Characterization of Recombinant Human PRG4 as an Ocular Surface Boundary Lubricant

Introduction: Dry-eye disease involves tear film instability that can result in surface-to-surface contact between the cornea and eyelid or contact lens, where boundary lubrication can be dominant1. Motivated by the recent discovery that proteoglycan 4 (PRG4, a mucin-like glycoprotein originally discovered in synovial fluid as a boundary lubricant2), functions as an ocular surface boundary lubricant3, advances in recombinant protein expression technology4, and PRG4’s potential use as a friction-reducing contact lens coating, the objectives of this study were to: 1) biochemically characterize recombinant human PRG4 (rh- PRG4); and 2) assess the boundary lubricating properties of rh-PRG4, both before and after autoclave sterilization, at a cornea-contact lens material (PDMS) biointerface. Methods: SDS-PAGE western blot analysis using a variety of anti-PRG4 antibodies and lectins was performed on native PRG4 (nPRG4) and rh-PRG4 samples, both nonreduced and reduced, with and without enzymatic removal of O-linked glycosylations. Human corneas and PDMS were articulated against each other, subject to physiological loads of 8-25 kPa, at effective sliding velocities of 0.3-30 mm/s. Test lubricant sequences were A) saline, rh-PRG4 @300μg/mL, nPRG4 @300μg/mL, and saline; and B) saline, autoclaved rh-PRG4 @300μg/mL, rh-PRG4 @300μg/mL, and saline. Static and kinetic coefficients of friction were calculated. Results: rh-PRG4 demonstrated similar immunoreactivity to nPRG4, and effectively lowered friction at the cornea-PDMS biointerface. Western blotting indicated immunoreactive rh-PRG4 bands had a similar apparent molecular weight (MW) to nPRG4, and decreased appropriately upon reduction as well as enzymatic removal of glycosylations. Kinetic friction coefficients, which were highest in saline (0.31±0.06 to 0.40±0.06, mean±SEM), were similar in rh-PRG4 (0.12±0.01 to 0.25±0.03) and nPRG4 (0.19±0.02 to 0.28±0.03) across all velocities. Autoclaved rh-PRG4 had similar values to rh-PRG4 as well (0.19±0.02 to 0.26±0.04, 0.16±0.02 to 0.26±0.02, respectively). Conclusions: rh-PRG4 demonstrates similar biochemical and ocular surface lubricating properties to nPRG4, and may function as an effective friction-reducing contact lens coating

The role of cell-cell adhesion in wound healing

We present a stochastic model which describes fronts of cells invading a wound. In the model cells can move, proliferate, and experience cell-cell adhesion. We find several qualitatively different regimes of front motion and analyze the transitions between them. Above a critical value of adhesion and for small proliferation large isolated clusters are formed ahead of the front. This is mapped onto the well-known ferromagnetic phase transition in the Ising model. For large adhesion, and larger proliferation the clusters become connected (at some fixed time). For adhesion below the critical value the results are similar to our previous work which neglected adhesion. The results are compared with experiments, and possible directions of future work are proposed.Comment: to appear in Journal of Statistical Physic

Mineralocorticoid receptors are indispensable for nongenomic modulation of hippocampal glutamate transmission by corticosterone

The adrenal hormone corticosterone transcriptionally regulates responsive genes in the rodent hippocampus through nuclear mineralocorticoid and glucocorticoid receptors. Via this genomic pathway the hormone alters properties of hippocampal cells slowly and for a prolonged period. Here we report that corticosterone also rapidly and reversibly changes hippocampal signaling. Stress levels of the hormone enhance the frequency of miniature excitatory postsynaptic potentials in CA1 pyramidal neurons and reduce paired-pulse facilitation, pointing to a hormone-dependent enhancement of glutamate-release probability. The rapid effect by corticosterone is accomplished through a nongenomic pathway involving membrane-located receptors. Unexpectedly, the rapid effect critically depends on the classical mineralocorticoid receptor, as evidenced by the effectiveness of agonists, antagonists, and brain-specific inactivation of the mineralocorticoid but not the glucocorticoid receptor gene. Rapid actions by corticosterone would allow the brain to change its function within minutes after stress-induced elevations of corticosteroid levels, in addition to responding later through gene-mediated signaling pathways

The role of ATP and adenosine in the brain under normoxic and ischemic conditions

By taking advantage of some recently synthesized compounds that are able to block ecto-ATPase activity, we demonstrated that adenosine triphosphate (ATP) in the hippocampus exerts an inhibitory action independent of its degradation to adenosine. In addition, tonic activation of P2 receptors contributes to the normally recorded excitatory neurotransmission. The role of P2 receptors becomes critical during ischemia when extracellular ATP concentrations increase. Under such conditions, P2 antagonism is protective. Although ATP exerts a detrimental role under ischemia, it also exerts a trophic role in terms of cell division and differentiation. We recently reported that ATP is spontaneously released from human mesenchymal stem cells (hMSCs) in culture. Moreover, it decreases hMSC proliferation rate at early stages of culture. Increased hMSC differentiation could account for an ATP-induced decrease in cell proliferation. ATP as a homeostatic regulator might exert a different effect on cell trophism according to the rate of its efflux and receptor expression during the cell life cycle. During ischemia, adenosine formed by intracellular ATP escapes from cells through the equilibrative transporter. The protective role of adenosine A1 receptors during ischemia is well accepted. However, the use of selective A1 agonists is hampered by unwanted peripheral effects, thus attention has been focused on A2A and A3 receptors. The protective effects of A2A antagonists in brain ischemia may be largely due to reduced glutamate outflow from neurones and glial cells. Reduced activation of p38 mitogen-activated protein kinases that are involved in neuronal death through transcriptional mechanisms may also contribute to protection by A2A antagonism. Evidence that A3 receptor antagonism may be protective after ischemia is also reported

Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci

Background: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). Results: We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an `autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. Conclusions: 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi

FGF4 Independent Derivation of Trophoblast Stem Cells from the Common Vole

The derivation of stable multipotent trophoblast stem (TS) cell lines from preimplantation, and early postimplantation mouse embryos has been reported previously. FGF4, and its receptor FGFR2, have been identified as embryonic signaling factors responsible for the maintenance of the undifferentiated state of multipotent TS cells. Here we report the derivation of stable TS-like cell lines from the vole M. rossiaemeridionalis, in the absence of FGF4 and heparin. Vole TS-like cells are similar to murine TS cells with respect to their morphology, transcription factor gene expression and differentiation in vitro into derivatives of the trophectoderm lineage, and with respect to their ability to invade and erode host tissues, forming haemorrhagic tumours after subcutaneous injection into nude mice. Moreover, vole TS-like cells carry an inactive paternal X chromosome, indicating that they have undergone imprinted X inactivation, which is characteristic of the trophoblast lineage. Our results indicate that an alternative signaling pathway may be responsible for the establishment and stable proliferation of vole TS-like cells