Characterization of UHRF1 expression in human induced pluripotent stem cells and in UHRF1 conditional knockout models

Thesis (M.A.)--Boston UniversityUHRFl (ubiquitin-like protein, containing PHD and RING finger domains 1) is essential in epigenetic modification and cell cycle progression. In vivo knockdown of UHRF1 is lethal and embryogenesis does not progress to produce viable knockout models. In this study, Cre recombinase adenovirus is used to generate an in vitro knockdown of UHRF1 in Mouse Embryo Fibroblasts. The study also proposes the use of Albumin-Cre mouse model and Cre-lox technology to make conditional UHRF1 knockdown in the liver. These models will prove to be essential in further experiments to understand the precise role of UHRF1 and its homologs. Unlike in non-mammals, a number of UHRF1 homologs have been identified in mammals but their exact function is disputed. This study measures the mRNA expression of UHRF1 and UHRF2 in human induced pluripotent stem cells as they differentiate into hepatocytes and show that mRNA expression of UHRF2 is significantly higher in differentiated cells as compared to the expression of UHRF1. There have been studies that show UHRF 1 expression at various cell cycle phases, but there is little known about the regulation of UHRF1 itself. To further understand the relation between DNA methylation and UHRFl expression, this study shows that hypomethylation of DNA with methylation inhibitor, 5-Azacytidine, induces an increase in UHRF1 expression, suggesting that UHRF1 not only regulates the gene expression by DNA methylation but may also play a role in self-regulating its own expression

Targeted Deletion of Fibrinogen Like Protein 1 Reveals a Novel Role in Energy Substrate Utilization

Fibrinogen like protein 1(Fgl1) is a secreted protein with mitogenic activity on primary hepatocytes. Fgl1 is expressed in the liver and its expression is enhanced following acute liver injury. In animals with acute liver failure, administration of recombinant Fgl1 results in decreased mortality supporting the notion that Fgl1 stimulates hepatocyte proliferation and/or protects hepatocytes from injury. However, because Fgl1 is secreted and detected in the plasma, it is possible that the role of Fgl1 extends far beyond its effect on hepatocytes. In this study, we show that Fgl1 is additionally expressed in brown adipose tissue. We find that signals elaborated following liver injury also enhance the expression of Fgl1 in brown adipose tissue suggesting that there is a cross talk between the injured liver and adipose tissues. To identify extra hepatic effects, we generated Fgl1 deficient mice. These mice exhibit a phenotype suggestive of a global metabolic defect: Fgl1 null mice are heavier than wild type mates, have abnormal plasma lipid profiles, fasting hyperglycemia with enhanced gluconeogenesis and exhibit differences in white and brown adipose tissue morphology when compared to wild types. Because Fgl1 shares structural similarity to Angiopoietin like factors 2, 3, 4 and 6 which regulate lipid metabolism and energy utilization, we postulate that Fgl1 is a member of an emerging group of proteins with key roles in metabolism and liver regeneration

Tuning structural durability of yeast-encapsulating alginate gel beads with interpenetrating networks for sustained bioethanol production

Microorganisms have become key components in many biotechnological processes to produce various chemicals and biofuels. The encapsulation of microbial cells in calcium cross-linked alginate gel beads has been extensively studied due to several advantages over using free cells. However, industrial use of alginate gel beads has been hampered by the low structural stability of the beads. In this study, we demonstrate that the incorporation of interpenetrating covalent cross-links in an ionically cross-linked alginate gel bead significantly enhances the bead's structural durability. The interpenetrating network (IPN) was prepared by first cross-linking alginate chemically modified with methacrylic groups, termed methacrylic alginate (MA), with calcium ions and subsequently conducting a photo cross-linking reaction. The resulting methacrylic alginate gel beads (IPN-MA) exhibited higher stiffness, ultimate strength and ultimate strain and also remained more stable in media either subjected to high shear or supplemented with chelating agents than calcium cross-linked alginate gel beads. Furthermore, yeast cells encapsulated in IPN-MA gel beads remained more metabolically active in ethanol production than those in calcium cross-linked alginate gel beads. Overall, the results of this study will be highly useful in designing encapsulation devices with improved structural durability for a broad array of prokaryotic and eukaryotic cells used in biochemical and industrial processes.close4

Length and tissue mass of <i>Fgl1^+/+</i> and <i>Fgl1^−/−</i> mice.

<p>(n = 5 for both cohorts except for liver (*) where n = 9).</p

Food intake and indirect calorimetry.

<p>Scatter plot of average food intake versus average weight for individually housed <i>Fgl1^+/+</i> (blue squares, n = 4) and <i>Fgl1^−/−</i>(red circles, n = 8) taken daily over an 18 day period. Note that <i>Fgl1^−/−</i> remain larger that wild types for the duration of the experiment. B and C) Indirect calorimetric values for VO₂ and VCO₂ respectively and D) RER. The RER is significant irrespective of day and night cycles (P = 0.04 and 0.016 respectively) and over the entire 24 h (P = 0.019). E) Heat generation is not significantly different and F) activity is not different between the Fgl1 containing and deficient mice. n = 6 for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058084#pone-0058084-g006" target="_blank">Figures 6B to 6F</a>.</p

Structure, content and activity of adipose tissues in the Fgl1 null mouse.

<p>A) Representative H&E stains (40× magnification) of brown adipose tissue. Lipid droplets are larger in <i>Fgl1^−/−</i> mice. B) Quantitation of lipid droplet size show significant difference between <i>Fgl1^+/+</i> and <i>Fgl1^−/−</i> mice (P = 0.011, n = 5 per group). C) Expression of brown adipose genes. Note the paradoxical up regulation of DiO2 and UCP1 (P = 0.002 and 0.0001 respectively. n = 11 per group except for Perilipin and HSL where n = 5 and 6 respectively). D) ¹⁸FDG incorporation into BAT. The % uptake represents the uptake of injected dose per gram of tissue. Note the marked decrease in radioisotope uptake in BAT (P = 0.05, n = 5 per group). E). Representative H&E stains (40× magnification) of white adipose tissue. Lipid droplets are larger in <i>Fgl1^−/−</i> mice. F) Quantitation of number of cells per HPF shows smaller number of white adipose cells in <i>Fgl1^−/−</i> (P = 0.005, n = 5). G) Expression of white adipose genes. Glut4, leptin and perilipin are significantly down regulated (*) with a P<0.04 for each. P for ATGL (#) is 0.06. n = 4–6 mice per group.</p

The liver in the Fgl1 knockout mouse.

<p>A) Fgl1 transcript is absent in the livers of the knockout mouse pre and post PH. Note the expected induction of Fgl1 in the wild type mouse after PH (P<0.01 for Fgl1 at baseline and 48 h after PH, n = 3 per group) B) Fgl1 protein is absent in the livers of the knockout mouse before and after PH while it is detectable at baseline and induced after PH in the wild type mouse. C) <i>Fgl1^−/−</i> mice are larger than wild type mates as early as three weeks after birth (P<0.0001, n = 5 for each group). D) Representative graph of change in weight over time for <i>Fgl1^+/+</i> (n = 8 for first 4 weeks, n = 4 for last three weeks) and <i>Fgl1^−/−</i> (n = 9 for first 4 weeks and n = 8 for last three weeks). E) Liver mass is not different between <i>Fgl1^+/+</i> and <i>Fgl1^−/−</i> mice (n = 9 for each group). F) Liver weight to body weight ratio is smaller for <i>Fgl1^−/−</i> mice (P = 0.008, n = 9). G) Marked lipid accumulation in the livers of <i>Fgl1^−/−</i> mice after PH. Top: gross images of representative livers from <i>Fgl1^+/+</i> (left) and <i>Fgl1^−/−</i> (right). Arrows indicate remnant liver lobes. Middle: H&E images at 40× magnification of liver sections from <i>Fgl1^+/+</i> and <i>Fgl1^−/−</i> mice at 48 h post PH and bottom: similar H&E images at 96 h post PH. Note the resolution of steatosis in the <i>Fgl1^−/−</i> mouse by 96 h after PH. H) Triglyceride (TG) content of liver extracts from <i>Fgl1^+/+</i> and <i>Fgl1^−/−</i> mice before and after PH. The difference between <i>Fgl1^−/−</i> and <i>Fgl1^+/+</i> at 48 h after PH is significant (P = 0.014, n = 3–5 per cohort for experiments). I) mRNA levels of lipid regulatory genes at 48 h after PH (P = 0.011, 0.014 and 0.037 respectively for PPARα, PPARδ and FATP5 but is otherwise non significant). n = 4 for <i>Fgl1^+/+</i> except FATP5 where n = 3 per group. n = 5 for <i>Fgl1^−/−</i>.</p