Perturbation in iron homeostasis is a hallmark of some hematologic diseases. Abnormal sideroblasts with accumulation of iron in the mitochondria are named ring sideroblasts (RS). RS is a cardinal feature of refractory anemia with RS (RARS) and RARS with marked thrombocytosis (RARS/-T). Mutations in SF3B1, a member of the RNA splicing machinery are frequent in RARS/-T and defects of this gene were linked to RS formation. Here we showcase the differences in iron architecture of SF3B1-mutant and wild-type (WT) RARS/-T and provide new mechanistic insights by which SF3B1 mutations lead to differences in iron. We found higher iron levels in SF3B1 mutant vs WT RARS/-T by transmission electron microscopy/spectroscopy/flow cytometry. SF3B1 mutations led to increased iron without changing the valence as shown by the presence of Fe(2+) in mutant and WT. Reactive oxygen species and DNA damage were not increased in SF3B1-mutant patients. RNA-sequencing and Reverse transcriptase PCR showed higher expression of a specific isoform of SLC25A37 in SF3B1-mutant patients, a crucial importer of Fe(2+) into the mitochondria. Our studies suggest that SF3B1 mutations contribute to cellular iron overload in RARS/-T by deregulating SLC25A37.

To examine the roles of FGF and ERK MAPK signaling in osteocyte differentiation and function, we performed microarray analyses using the osteocyte cell line MLO-Y4. This experiment identified a number of mineralization-related genes that were regulated by FGF2 in an ERK MAPK dependent manner. Real-time PCR analysis indicated that FGF2 upregulates Ank, Enpp1, Mgp, Slc20a1, and Dmp1 in MLO-Y4 cells. Consistent with this observation, the selective FGF receptor inhibitor PD173074 decreased Ank, Enpp1, Slc20a1, and Dmp1 mRNA expression in mouse calvaria in organ culture. Since Dmp1 plays a central role in osteocyte differentiation and mineral homeostasis, we further analyzed FGF regulation of Dmp1. Similar to FGF2, FGF23 upregulated Dmp1 expression in MLO-Y4 cells in the presence of Klotho. Furthermore, increased extracellular phosphate levels partially inhibited FGF2-induced upregulation of Dmp1 mRNA expression, suggesting a coordinated regulation of Dmp1 expression by FGF signaling and extracellular phosphate. In MLO-Y4 osteocytes and in MC3T3E1 and primary calvaria osteoblasts, U0126 strongly inhibited both basal expression of Dmp1 mRNA and FGF2-induced upregulation. Consistent with the in vitro observations, real-time PCR and immunohistochemical analysis showed a strong decrease in Dmp1 expression in the skeletal elements of ERK1−/−; ERK2 flox/flox; Prx1-Cre mice. Furthermore, scanning electron microscopic analysis revealed that no osteocytes with characteristic dendritic processes develop in the limbs of ERK1−/−; ERK2 flox/flox; Prx1-Cre mice. Collectively, our observations indicate that FGF signaling coordinately regulates mineralization-related genes in the osteoblast lineage and that ERK signaling is essential for Dmp1 expression and osteocyte differentiation.

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In order to evaluate the applicability of different measurement parameters employed in the comet assay for analyzing environmental samples, fish hepatoma (RTH-149) cells were exposed to concentrations of the model genotoxic agent hydrogen peroxide (H(2)O(2); 1, 5, and 10 microM) and to five water samples from sites along the Kishon River, the most polluted river in Israel. DNA damage was scored in parallel by visual and computer-image (Viscomet) analyses using 12 different parameters. Each parameter exhibited a different profile of responses. The four visual parameters were highly sensitive to the lowest (1 microM) H(2)O(2) concentration (1.8-7.0-fold of the control). At 10 microM H(2)O(2) exposure, the visual parameter, percentage severe damage, showed the highest (40.3-fold) response while four other parameters, tail area, tail extent moment (Viscomet), mean actual tail length and cumulative tail length (visual analysis), also had substantially elevated responses (8-11-fold). We found that the DNA damage induced by field samples was similar in magnitude to the damage induced by 1 microM H(2)O(2), with only some of the parameters being highly sensitive to the damage. Only about one-half of the parameters could distinguish four significant levels of genotoxicity among the five sampling sites, while the remaining parameters detected only three levels. It is concluded that the choice of parameters for analyzing genotoxicity in ecotoxicological studies should be made in accordance with the characteristics of each parameter.

-2 ) and subjected to the comet assay. UVB radiation, at levels that induced a moderate DNA breakage to the non-symbiotic coral and algal cell compartments, caused dramatic increase in DNA breakage to the holobiont entities. After a 1·h repair period, DNA breakage levels in the algal and animal cell fractions were augmented as compared with a reduction in DNA breakage in the holobiont fraction. This discordancy in DNA breakage between the three cellular compartments reveals that the holobiont cell fraction is more vulnerable to increased natural UV irradiation and associated anthropogenic genotoxic impacts, providing another possible explanation for recent increase in worldwide coral bleaching events.