The Roles of Matrix-Associated Periostin in an In Vitro Model of Hypertrophic Scarring

Abnormal scarring is a type of benign fibrosis of the skin that can restrict mobility, dexterity and quality of life. There are few, if any, truly effective treatment options for these conditions. Hypertrophic scarring is a common form of abnormal scarring characterized by increased fibroblast proliferation and differentiation of apoptosis-resistant and hyper-contractile myofibroblasts that promote excessive deposition and contracture of the extracellular matrix (ECM). Periostin, a secreted ECM protein, is transiently expressed during normal cutaneous wound repair, but is abnormally abundant and persistent in abnormal scars and other benign fibroses that display enhanced fibroblast proliferation and myofibroblast differentiation. The objectives of this study were to elucidate the effects of periostin on fibroblast proliferation, myofibroblast differentiation and myofibroblast persistence in an in vitro human fibroblast model of hypertrophic scarring. Primary fibroblasts derived from patients with hypertrophic scars (HTS) or normal skin (NS) were cultured in two- and three-dimensional collagen cultures to more closely mimic their in vivo microenvironment. A series of in vitro techniques, including adenoviral transduction, WST-1 assays, stressed fibroblast populated collagen lattices (sFPCLs), western immunoblotting and immunofluorescence confocal microscopy were employed to assess periostin effects on fibroblast proliferation and myofibroblast differentiation under these culture conditions. Periostin treatment was shown to enhance HTS fibroblast proliferation in compliant 2D collagen cultures through Akt and Rho kinase dependent pathways. When subjected to isometric tension in sFPCLs, periostin enhanced myofibroblast differentiation, as evidenced by increases in collagen contraction, alpha smooth muscle actin (αSMA) and the formation of supermature focal adhesions. In contrast, no discernible effects of periostin treatment were evident in NS fibroblasts. Periostin signalling maintained αSMA levels in HTS fibroblasts in an environment of decreasing ECM tension, and this was correlated with changes in focal adhesion kinase, cofilin and myosin light chain activities. These studies are the first to identify periostin as a mediator of excessive proliferation, myofibroblast differentiation and persistence in human fibroblasts derived from hypertrophic scars. The findings reported here suggest that novel therapeutic interventions designed to deplete periostin levels in the dermis of hypertrophic scars may have utility for attenuating fibroblast proliferation and depleting myofibroblast populations, and thereby enhance scar resolution

High-sensitivity spectral fluorescence lifetime imaging for resolving spectroscopically overlapping species

The capability to resolve the contributions from spectroscopically overlapping fluorophores has enabled significant breakthroughs in cellular imaging. However, commercial microscopes for this purpose use analog light detection with least squares curve-fitting analysis and improvements in sensitivity are needed. To this end, a microscope has been constructed with high throughput and single-photon detection capability. The fluorescence is separated through use of a prism spectrometer or a series of dichroic mirrors into four spectral bands and detected using four single-photon avalanche diode (SPAD) detectors, which provide high-quantum efficiency in the red spectral region. The detectors are connected to a time-correlated single photon counting module to provide sub-nanosecond temporal resolution for distinguishing fluorophores with different fluorescence lifetimes. Maximum-likelihood (ML) methods have been developed for analyzing the temporally and spectrally resolved photon count data from the SPADs to find the contributions from different fluorescent species and from background. Commercially available SPADs exhibit a count-rate dependent time shift in the impulse response function, and hence the instrument incorporates custom modified SPADs with improved timing stability. Nevertheless, there is still some time shift, and hence the ML-analysis has been extended to include this as an adjustable parameter for each individual SPAD. Monte Carlo simulations have also been developed to enable studies of the number of photons needed to resolve specific fluorophores

Survival, movements and habitat selection of pygmy rabbits (Brachylagus idahoensis) on the Great Basin of southeastern Oregon and northwestern Nevada

I investigated survival, movements, home range sizes, and habitat selection of pygmy rabbits (Brachylagus idahoensis) in southeastern Oregon and northwestern Nevada from June 2005 to June 2007. I trapped 298 rabbits on four sites and fitted each with radio transmitters. More than 13,000 locations of telemetered rabbits were recorded. I used known fate models in program MARK to estimate survival of radio-marked pygmy rabbits from September 2005 – August 2006. The most appropriate model, based on Akaike's Information Criterion (AICc), indicated survival rates varied among study sites, sexes, and with monthly interval in a parallel pattern [model S(area * sex + t)]. The estimated annual survival rate on the four study sites was notably low, varying from 0.003 (SE = 0.003) to 0.173 (SE = 0.066). Predation on collared rabbits was high for both adult (88.6%) and juvenile (89.4%) rabbits. When the cause of mortality could be determined, the most common predators of pygmy rabbits were coyotes (Canis latrans)–19.6%, avian predators–18.5%, and weasels (Mustela spp.)–9.8%. Spatial use by pygmy rabbits was strongly influenced by sex and season. Males used larger annual (mean = 1.70 ha ± 0.69 SE) and breeding (1.67 ± 0.27 ha) home ranges than females (Annual: 0.90 ± 0.26 ha; Breeding: 0.92 ± 0.16 ha), likely influenced by mate-searching behaviors exhibited by males. Both sexes utilized larger home ranges during the breeding season than during the nonbreeding season (Male: 0.63 ± 0.11 ha; Female: 0.50 ± 0.09 ha). Core area sizes of males (0.10 ± 0.04 ha) also were larger than females (0.07 ± 0.03 ha). Twenty-four radio-marked individuals dispersed greater than 0.5 km, with a maximal observed dispersal distance of 8.5 km. The majority (62.5%) of these long distance movements were by juvenile males, which likely represented dispersal from natal areas. Many of these individuals apparently crossed low sagebrush (Artemisia arbuscula) communities and relatively open areas. The ability of this species to cross unsuitable habitats, previously considered barriers to movement, may suggest that fragmented populations of pygmy rabbits may not be as isolated as once thought. To examine resource selection by pygmy rabbits, I sampled vegetative and soil characteristics at locations used by radio-marked rabbits (n = 178) and available (n = 100) sites and compared them using logistic regression. The top model, based on Akaike's Information Criterion (AICc), indicated that the increased density and height of live shrubs, higher silt content of soil, and lower clay content of soil were the primary variables related to pygmy rabbit occupation relative to available habitat. Habitat-use patterns also varied among study sites as I did not find a consensus model that described habitat selection among pygmy rabbits for each study site. Further, my analysis demonstrated that soils at pygmy rabbit burrows were deeper, had greater subsurface strengths, lower strengths at the surface, and lower clay composition than was randomly available in the study area. The findings in this study provide needed information to management agencies making land-use decisions. Given the concern over the status of this species throughout its range, a more thorough understanding of survival rates, spatial use, dispersal capabilities, and habitat associations of this sagebrush obligate species is essential to identify factors influencing their conservation

A climatological analysis of heatbursts in Oklahoma (1994–2009)

Heatbursts are characterized by a sudden and highly localized increase in air temperature, a simultaneous decrease in relative humidity and dewpoint temperature, and strong gusty winds, typically associated with decaying thunderstorms. The small spatial extent and short duration of most heatbursts makes detailed study of these events difficult using the standard federal observation network (e.g. hourly observations) established in most countries. Thus, many discussions of heatbursts note that they are ‘rare’ phenomena. However, observations from the Oklahoma Mesonet indicate that although heatbursts are meso-alpha scale phenomena, they are not rare. Using multi-criteria analysis, 207 heatburst events of various magnitudes, areal coverage and duration were identified between 1994 and 2009 across Oklahoma. Significantly more heatbursts were detected in the western two-thirds of the state as compared to the eastern third. Heatbursts were primarily nocturnal events, with 70% of heatburst events initiated between 0000 and 0800 UTC (i.e. between 6 p.m. and 2 a.m. Central Standard Time). With 62 of the 207 events, June was the most active month for heatbursts. At the other extreme, no heatbursts were detected during February and November, and only one was observed during January and December. Almost all of the heatbursts occurred underneath or adjacent to weak radar echoes at the time of the event. Four basic radar patterns were associated with the events: (1) radar echoes associated with rapidly weakening convection, (2) weak reflectivity not produced by or associated with deep moist convection, (3) weak radar echoes to the rear of a dissipating mesoscale convective system (MCS) and (4) weak reflectivity along the periphery of intense convection that did not appear to be weakening

A method for high-throughput gene expression signature analysis

Genome-wide transcriptional profiling has shown that different biologic states (for instance, disease and response to pharmacologic manipulation) can be recognized by the expression pattern of relatively small numbers of genes. However, the lack of a practical and cost-effective technology for detection of these gene expression 'signatures' in large numbers of samples has severely limited their exploitation in important medical and pharmaceutical discovery applications. Here, we describe a solution based on the combination of ligation-mediated amplification with an optically addressed microsphere and flow cytometric detection system

Fear of the human ‘super predator’ reduces feeding time in large carnivores

Large carnivores’ fear of the human ‘super predator’ has the potential to alter their feeding behaviour and result in human-induced trophic cascades. However, it has yet to be experimentally tested if large carnivores perceive humans as predators and react strongly enough to have cascading effects on their prey. We conducted a predator playback experiment exposing pumas to predator (human) and non-predator control (frog) sounds at puma feeding sites to measure immediate fear responses to humans and the subsequent impacts on feeding. We found that pumas fled more frequently, took longer to return, and reduced their overall feeding time by more than half in response to hearing the human ‘super predator’. Combined with our previous work showing higher kill rates of deer in more urbanized landscapes, this study reveals that fear is the mechanism driving an ecological cascade from humans to increased puma predation on deer. By demonstrating that the fear of humans can cause a strong reduction in feeding by pumas, our results support that non-consumptive forms of human disturbance may alter the ecological role of large carnivores

FunnyBase: a systems level functional annotation of Fundulus ESTs for the analysis of gene expression

BACKGROUND: While studies of non-model organisms are critical for many research areas, such as evolution, development, and environmental biology, they present particular challenges for both experimental and computational genomic level research. Resources such as mass-produced microarrays and the computational tools linking these data to functional annotation at the system and pathway level are rarely available for non-model species. This type of "systems-level" analysis is critical to the understanding of patterns of gene expression that underlie biological processes. RESULTS: We describe a bioinformatics pipeline known as FunnyBase that has been used to store, annotate, and analyze 40,363 expressed sequence tags (ESTs) from the heart and liver of the fish, Fundulus heteroclitus. Primary annotations based on sequence similarity are linked to networks of systematic annotation in Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and can be queried and computationally utilized in downstream analyses. Steps are taken to ensure that the annotation is self-consistent and that the structure of GO is used to identify higher level functions that may not be annotated directly. An integrated framework for cDNA library production, sequencing, quality control, expression data generation, and systems-level analysis is presented and utilized. In a case study, a set of genes, that had statistically significant regression between gene expression levels and environmental temperature along the Atlantic Coast, shows a statistically significant (P < 0.001) enrichment in genes associated with amine metabolism. CONCLUSION: The methods described have application for functional genomics studies, particularly among non-model organisms. The web interface for FunnyBase can be accessed at . Data and source code are available by request at [email protected]