IκB Kinase β Regulates Epithelium Migration during Corneal Wound Healing

The IKKβ is known to regulate transcription factor NF-κB activation leading to inflammatory responses. Recent gene knockout studies have shown that IKKβ can orchestrate local inflammatory responses and regulate homeostasis of epithelial tissues. To investigate whether IKKβ has an intrinsic role in epithelial cells, we established an in vivo system in the immune privileged corneal epithelium. We generated triple transgenic Krt12rtTA/rtTAt/tet-O-Cre/IkkβF/F (IkkβΔCE/ΔCE) mice by crossing the Krt12-rtTA knock-in mice, which express the reverse tetracycline transcription activator in corneal epithelial cells, with the tet-O-Cre and IkkβF/F mice. Doxcycline-induced IKKβ ablation occurred in corneal epithelial cells of triple transgenic IkkβΔCE/ΔCE mice, but loss of IKKβ did not cause ocular abnormalities in fetal development and postnatal maintenance. Instead, loss of IKKβ significantly delayed healing of corneal epithelial debridement without affecting cell proliferation, apoptosis or macrophage infiltration. In vitro studies with human corneal epithelial cells (HCEpi) also showed that IKKβ was required for cytokine-induced cell migration and wound closure but was dispensable for cell proliferation. In both in vivo and in vitro settings, IKKβ was required for optimal activation of NF-κB and p38 signaling in corneal epithelial cells, and p38 activation is likely mediated through formation of an IKKβ-p38 protein complex. Thus, our studies in corneal epithelium reveal a previously un-recognized role for IKKβ in the control of epithelial cell motility and wound healing

Dynamic Sulfur Tolerant Process and System with Inline Acid Gas-Selective Removal for Generating Hydrogen for Fuel Cells

This invention relates to a sulfur tolerant, dynamic, compact, lightweight fuel process and system that is capable of converting sulfur bearing carbonaceous fuels to hydrogen rich gases suitable for fuel cells or chemical processing applications. The process and system is based on the AHR and WGS reactions, followed by cleanup of byproduct sulfur-containing gases and carbon oxides that would otherwise poison the fuel cell electrocatalyst. Advantageously, this is accomplished via an ASMS and a methanator or an AWMR. The process and system preferably uses a special sulfur tolerant catalysts and hardware designs that enable the conversion in an energy efficient manner while maintaining desirable performance characteristics such as rapid start-stop and fast response to load change capabilities

Role of SH2-containing tyrosine phosphatase Shp2 in mouse corneal epithelial stratification.

PURPOSE: Shp2 protein tyrosine phosphatase mediates a wide variety of receptor tyrosine kinases (RTK) cell signaling. Herein, we investigate the role of Shp2 in corneal morphogenesis and homeostasis. METHODS: Shp2 was conditionally knocked out (Shp2(cko)) in Krt14-rtTA;tet-O-Cre;Shp2(f/f) triple transgenic mice administrated with doxycycline (Dox) from postnatal day 1 (P1) to P10, P15, and P25, respectively. In addition, corneal epithelial debridement was performed in adult (P42) mice treated with or without Dox for 8 days (from P42-P50). Mouse eyes were then subjected to histology and immunohistochemistry. RESULTS: Shp2(cko) revealed impaired stratification of conjunctival and corneal epithelia during morphogenesis. Likewise, Shp2(cko) failed to restore epithelial stratification after a corneal epithelial wound in adult Shp2(cko). At the cellular level, the ratio of proliferating cell nuclear antigen (PCNA-positive)/total basal cells remained unchanged, but cells in G2/M (survivin-positive) phase was significantly increased in Shp2(cko) as compared with those in the control littermate. Interestingly, deltaN-p63 (ΔNp63) expression and the asymmetric division of the basal cells were coincidentally dampened in Shp2(cko). Transmission electron microscopic study showed that desmosome and hemidesmosome densities were reduced in the corneal epithelium of Shp2(cko). Immunohistochemistry also demonstrated that expression of E-cadherin/β-catenin junction and laminin-β1 was extensively downregulated in Shp2(cko). On the other hand, corneal epithelium lacking Shp2 remained positive for K14, Pax-6, and keratin 12 (K12), suggesting that Shp2 was dispensable for the corneal epithelial-type differentiation. CONCLUSIONS: These data argued that Shp2 deficiency predominantly impacted p63-dependent cell division and cell adhesive ability, which resulted in the impairment of stratification during corneal epithelial development and wound healing

Lumican Peptides: Rational Design Targeting ALK5/TGFBRI

Lumican, a small leucine rich proteoglycan (SLRP), is a component of extracellular matrix which also functions as a matrikine regulating multiple cell activities. In the cornea, lumican maintains corneal transparency by regulating collagen fibrillogenesis, promoting corneal epithelial wound healing, regulating gene expression and maintaining corneal homeostasis. We have recently shown that a peptide designed from the 13 C-terminal amino acids of lumican (LumC13) binds to ALK5/TGFBR1 (type1 receptor of TGF beta) to promote wound healing. Herein we evaluate the mechanism by which this synthetic C-terminal amphiphilic peptide (LumC13), binds to ALK5. These studies clearly reveal that LumC13-ALK5 form a stable complex. In order to determine the minimal amino acids required for the formation of a stable lumican/ALK5 complex derivatives of LumC13 were designed and their binding to ALK5 investigated in silico. These LumC13 derivatives were tested both in vitro and in vivo to evaluate their ability to promote corneal epithelial cell migration and corneal wound healing, respectively. These validations add to the therapeutic value of LumC13 (Lumikine) and aid its clinical relevance of promoting the healing of corneal epithelium debridement. Moreover, our data validates the efficacy of our computational approach to design active peptides based on interactions of receptor and chemokine/ligand.NIH/NEI grantsResearch to Prevent BlindnessOhio Eye Research FoundationUniv Cincinnati, Dept Ophthalmol, Cincinnati, OH 45267 USAUniv Fed Sao Paulo, Dept Bioquim, Sao Paulo, BrazilUniv Houston, Coll Optometry, Ocular Surface Inst, Houston, TX 77204 USAUniv Fed Sao Paulo, Dept Bioquim, Sao Paulo, BrazilNIH/NEI grants: RO1 EY011845NIH/NEI grants: R01 021768Web of Scienc

The development of meibomian glands in mice

PurposeThe purpose of this study was to characterize the natural history of meibomian gland morphogenesis in the mouse.MethodsEmbryonic (E) and post natal (P) C57Bl/6 mouse pups were obtained at E18.5, P0, P1, P3, P5, P8, P15, and P60. Eyelids were fixed and processed for en bloc staining with Phalloidin/DAPI to identify gland morphogenesis, or frozen for immunohistochemistry staining with Oil red O (ORO) to identify lipid and antibodies specific against peroxisome proliferator-activated receptor gamma (PPARγ) to identify meibocyte differentiation. Samples were then evaluated using a Zeiss 510 Meta laser scanning confocal microscope or Nikon epi-fluorescent microscope. Tissues from adult mice (2 month-old) were also collected for western blotting.ResultsMeibomian gland morphogenesis was first detected at E18.5 with the formation of an epithelial placode within the fused eyelid margin. Invagination of the epithelium into the eyelid was detected at P0. From P1 to P3 there was continued extension of the epithelium into the eyelid. ORO and PPARγ staining was first detected at P3, localized to the central core of the epithelial cord thus forming the presumptive ductal lumen. Ductal branching was first detected at P5 associated with acinar differentiation identified by ORO and PPARγ staining. Adult meibomian glands were observed by P15. Western blotting of meibomian gland proteins identified a 50 kDa and a 72 kDa band that stained with antibodies specific to PPARγ.ConclusionsWe have demonstrated that meibomian gland development bears distinct similarities to hair development with the formation of an epithelial placode and expression of PPARγ co-incident with lipid synthesis and meibocyte differentiation

Dopamine Gene Profiling to Predict Impulse Control and Effects of Dopamine Agonist Ropinirole

Dopamine agonists can impair inhibitory control and cause impulse control disorders for those with Parkinson disease (PD), although mechanistically this is not well understood. In this study, we hypothesized that the extent of such drug effects on impulse control is related to specific dopamine gene polymorphisms. This double-blind, placebo-controlled study aimed to examine the effect of single doses of 0.5 and 1.0 mg of the dopamine agonist ropinirole on impulse control in healthy adults of typical age for PD onset. Impulse control was measured by stop signal RT on a response inhibition task and by an index of impulsive decision-making on the Balloon Analogue Risk Task. A dopamine genetic risk score quantified basal dopamine neurotransmission from the influence of five genes: catechol-O-methyltransferase, dopamine transporter, and those encoding receptors D1, D2, and D3. With placebo, impulse control was better for the high versus low genetic risk score groups. Ropinirole modulated impulse control in a manner dependent on genetic risk score. For the lower score group, both doses improved response inhibition (decreased stop signal RT) whereas the lower dose reduced impulsiveness in decision-making. Conversely, the higher score group showed a trend for worsened response inhibition on the lower dose whereas both doses increased impulsiveness in decision-making. The implications of the present findings are that genotyping can be used to predict impulse control and whether it will improve or worsen with the administration of dopamine agonists