Identification of DUOX1-dependent redox signaling through protein S-glutathionylation in airway epithelial cells

The NADPH oxidase homolog dual oxidase 1 (DUOX1) plays an important role in innate airway epithelial responses to infection or injury, but the precise molecular mechanisms are incompletely understood and the cellular redox-sensitive targets for DUOX1-derived H2O2 have not been identified. The aim of the present study was to survey the involvement of DUOX1 in cellular redox signaling by protein S-glutathionylation, a major mode of reversible redox signaling. Using human airway epithelial H292 cells and stable transfection with DUOX1-targeted shRNA as well as primary tracheal epithelial cells from either wild-type or DUOX1-deficient mice, DUOX1 was found to be critical in ATP-stimulated transient production of H2O2 and increased protein S-glutathionylation. Using cell pre-labeling with biotin-tagged GSH and analysis of avidin-purified proteins by global proteomics, 61 S-glutathionylated proteins were identified in ATP-stimulated cells compared to 19 in untreated cells. Based on a previously established role of DUOX1 in cell migration, various redox-sensitive proteins with established roles in cytoskeletal dynamics and/or cell migration were evaluated for S-glutathionylation, indicating a critical role for DUOX1 in ATP-stimulated S-glutathionylation of β-actin, peroxiredoxin 1, the non-receptor tyrosine kinase Src, and MAPK phosphatase 1. Overall, our studies demonstrate the importance of DUOX1 in epithelial redox signaling through reversible S-glutathionylation of a range of proteins, including proteins involved in cytoskeletal regulation and MAPK signaling pathways involved in cell migration. © 2014 The Authors

Calcium flashes orchestrate the wound inflammatory response through DUOX activation and hydrogen peroxide release

A crucial early wound response is the recruitment of inflammatory cells drawn by danger cues released by the damaged tissue. Hydrogen peroxide (H2O2) has recently been identified as the earliest wound attractant in Drosophila embryos and zebrafish larvae. The H2O2 signal is generated by activation of an NADPH oxidase, DUOX, and as a consequence, the first inflammatory cells are recruited to the wound within minutes. To date, nothing is known about how wounding activates DUOX. Here, we show that laser wounding of the Drosophila embryo epidermis triggers an instantaneous calcium flash, which travels as a wave via gap junctions several cell rows back from the wound edge. Blocking this calcium flash inhibits H2O2 release at the wound site and leads to a reduction in the number of immune cells migrating to the wound. We suggest that the wound-induced calcium flash activates DUOX via an EF hand calcium-binding motif and thus triggers the production of the attractant damage cue H2O2. Therefore, calcium represents the earliest signal in the wound inflammatory response

Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4(-/-)) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4(-/-) mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy

The anatomy, physiology, functional significance and evolution of specialized hearing organs of gerbilline rodents

Middle and inner ear anatomy correlates with neurophysiological responses to a wide range of sound frequencies for species of the Gerbillinae representing generalized, intermediate, and specialized anatomical conditions. Neurophysiological data were recorded from 81 specimens of 13 species representing six genera. Anatomical parameters involved in the process of hearing were correlated with the neurophysiological data to assess the effects of different degrees of anatomical specialization on hearing. The 13 species tested in this manner have graphic curves of auditory sensitivity of remarkably similar disposition over the frequencies tested and to those published for Kangaroo Rats. Ears with anatomical specializations show greater auditory sensitivity. The natural history of the Gerbillinae, particularly the kinds of predators, degree of predation, and habitat is reviewed and utilized to interpret the significance of the degree of auditory specialization in the forms studied and to evaluate the prevailing hypothesis that these specializations enhance the ability of these rodents to survive in open desert situations by detecting and evading predators. The middle ear anatomy of five additional genera and species was also studied. Thus, data on the entire spectrum of gerbilline middle ear morphology provide an evolutionary sequence. Certain anatomical parameters of the organ of Corti show a degree of specialization parallel to that of features of the middle ear. The morphological changes and possible functional roles of these features are considered. A very high correlation exists for degree of specialization and aridity of habitat, thus specialization increases with increasing aridity. This increased specialization may result from more effective predation in open xeric environments. Auditory acuity for a wide range of low frequency sounds augmented by auditory specialization is hence more advantageous here. There does not appear to be selection for hearing at particular frequencies in this range. The peaks of greatest auditory sensitivity appear to correspond to the resonant frequencies of the different components of the middle ear transformer and cavity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50256/1/1051380103_ftp.pd

Habitat preference of geese is affected by livestock grazing:Seasonal variation in an experimental field evaluation

The number of staging geese in northwestern Europe has increased dramatically. Growing goose numbers put strong grazing pressure on agricultural pastures. Damage to agricultural land may be mitigated by managing nature reserves in order to optimally accommodate large numbers of grazing geese. Livestock grazing has been shown to facilitate foraging geese; we take the novel approach of determining the effects of four different livestock grazing treatments in a replicated experiment on the distribution of geese. We present experimental field evidence that livestock grazing of a salt marsh in summer affects the habitat preference of foraging geese during autumn and spring staging. In an experimental field set-up with four different livestock grazing treatments we assessed goose visitation through dropping counts, in both autumn and spring. Grazing treatments included 0.5 or 1 horse ha(-1) and 0.5 or 1 cattle ha(-1) during the summer season. The livestock grazing regime affected goose distribution in autumn, just after livestock had been removed from the salt marsh. In autumn, goose visitation was highest in the 1 head ha(-1) grazing treatments, where grazing intensity by livestock was also highest. In line with this result, goose visitation was lowest in the 0.5 head ha(-1) livestock grazing treatments, where the grazing intensity by livestock was lowest. The differences in goose visitation among the experimental treatments in autumn could not be explained by the canopy height. In spring we did not find any effect of livestock grazing treatment on goose visitation. Differences in the distribution of geese over the experiment between autumn and spring may be explained by changes in the availability of nutrient-rich vegetation. Livestock summer grazing with a high stocking density, especially with horses, can be used to attract geese to salt marshes in autumn and potentially reduces damage caused by geese to inland farmland. From a nature conservation interest point of view, however, variation in structure of the vegetation is a prerequisite for other groups of organisms. Hence, we recommend grazing of salt marshes with densities of 0.5 head ha(-1) of livestock when goose conservation is not the only management issue

Tissue-specific contribution of macrophages to wound healing

AbstractMacrophages are present in all tissues, either as resident cells or monocyte-derived cells that infiltrate into tissues. The tissue site largely determines the phenotype of tissue-resident cells, which help to maintain tissue homeostasis and act as sentinels of injury. Both tissue resident and recruited macrophages make a substantial contribution to wound healing following injury. In this review, we evaluate how macrophages in two fundamentally distinct tissues, i.e. the lung and the skin, differentially contribute to the process of wound healing. We highlight the commonalities of macrophage functions during repair and contrast them with distinct, tissue-specific functions that macrophages fulfill during the different stages of wound healing

Cell-based tissue engineering strategies used in the clinical repair of articular cartilage

One of the most important issues facing cartilage tissue engineering is the inability to move technologies into the clinic. Despite the multitude of review articles on the paradigm of biomaterials, signals, and cells, it is reported that 90% of new drugs that advance past animal studies fail clinical trials (1). The intent of this review is to provide readers with an understanding of the scientific details of tissue engineered cartilage products that have demonstrated a certain level of efficacy in humans, so that newer technologies may be developed upon this foundation. Compared to existing treatments, such as microfracture or autologous chondrocyte implantation, a tissue engineered product can potentially provide more consistent clinical results in forming hyaline repair tissue and in filling the entirety of the defect. The various tissue engineering strategies (e.g., cell expansion, scaffold material, media formulations, biomimetic stimuli, etc.) used in forming these products, as collected from published literature, company websites, and relevant patents, are critically discussed. The authors note that many details about these products remain proprietary, not all information is made public, and that advancements to the products are continuously made. Nevertheless, by fully understanding the design and production processes of these emerging technologies, one can gain tremendous insight into how to best use them and also how to design the next generation of tissue engineered cartilage products

Autologous chondrocyte implantation in the knee : systematic review and economic evaluation

Background: The surfaces of the bones in the knee are covered with articular cartilage, a rubber-like substance that is very smooth, allowing frictionless movement in the joint and acting as a shock absorber. The cells that form the cartilage are called chondrocytes. Natural cartilage is called hyaline cartilage. Articular cartilage has very little capacity for self-repair, so damage may be permanent. Various methods have been used to try to repair cartilage. Autologous chondrocyte implantation (ACI) involves laboratory culture of cartilage-producing cells from the knee and then implanting them into the chondral defect. Objective: To assess the clinical effectiveness and cost-effectiveness of ACI in chondral defects in the knee, compared with microfracture (MF). Data sources: A broad search was done in MEDLINE, EMBASE, The Cochrane Library, NHS Economic Evaluation Database and Web of Science, for studies published since the last Health Technology Assessment review. Review methods: Systematic review of recent reviews, trials, long-term observational studies and economic evaluations of the use of ACI and MF for repairing symptomatic articular cartilage defects of the knee. A new economic model was constructed. Submissions from two manufacturers and the ACTIVE (Autologous Chondrocyte Transplantation/Implantation Versus Existing Treatment) trial group were reviewed. Survival analysis was based on long-term observational studies. Results: Four randomised controlled trials (RCTs) published since the last appraisal provided evidence on the efficacy of ACI. The SUMMIT (Superiority of Matrix-induced autologous chondrocyte implant versus Microfracture for Treatment of symptomatic articular cartilage defects) trial compared matrix-applied chondrocyte implantation (MACI®) against MF. The TIG/ACT/01/2000 (TIG/ACT) trial compared ACI with characterised chondrocytes against MF. The ACTIVE trial compared several forms of ACI against standard treatments, mainly MF. In the SUMMIT trial, improvements in knee injury and osteoarthritis outcome scores (KOOSs), and the proportion of responders, were greater in the MACI group than in the MF group. In the TIG/ACT trial there was improvement in the KOOS at 60 months, but no difference between ACI and MF overall. Patients with onset of symptoms < 3 years’ duration did better with ACI. Results from ACTIVE have not yet been published. Survival analysis suggests that long-term results are better with ACI than with MF. Economic modelling suggested that ACI was cost-effective compared with MF across a range of scenarios. Limitations: The main limitation is the lack of RCT data beyond 5 years of follow-up. A second is that the techniques of ACI are evolving, so long-term data come from trials using forms of ACI that are now superseded. In the modelling, we therefore assumed that durability of cartilage repair as seen in studies of older forms of ACI could be applied in modelling of newer forms. A third is that the high list prices of chondrocytes are reduced by confidential discounting. The main research needs are for longer-term follow-up and for trials of the next generation of ACI. Conclusions: The evidence base for ACI has improved since the last appraisal by the National Institute for Health and Care Excellence. In most analyses, the incremental cost-effectiveness ratios for ACI compared with MF appear to be within a range usually considered acceptable. Research is needed into long-term results of new forms of ACI