Macrophages are distributed throughout the body and are crucial for the restoration of damaged tissues. However, their characteristics in the cornea and roles in the repair of corneal injures are unclear. Here we show that corneal macrophages can be classified as CCR2- macrophages, which already exist in the cornea at embryonic day 12.5 (E12.5) and are similar to yolk sac-derived macrophages, microglia, in phenotype and gene expression, and CCR2+ macrophages, which do not appear in the cornea until E17.5. At a steady state, CCR2- corneal macrophages have local proliferation capacity and are rarely affected by monocytes; however, following corneal epithelial abrasion, most CCR2- corneal macrophages are replaced by monocytes. In contrast, CCR2+ macrophages are repopulated by monocytes under both a steady-state condition and following corneal wounding. Depletion of CCR2+ macrophages decreases corneal inflammation after epithelial abrasion, whereas depletion of CCR2- macrophages increases inflammation of the injured cornea. Loss of either cell type results in a delay in corneal healing. These data indicate that there are two unique macrophage populations present in the cornea, both of which participate in corneal wound healing by balancing the inflammatory response.

Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20-30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue.

Despite its promise as a highly useful therapy for pancreatic cancer (PC), the addition of external beam radiation therapy to PC treatment has shown varying success in clinical trials. Understanding PC radioresistance and discovery of methods to sensitise PC to radiation will increase patient survival and improve quality of life. In this study, we identified PC radioresistance-associated pathways using global, unbiased techniques.

Occupational and environmental Pb-exposure is associated with protein aggregation diseases which typically present in elderly populations (Parkinsons and cataract). Post-translational processing of crystallins, the major structural proteins of the lens, is altered with short-term Pb-exposure in Fisher 344 rats. In addition, lenses from aged rats become opaque upon long-term exposure to Pb in organ culture. To explore the route to lens opacification in the presence of Pb, cultured lenses from young rats which exhibit higher metabolic activity in lens culture and are more susceptible to experimental cataract in vivo and in vitro were exposed to Pb and evaluated for morphological and biochemical alterations.

Using the structural equation modeling (SEM) method, the present study examined the role of large-scale neural interactions in developmental stuttering while 10 stuttering and nine non-stuttering subjects performed a covert picture-naming task. Results indicated that the connection patterns were significantly different between stuttering and non-stuttering speakers in both omnibus connection pattern and individual connection path coefficient. Specifically, stuttering speakers showed functional disconnection from the left inferior frontal gyrus to the left motor areas, and altered connectivity in the basal ganglia-thalamic-cortical circuit, and abnormal integration of supramodal information across the cerebellum and several frontal-parietal regions. These results indicate that the large-scale dysfunctional neural interactions may be involved in stuttering speakers' difficulties in planning, execution, and self-monitoring of speech motor sequence during word production.

Visceral hypersensitivity in irritable bowel syndrome (IBS) is still poorly understood, despite that chronic abdominal pain is the most common symptoms in IBS patients. To study effects of BK channels on visceral hypersensitivity in IBS rats and the underlying mechanisms, IBS rats were established by colorectal distention (CRD) in postnatal rats. The expression of large-conductance calcium and voltage-dependent potassium ion channels (BK channels) of the thoracolumbar spinal cord was examined in IBS and control rats. The effects of BK channel blockade on visceral hypersensitivity were evaluated. The interaction of BK channels and N-methyl-D-aspartate acid (NMDA) receptors was explored, and synaptic transmission at superficial dorsal horn (SDH) neurons of the thoracolumbar spinal cord was recorded by whole-cell patch clamp in IBS rats.

Desmogleins are desmosomal cadherins that mediate cell-cell adhesion. In stratified squamous epithelia there are two major isoforms of desmoglein, 1 and 3, with different distributions in epidermis and mucous membrane. Since either desmoglein isoform alone can mediate adhesion, the reason for their differential distribution is not known. To address this issue, we engineered transgenic mice with desmoglein 3 under the control of the involucrin promoter. These mice expressed desmoglein 3 with the same distribution in epidermis as found in normal oral mucous membranes, while expression of other major differentiation molecules was unchanged. Although the nucleated epidermis appeared normal, the epidermal stratum corneum was abnormal with gross scaling, and a lamellar histology resembling that of normal mucous membrane. The mice died shortly after birth with severe dehydration, suggesting excessive transepidermal water loss, which was confirmed by in vitro and in vivo measurement. Ultrastructure of the stratum corneum showed premature loss of cohesion of corneocytes. This dysadhesion of corneocytes and its contribution to increased transepidermal water loss was confirmed by tape stripping. These data demonstrate that differential expression of desmoglein isoforms affects the major function of epidermis, the permeability barrier, by altering the structure of the stratum corneum.

Our previous studies showed that bovine respiratory syncytial virus (BRSV) followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2) epithelial cells with H. somni concentrated culture supernatant (CCS) stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2--RSAD2) and ISG15 (IFN-stimulated gene 15--ubiquitin-like modifier) were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT) upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide) but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt) does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo.

Hemoglobin (Hb) is a major constituent of blood and a potent mediator of oxidative or nitrative stress after intracerebral hemorrhage (ICH). Our previous study demonstrated that Hb could induce abundant peroxynitrite (ONOO(-)) formation in vivo, which may be involved in the blood-brain barrier (BBB) disruption, however, the drug intervention is absent and also the underlying mechanism. Using an experimental stroke model by injecting Hb into the caudate nucleus of male Sprague-Dawley rats, we assessed the role of ONOO(-) decomposition catalyst, 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron(III) [FeTPPS] in the activation of MMP-9 and Hb-induced neurovascular injuries. 3-Nitrotyrosine (3-NT, as an index of ONOO(-) formation) and NF-κB expression was measured by western blot (WB) and immunohistochemistry (IHC)/immunofluorescence (IF). Activity of MMP was evaluated by in situ zymography. Neurovascular injury was assessed using zonula occludens-1 (ZO-1) by WB and IF, fibronectin (FN) and neuron-specific nuclear protein (NeuN) IHC. Perihematomal cell death was determined by TUNEL assay. Behavioral outcome was measured by modified neurological severity score (mNSS) test. At the injured striata, profuse 3-NT was produced and mainly expressed in neutrophils and microglia/macrophages. 3-NT formation significantly colocalized with nuclear factor-κB (NF-κB) expression. In situ zymography showed that gelatinase activity was mostly co-localized with neurons and blood vessel walls and partly with neutrophils and microglia/macrophages. Enhanced 3-NT production, NF-κB induction and MMP-9 activation were obviously reduced after FeTPPS treatment. Hb-induced injury to tight junction protein (ZO-1), basal lamina of FN-immunopositive microvasculature and neural cells was evidently ameliorated by FeTPPS. In addition, apoptotic cell numbers as well as behavioral deficits were also improved. The present study shows that the administration of the ONOO(-) decomposition catalyst FeTPPS protects against Hb-induced neurovascular injuries and improves neurological function, which possibly in part by suppressing MMP-9 activation.

Neural stem cells in the subventricular zone (SVZ) generate progenitors which in turn give rise to neuroblasts. These neuroblasts then migrate along the rostral migratory stream (RMS) in chains and reach the olfactory bulb (OB), where they mature into local interneurons. Interneurons in the OB are heterogeneous, which are mainly located in granular cell layer (GCL), external plexiform layer (EPL) and glomerular layer (GL). In this study, we show that green fluorescent protein-expressing (GFP+) cells in the SVZ and RMS of the 5HT3aR-BAC(EGFP) transgenic mouse exclusively express transcription factor Sp8 which is expressed in lateral ganglionic embryonic eminence (LGE) and postnatal SVZ. These GFP+ neuroblasts of the 5HT3aR-BAC(EGFP) transgenic mouse migrate along the RMS to the OB where they differentiate into calretinin+ (CR+), parvabumin+ (PV+), vasoactive intestinal peptide+ (VIP+), somatostatin+ (Som+) and tyrosine hydroxylase+ (TH+), but not calbindin+ (CB+) interneurons. These GFP+ interneurons continuously express Sp8 in the OB. Furthermore, these results suggest that GFP-expressing cells are derived from LGE, and this transgenic mouse line will be a useful tool for studying the development and function of interneurons in both neocortex and OB.

The nuclear p68 RNA helicase is a prototypical member of the DEAD-box family of RNA helicases. p68 RNA helicase has been implicated in cell proliferation and early organ development and maturation. However, the functional role of p68 RNA helicase in these biological processes at the molecular level is not well understood. We previously reported that tyrosine phosphorylation of p68 RNA helicase mediates the effects of platelet-derived growth factor (PDGF) in induction of epithelial mesenchymal transition by promoting β-catenin nuclear translocation. Here, we report that phosphorylation of p68 RNA helicase at Y593 upregulates transcription of the Snail1 gene. The phosphorylated p68 activates transcription of the Snail1 gene by promoting histone deacetylase (HDAC)1 dissociation from the Snail1 promoter. Our results showed that p68 interacted with the nuclear remodeling and deacetylation complex MBD3:Mi-2/NuRD. Thus, our data suggested that a DEAD-box RNA unwindase could potentially regulate gene expression by functioning as a protein 'displacer' to modulate protein-protein interactions at the chromatin-remodeling complex.