription and processing. Our study reveals a novel mechanism of rapid miRNA activation in response to environmental stimuli occurring at the mature miRNA level

miR-34 activity is modulated through 5′-end phosphorylation in response to DNA damage

MicroRNA (miRNA) expression is tightly regulated by several mechanisms, including transcription and cleavage of the miRNA precursor RNAs, to generate a mature miRNA, which is thought to be directly correlated with activity. MiR-34 is a tumour-suppressor miRNA important in cell survival, that is transcriptionally upregulated by p53 in response to DNA damage. Here, we show for the first time that there is a pool of mature miR-34 in cells that lacks a 5′-phosphate and is inactive. Following exposure to a DNA-damaging stimulus, the inactive pool of miR-34 is rapidly activated through 5′-end phosphorylation in an ATM- and Clp1-dependent manner, enabling loading into Ago2. Importantly, this mechanism of miR-34 activation occurs faster than, and independently of, de novo p53-mediated transcription and processing. Our study reveals a novel mechanism of rapid miRNA activation in response to environmental stimuli occurring at the mature miRNA level