Faculty Bibliography

For future application of induced pluripotent stem cell (iPSC) technology, the ability to assess the overall quality of iPSC clones will be an important issue. Here we show that the histone variant H2A.X is a functional marker that can distinguish the developmental potentials of mouse iPSC lines. We found that H2A.X is specifically targeted to and negatively regulates extraembryonic lineage gene expression in embryonic stem cells (ESCs) and prevents trophectoderm lineage differentiation. ESC-specific H2A.X deposition patterns are faithfully recapitulated in iPSCs that support the development of "all-iPS" animals via tetraploid complementation, the most stringent test available of iPSC quality. In contrast, iPSCs that fail to support all-iPS embryonic development show aberrant H2A.X deposition, upregulation of extraembryonic lineage genes, and a predisposition to extraembryonic differentiation. Thus, our work has highlighted an epigenetic mechanism for maintaining cell lineage commitment in ESCs and iPSCs that can be used to distinguish the quality of iPSC lines.

Vanishing white matter disease (VWMD) is an inherited autosomal-recessive hypomyelinating disease caused by mutations in eukaryotic translation initiation factor 2B (eIF2B). eIF2B mutations predominantly affect the brain white matter, and the characteristic features of VWMD pathology include myelin loss and foamy oligodendrocytes. Activation of pancreatic endoplasmic reticulum kinase (PERK) has been observed in oligodendrocytes in VWMD. PERK activation in response to endoplasmic reticulum stress attenuates eIF2B activity by phosphorylating eIF2alpha, suggesting that impaired eIF2B activity in oligodendrocytes induced by VWMD mutations or PERK activation exploit similar mechanisms to promote selective white matter pathology in VWMD. Using transgenic mice that allow for temporally controlled activation of PERK specifically in oligodendrocytes, we discovered that strong PERK activation in oligodendrocytes during development suppressed eIF2B activity and reproduced the characteristic features of VWMD in mice, including hypomyelinating phenotype, foamy oligodendrocytes, and myelin loss. Notably, impaired eIF2B activity induced by PERK activation in oligodendrocytes of fully myelinated adult mice had minimal effects on morphology or function. Our observations point to a cell-autonomous role of impaired eIF2B activity in myelinating oligodendrocytes in the pathogenesis of VWMD.

Transforming growth factor-beta (TGFbeta) is a well-known master regulator of cellular proliferation and is a critical factor in the maintenance of tissue homeostasis. TGFbeta is classically defined as a tumor suppressor that functions in the early stages of carcinogenesis, yet paradoxically it functions as a tumor promoter in established cancers. Less well studied is its role in maintaining genomic stability through its participation in the DNA damage response (DDR). Deletion of Tgfb1 in murine epithelium increases genomic instability (GIN) as measured by gene amplification, aneuploidy, and centrosome aberrations; likewise, GIN is increased by depleting the TGFbeta ligand or inhibiting TGFbeta pathway signaling in human epithelial cells. Subsequent studies demonstrated that TGFbeta depletion compromises cell survival in response to radiation and impairs activation of the DDR because of severely reduced activity of ataxia telangiectasia mutated (ATM), a serine/threonine protein kinase that is rapidly activated by DNA double-strand breaks. The SMAD transcription factors are intermediaries in the crosstalk between the TGFbeta and ATM pathways in the DDR. Recent studies have shown that SMAD2 and SMAD7 participate in the DDR in a manner dependent on ATM or TGFbeta receptor type I, respectively, in human fibroblasts and epithelial cells. Understanding the role of TGFbeta in the DDR and suppressing GIN is important to understanding its seemingly paradoxical roles in tumorigenesis and thus has therapeutic implications for improving the response to DNA damage-inducing therapy.

There are various procedures for isolating microsomal fractions from tissue culture cells. The essential conditions for each step of one procedure are described here. Notes for special circumstances are included so that the procedure can be modified according to the experimental purpose.

When eukaryotic cells are homogenized, the rough endoplasmic reticula are converted into small vesicles, called rough microsomes. Strategies for the isolation of rough microsomes are introduced here, as are methods for evaluating the purity and intactness of an isolated rough microsomal fraction.

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Forster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases.

STRUCTURED ABSTRACT: Study Design. Animal modelObjective. Determine whether Amicar and TXA inhibit spine fusion volumeSummary of Background Data. Amicar and TXA are antifibrinolytics used to reduce perioperative bleeding. Prior in vitro data showed that antifibrinolytics reduce osteoblast bone mineralization. This study tested whether antifibrinolytics Amicar and TXA inhibit spine fusion.Methods. Posterolateral L4-L6 fusion was performed in fifty mice, randomized into groups of ten, that received the following treatment before and after surgery: (1) Saline; (2) TXA 100mg/kg; (3) TXA 1000mg/kg; (4) Amicar 100 mg/kg; (5) Amicar 1000 mg/kg. High-resolution plane radiography was performed after 5 weeks and micro-CT was performed at the end of the 12-week study. Radiographs were graded using the Lenke scale. Micro-CT was used to quantify fusion mass bone volume. One-way analysis of variance (ANOVA) by ranks with Kruskal-Wallis testing was used to compare the radiographic scores. One-way ANOVA with least-significant differences (LSD) post-hoc testing was used to compare the micro-CT bone volume.Results. The average (+/- SD) bone volume/total volume (%) measured in the saline, TXA 100 mg/kg, TXA 1000 mg/kg, Amicar 100 mg/kg and Amicar 1000 mg/kg groups were 10.8+/-2.3, 9.7+/-2.2, 13.4+/-3.2, 15.5+/-5.2 and 17.9+/-3.5%, respectively. There was a significant difference in the Amicar 100 mg/kg (p < 0.05) and Amicar 1000 mg/kg (p < 0.001) groups compared to saline. There was greater bone volume in the Amicar groups compared to the TXA group (p < 0.001). There was more bone volume in the TXA 1000 mg/kg group compared to TXA 100 mg/kg (p < 0.05) but the bone volume in neither of the TXA groups was different to saline (p = 0.49). There were no between-group differences observed using plane radiographic scoring.Conclusions. Amicar significantly enhanced the fusion bone mass in a dose-dependent manner while TXA did not have a significant effect on fusion compared to saline control.These data are in contrast to prior in vitro data that antifibrinolytics inhibit osteoblast bone mineralization.

Liver X receptors (LXRs) attenuate inflammation by modulating the expression of key inflammatory genes, making LXRs and their ligands particularly attractive candidates for therapeutic intervention in cardiovascular, metabolic, and/or inflammatory diseases. Herein, enhanced proresolving activity of polymeric nanoparticles (NPs) containing the synthetic LXR agonist GW3965 (LXR-NPs) is demonstrated, developed from a combinatorial library of more than 70 formulations with variations in critical physicochemical parameters. In vitro studies on peritoneal macrophages confirm that LXR-NPs are significantly more effective than the free agonist at downregulating pro-inflammatory mediators (MCP-1 and TNFalpha), as well as inducing the expression of LXR target genes (ABCA1 and SREBP1c). Through a zymosan-induced acute peritonitis in vivo model, LXR-NPs are found to be more efficient than free GW3965 at limiting the recruitment of polymononuclear neutrophils (50% vs 17%), suppressing the gene expression and secretion of pro-inflammatory factors MCP-1 and TNFalpha in peritoneal macrophages, and decreasing the resolution interval up to 4 h. Furthermore, LXR-NPs suppress the secretion of MCP-1 and TNFalpha by monocytes and macrophages more efficiently than the commercial drug dexamethasone. Overall, these findings demonstrate that LXR-NPs are capable of promoting resolution of inflammation and highlight the prospect of LXR-based nanotherapeutics for inflammatory diseases.

The principal function of an epithelium is to form a dynamic barrier that regulates movement between body compartments. Each epithelium is specialized with barrier functions that are specific for the tissues it serves. The apical surface commonly faces a lumen, but the retinal pigment epithelium (RPE) appears to be unique by a facing solid tissue, the sensory retina. Nonetheless, there exists a thin (subretinal) space that can become fluid filled during pathology. RPE separates the subretinal space from the blood supply of the outer retina, thereby forming the outer blood-retinal barrier. The intricate interaction between the RPE and sensory retina presents challenges for learning how accurately culture models reflect native behavior. The challenge is heightened by findings that detail the variation of RPE barrier proteins both among species and at different stages of the life cycle. Among the striking differences is the expression of claudin family members. Claudins are the tight junction proteins that regulate ion diffusion across the spaces that lie between the cells of a monolayer. Claudin expression by RPE varies with species and life-stage, which implies functional differences among commonly used animal models. Investigators have turned to transcriptomics to supplement functional studies when comparing native and cultured tissue. The most detailed studies of the outer blood-retinal barrier have focused on human RPE with transcriptome and functional studies reported for human fetal, adult, and stem-cell derived RPE.

Insulin and insulin-like growth factors (IGFs) are important regulators of growth and metabolism. In both vertebrates and invertebrates, insulin/IGFs are made available to various organs, including the brain, through two routes: the circulating systemic insulin/IGFs act on distant organs via endocrine signalling, whereas insulin/IGF ligands released by local tissues act in a paracrine or autocrine fashion. Although the mechanisms governing the secretion and action of systemic insulin/IGF have been the focus of extensive investigation, the significance of locally derived insulin/IGF has only more recently come to the fore. Local insulin/IGF signalling is particularly important for the development and homeostasis of the central nervous system, which is insulated from the systemic environment by the blood-brain barrier. Local insulin/IGF signalling from glial cells, the blood-brain barrier and the cerebrospinal fluid has emerged as a potent regulator of neurogenesis. This review will address the main sources of local insulin/IGF and how they affect neurogenesis during development. In addition, we describe how local insulin/IGF signalling couples neural stem cell proliferation with systemic energy state in Drosophila and in mammals.