The MexTAg collaborative cross: host genetics affects asbestos related disease latency, but has little influence once tumours develop.

Objectives: This study combines two innovative mouse models in a major gene discovery project to assess the influence of host genetics on asbestos related disease (ARD). Conventional genetics studies provided evidence that some susceptibility to mesothelioma is genetic. However, the identification of host modifier genes, the roles they may play, and whether they contribute to disease susceptibility remain unknown. Here we report a study designed to rapidly identify genes associated with mesothelioma susceptibility by combining the Collaborative Cross (CC) resource with the well-characterised MexTAg mesothelioma mouse model. Methods: The CC is a powerful mouse resource that harnesses over 90% of common genetic variation in the mouse species, allowing rapid identification of genes mediating complex traits. MexTAg mice rapidly, uniformly, and predictably develop mesothelioma, but only after asbestos exposure. To assess the influence of host genetics on ARD, we crossed 72 genetically distinct CC mouse strains with MexTAg mice and exposed the resulting CC-MexTAg (CCMT) progeny to asbestos and monitored them for traits including overall survival, the time to ARD onset (latency), the time between ARD onset and euthanasia (disease progression) and ascites volume. We identified phenotype-specific modifier genes associated with these traits and we validated the role of human orthologues in asbestos-induced carcinogenesis using human mesothelioma datasets. Results: We generated 72 genetically distinct CCMT strains and exposed their progeny (2,562 in total) to asbestos. Reflecting the genetic diversity of the CC, there was considerable variation in overall survival and disease latency. Surprisingly, however, there was no variation in disease progression, demonstrating that host genetic factors do have a significant influence during disease latency but have a limited role once disease is established. Quantitative trait loci (QTL) affecting ARD survival/latency were identified on chromosomes 6, 12 and X. Of the 97-protein coding candidate modifier genes that spanned these QTL, eight genes (CPED1, ORS1, NDUFA1, HS1BP3, IL13RA1, LSM8, TES and TSPAN12) were found to significantly affect outcome in both CCMT and human mesothelioma datasets. Conclusion: Host genetic factors affect susceptibility to development of asbestos associated disease. However, following mesothelioma establishment, genetic variation in molecular or immunological mechanisms did not affect disease progression. Identification of multiple candidate modifier genes and their human homologues with known associations in other advanced stage or metastatic cancers highlights the complexity of ARD and may provide a pathway to identify novel therapeutic targets.

Optimising multiplex immunofluorescence staining for characterising the tumour immune micro-environment.

Exploring the tumour microenvironment provides insight into the unique interaction between the host and tumour. Ultimately, its study improves understanding of how an individual mounts and achieves an anti-tumour immune response. In the context of colorectal cancer, immune biomarkers within the tumour microenvironment outperform traditional histopathological staging in predicting disease recurrence. Multiplex immunofluorescence enables simultaneous assessment of multiple markers to provide a highly accurate classification of immune cells and their spatial characterisation relative to tumour tissue. Further, automated slide staining provides staining consistency and reduces labour costs. Image acquisition using a non-spectral scanner allows more researchers to utilise multiplexed immunofluorescence for translational research. Herein we describe the optimisation process of conducting automated staining using a five-colour, tyramide signal amplification-based multiplex immunofluorescence panel. Using antibodies against CD3, CD8, CD103 and cytokeratin, the panel characterises T cell populations within human colorectal adenocarcinoma tissue. We provide an overview of primary antibody titration and the development of tyramide signal amplification immunofluorescence monoplex assays. We detail the processes of antibody stripping and the role of exogenous horseradish peroxidase inhibition to facilitate multiplexing. An account of determining the staining sequence and fluorophore assignment is provided. We describe image acquisition using a standard fluorescence microscope slide scanner and the management of spectral crosstalk using this system. Finally, we briefly document the digital image analysis required to characterise cells and determine their spatial distribution within the colorectal tumour microenvironment.

PD-L1+ dendritic cells in the tumor microenvironment correlate with good prognosis and CD8+ T cell infiltration in colon cancer.

BACKGROUND: The prognostic value of tumor-associated dendritic cells (DC) in colon cancer remains poorly understood. This may be in part due to the interchangeable expression of immunostimulatory and immunoinhibitory molecules on DC. Here we investigated the prognostic impact of CD11c+ DC co-expressing the immunoinhibitory molecule PD-L1 and their spatial relationship with CD8+ T-cells in patients treated for stage III colon cancer. METHODS: Tissue microarrays containing representative cores of central tumor, leading edge, and adjacent normal tissue from 221 patients with stage III colon cancer were immunostained for CD8, CD11c, PD-L1, and cytokeratin using immunofluorescent probes. Cells were quantified using StrataQuest digital image analysis software, with intratumoral and stromal regions analyzed separately. Kaplan-Meier estimates and Cox regression were used to assess survival. RESULTS: Intratumoral CD8+ cell density (HR = .52, 95% confidence interval [CI] .33-.83, P = .007), stromal CD11c+ cell density (HR = .52, 95% CI .33-.83, P = .006), intratumoral CD11c+ PD-L1+ cell density (HR = .57, 95% CI .35-.92, P = .021), and stromal CD11c+ PD-L1+ cell density (HR = .48, 95% CI .30-.77, P = .003) on leading-edge cores were all significantly associated with good survival. CD8+ cell density was positively correlated with both CD11c+ cell density and CD11c+ PD-L1+ cell density in tumor epithelium and stromal compartments. CONCLUSION: Here we showed that PD-L1-expressing DC in the tumor microenvironment are associated with improved survival in stage III colon cancer and likely reflect an immunologically "hot" tumor microenvironment. Further investigation into the expression of immunomodulatory molecules by tumor-associated DC may help to further elucidate their prognostic value.

Optimisation of multiplex immunofluorescence for a non-spectral fluorescence scanning system.

The use of multi-colour immunofluorescence (IF) for immunophenotyping in formalin-fixed paraffin-embedded tissue sections is gaining popularity worldwide. This technique allows for the simultaneous detection of multiple markers on the same tissue section, thereby yielding more complex information than is possible by chromogenic immunohistochemistry (IHC). However, many commercially-available multiplex IF kits are designed for use in conjunction with a multispectral imaging system, to which many research groups have limited access. Here we present two 5-colour IF panels designed for T cell characterisation in human colorectal tissue, which can be imaged using a non-spectral fluorescence slide scanner with standard band-pass filters. We describe the optimisation process and the key considerations in developing a multiplex fluorescence assay, and discuss some of the advantages and disadvantages of using multiplex IF with a non-spectral imaging system.

Effects of wildfire on floral display size and pollinator community reduce outcrossing rate in a plant with a mixed mating system.

PREMISE OF THE STUDY: Wildfire changes the demography, morphology, and behavior of plants, and may alter the pollinator community. Such trait changes may drastically alter the outcome of pollination mutualisms on plants; however, the direct role of fire on these mutualisms is poorly known. METHODS: Following a pair of fires in the northern California coast range chaparral, we censused floral visitor communities of Trichostema laxum (Lamiaceae), quantified visiting bee behavior, and estimated outcrossing rates using a widespread Mendelian recessive floral polymorphism across a matrix of populations in burned and unburned sites. We also compared pre- and postfire floral visitation in two populations. RESULTS: Outcrossing rates were significantly lower in burned areas; however, our data suggest that the much larger size of plants in burned areas, not burn status itself, drove this pattern. Large-bodied bees dominated floral visitor communities after fire, likely recruiting to the abundant postfire floral resources. These bees visited more flowers per plant than did the smaller bees prevalent before fire and in unburned areas, likely increasing selfing through geitonogamy (within-plant pollination), an effect made possible by the far larger size of plants in burned areas. CONCLUSIONS: Outcrossing rates dropped substantially after wildfires because of changes in the pollinators, plant display size, and their interactions. Reductions in outcrossing following fire may have important implications for population resilience and evolution in a changing climate with more frequent fires.

Tumour-infiltrating regulatory T cell density before neoadjuvant chemoradiotherapy for rectal cancer does not predict treatment response.

Neoadjuvant (preoperative) chemoradiotherapy (CRT) decreases the risk of rectal cancer recurrence and reduces tumour volume prior to surgery. However, response to CRT varies considerably between individuals and factors associated with response are poorly understood. Foxp3+ regulatory T cells (Tregs) inhibit anti-tumour immunity and may limit any response to chemotherapy and radiotherapy. We have previously reported that a low density of Tregs in the tumour stroma following neoadjuvant CRT for rectal cancer is associated with improved tumour regression. Here we have examined the association between Treg density in pre-treatment diagnostic biopsy specimens and treatment response, in this same patient cohort. We aimed to determine whether pre-treatment tumour-infiltrating Treg density predicts subsequent response to neoadjuvant CRT. Foxp3+, CD8+ and CD3+ cell densities in biopsy samples from 106 patients were assessed by standard immunohistochemistry (IHC) and evaluated for their association with tumour regression grade and survival. We found no association between the density of any T cell subset pre-treatment and clinical outcome, indicating that tumour-infiltrating Treg density does not predict response to neoadjuvant CRT in rectal cancer. Taken together with the findings of the previous study, these data suggest that in the context of neoadjuvant CRT for rectal cancer, the impact of chemotherapy and/or radiotherapy on anti-tumour immunity may be more important than the state of the pre-existing local immune response.

Nanoparticle layer deposition for highly controlled multilayer formation based on high- coverage monolayers of nanoparticles.

This paper establishes a strategy for chemical deposition of functionalized nanoparticles onto solid substrates in a layer-by-layer process based on self-limiting surface chemical reactions leading to complete monolayer formation within the multilayer system without any additional intermediate layers - nanoparticle layer deposition (NPLD). This approach is fundamentally different from previously established traditional layer-by-layer deposition techniques and is conceptually more similar to well-known atomic and molecular - layer deposition processes. The NPLD approach uses efficient chemical functionalization of the solid substrate material and complementary functionalization of nanoparticles to produce a nearly 100% coverage of these nanoparticles with the use of "click chemistry". Following this initial deposition, a second complete monolayer of nanoparticles is deposited using a copper-catalyzed "click reaction" with the azide-terminated silica nanoparticles of a different size. This layer-by-layer growth is demonstrated to produce stable covalently-bound multilayers of nearly perfect structure over macroscopic solid substrates. The formation of stable covalent bonds is confirmed spectroscopically and the stability of the multilayers produced is tested by sonication in a variety of common solvents. The 1-, 2- and 3-layer structures are interrogated by electron microscopy and atomic force microscopy and the thickness of the multilayers formed is fully consistent with that expected for highly efficient monolayer formation with each cycle of growth. This approach can be extended to include a variety of materials deposited in a predesigned sequence on different substrates with a highly conformal filling.

Reduction in Size and Number of Plantar Verrucae in Human Immunodeficiency Virus-Infected Individuals After the Implementation of Highly Active Antiretroviral Therapy.

BACKGROUND: Implementation of highly active antiretroviral therapy (HAART) significantly increased the life expectancy of those living with human immunodeficiency virus (HIV). Except for prevalence, scientific reports regarding clinical manifestations of plantar verrucae in the post-HAART era are lacking. The objective of this study was to compare clinical manifestations of plantar verrucae between HIV-infected and noninfected individuals and then to compare these findings with those observed before the implementation of HAART. METHODS: Nineteen patients with plantar verrucae (ten with HIV and nine without HIV) were examined to determine the size, number, and clinical type of verrucae present. The two groups were first compared with each other and then with previously collected data from a similar analysis conducted in 1995, before the implementation of HAART. Statistical significance was determined using the Fisher exact test or the Wilcoxon rank sum test. RESULTS: No significant differences were observed in the size, number, or clinical type of verrucae between HIV-negative and HIV-positive patients. Compared with the 1995 data, there was a significant decrease in the number of verrucae lesions per individual and a nonsignificant decrease in the average size of verrucae in HIV-positive patients. CONCLUSIONS: Study results indicate that the implementation of HAART has impacted the clinical manifestations of plantar verrucae in HIV-positive individuals. Further analyses with a larger number of patients are required to confirm and substantiate these findings.

Posterior Facet Settling and Changes in Bohler's Angle in Operatively and Nonoperatively Treated Calcaneus Fractures.

BACKGROUND: Patients with calcaneus fractures often exhibit settling of the posterior facet with a corresponding decrease in Bohler's angle (BA) following either operative or nonoperative treatment. Both injury BA and postoperative BA have been shown to be prognostic for outcomes; however, the demographic and surgeon-specific factors that may contribute to settling have not been critically examined in the literature. The purpose of this study was to identify these causative factors. METHODS: 234 patients with intra-articular calcaneus fractures were analyzed. All patients had preoperative plain radiographs, at least 5 months of orthopedic follow-up, and computed tomography scanning performed. BA was measured on the injury radiographs for all patients. For operatively treated patients, BA was measured on the immediate postoperative radiographs and compared with the last available radiograph. For nonoperatively treated patients, BA was measured on the last available radiograph. All patients were fully weightbearing at the time of final follow-up but not on initial radiographs due to their recent injury. Demographic data including age, gender, energy of injury mechanism, tobacco use, diabetes, osteoporosis, rheumatoid arthritis, and substance/alcohol abuse were retrospectively collected. Fractures were classified using the Essex-Lopresti and Sanders classifications. Time to full weightbearing was documented, as were any reports of noncompliance with weightbearing restrictions. For patients treated operatively, type of fixation (calcaneal-specific perimeter plate, nonperimeter plate, screw fixation), use of locking screws, use of bone graft or graft substitutes, and the number of screws supporting the posterior facet were documented. RESULTS: There was a statistically significant amount of settling within the operative and nonoperative groups, but there was no statistically significant difference in settling of BA between the groups. The average settling of BA for the operative and nonoperative group was 8 degrees. Age greater than 50 years, diabetes, and alcohol abuse were all statistically significant and independent predictors of BA settling irrespective of treatment. CONCLUSION: The amount of BA settling between the operative and nonoperative group was not significant and showed an average decrease of 8 degrees in each group. However, the amount of settling that we found, irrespective of treatment, increased with patient age, alcohol abuse, and diabetes. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

A cadaver study revisiting the original methodology of Lauge-Hansen and a commentary on modern usage.

BACKGROUND: The study by Lauge-Hansen published in the Archives of Surgery in 1950 still stands as the seminal work for our understanding of the pathomechanics of ankle fractures. The purpose of the present study was to recreate Lauge-Hansen's experiments for the supination-external rotation (SER) fracture mechanism and to determine whether the predicted sequence of osseous and soft-tissue injury is reproducible on the basis of his originally described methodology. METHODS: Ten fresh-frozen cadaver specimens amputated above the knee were utilized. The foot was axially loaded in a position of neutral dorsiflexion and supination. External rotation was applied manually in accordance with Lauge-Hansen's description until osseous and/or soft-tissue injury occurred. Fluoroscopic images were made and anatomic dissection was performed. RESULTS: Although several specimens exhibited findings consistent with certain stages of the SER injury pattern, no specimen demonstrated the complete sequence of predicted osseous and soft-tissue injury. CONCLUSIONS: Loading cadaver specimens with an SER mechanism utilizing a methodology similar to that in the original experiments by Lauge-Hansen does not reliably produce the sequence of osseous and soft-tissue injury predicted by Lauge-Hansen.

An ultraviolet floral polymorphism associated with life history drives pollinator discrimination in Mimulus guttatus.

UNLABELLED: • PREMISE OF THE STUDY: Ultraviolet (UV) floral patterns are common in angiosperms and mediate pollinator attraction, efficiency, and constancy. UV patterns may vary within species, yet are cryptic to human observers. Thus, few studies have explicitly described the distribution or ecological significance of intraspecific variation in UV floral patterning. Here, we describe the geographic distribution and pattern of inheritance of a UV polymorphism in the model plant species Mimulus guttatus (Phrymaceae). We then test whether naturally occurring UV phenotypes influence pollinator interactions within M. guttatus.• METHODS: We document UV patterns in 18 annual and 19 perennial populations and test whether UV pattern is associated with life history. To examine the pattern of inheritance, we conducted crosses within and between UV phenotypes. Finally, we tested whether bee pollinators discriminate among naturally occurring UV phenotypes in two settings: wild bee communities and captive Bombus impatiens.• KEY RESULTS: Within M. guttatus, perennial populations exhibit a small bulls-eye pattern, whereas a bilaterally symmetric runway pattern occurs mainly in annual populations. Inheritance of UV patterning is consistent with a single-locus Mendelian model in which the runway phenotype is dominant. Bee pollinators discriminate against unfamiliar UV patterns in both natural and controlled settings.• CONCLUSIONS: We describe a widespread UV polymorphism associated with life history divergence within Mimulus guttatus. UV pattern influences pollinator visitation and should be considered when estimating reproductive barriers between life history ecotypes. This work develops a new system to investigate the ecology and evolution of UV floral patterning in a species with extensive genomic resources.

The "joint-elevation" calcaneus fracture: a rare variant of the intra-articular calcaneus fracture-dislocation.

UNLABELLED: Calcaneus fractures are the most commonly fractured tarsal bone with approximately 75% being intra-articular in nature. Böhler's angle has been found to be reliable and prognostic, and it has been used as a proxy for joint depression and articular involvement. It often guides the need for advanced imaging and/or operative intervention. We describe a rare variant of intra-articular calcaneus fracture-dislocation that results in elevation of a portion of the posterior facet above the posterior talus and a seemingly normal or increased Böhler's angle, which we call the "joint-elevation" calcaneus fracture. Orthopaedic surgeons should be aware of this previously undescribed variant in order to avoid inappropriate treatment or misdiagnosis. LEVEL OF EVIDENCE: Therapeutic Level IV: Case Series.

Leatherback turtle movements, dive behavior, and habitat characteristics in ecoregions of the Northwest Atlantic Ocean.

Leatherback sea turtles, Dermochelys coriacea, are highly migratory predators that feed exclusively on gelatinous zooplankton, thus playing a unique role in coastal and pelagic food webs. From 2007 to 2010, we used satellite telemetry to monitor the movements and dive behavior of nine adult and eleven subadult leatherbacks captured on the Northeast USA shelf and tracked throughout the Northwest Atlantic. Leatherback movements and environmental associations varied by oceanographic region, with slow, sinuous, area-restricted search behavior and shorter, shallower dives occurring in cool (median sea surface temperature: 18.4°C), productive (median chlorophyll a: 0.80 mg m(-3)), shallow (median bathymetry: 57 m) shelf habitat with strong sea surface temperature gradients (median SST gradient: 0.23°C km(-1)) at temperate latitudes. Leatherbacks were highly aggregated in temperate shelf and slope waters during summer, early fall, and late spring and more widely dispersed in subtropical and tropical oceanic and neritic habitat during late fall, winter and early spring. We investigated the relationship of ecoregion, satellite-derived surface chlorophyll, satellite-derived sea surface temperature, SST gradient, chlorophyll gradient and bathymetry with leatherback search behavior using generalized linear mixed-effects models. The most well supported model showed that differences in leatherback search behavior were best explained by ecoregion and regional differences in bathymetry and SST. Within the Northwest Atlantic Shelves region, leatherbacks increased path sinuosity (i.e., looping movements) with increasing SST, but this relationship reversed within the Gulf Stream region. Leatherbacks increased path sinuosity with decreasing water depth in temperate and tropical shelf habitats. This relationship is consistent with increasing epipelagic gelatinous zooplankton biomass with decreasing water depth, and bathymetry may be a key feature in identifying leatherback foraging habitat in neritic regions. High-use habitat for leatherbacks in our study occurred in coastal waters of the North American eastern seaboard and eastern Caribbean, putting turtles at heightened risk from land- and ocean-based human activity.

Novel adaptation to hawkmoth pollinators in Clarkia reduces efficiency, not attraction of diurnal visitors.

BACKGROUND AND AIMS: Plant populations experiencing divergent pollination environments may be under selection to modify floral traits in ways that increase both attractiveness to and efficiency of novel pollinators. These changes may come at the cost of reducing overall effectiveness of other pollinators. The goal of this study was to examine differences in attractiveness and efficiency between Clarkia concinna and C. breweri, sister species of annual plants with parapatric distributions. METHODS: An assessment was made as to whether observed differences in visitors between natural populations are driven by differences in floral traits or differences in the local pollination environment. Differences in floral attractiveness were quantified by setting out arrays of both species in the geographical range of each species and exposing both species to nocturnal hawkmoths (Hyles lineata) in flight cages. Differences in visitor efficiency were estimated by measuring stigma-visitor contact frequency and pollen loads for diurnal visitors, and pollen deposition on stigmas for hawkmoths. KEY RESULTS: The composition of visitors to arrayed plants was similar between plant species at any particular site, but highly divergent among sites, and reflected differences in visitors to natural populations. Diurnal insects visited both species, but were more common at C. concinna populations. Hummingbirds and hawkmoths were only observed visiting within the range of C. breweri. Despite attracting similar species when artificially presented together, C. concinna and C. breweri showed large differences in pollinator efficiency. All visitors except hawkmoths pollinated C. concinna more efficiently. CONCLUSIONS: Differences in the available pollinator community may play a larger role than differences in floral traits in determining visitors to natural populations of C. concinna and C. breweri. However, floral traits mediate differences in pollinator efficiency. Increased effectiveness of the novel hawkmoth pollinator on C. breweri comes at relatively little cost in attractiveness to other visitors, but at large cost in their efficiency as pollinators.

Metacommunity dynamics over 16 years in a pyrogenic shrubland.

Metacommunity theory allows predictions about the dynamics of potentially interacting species' assemblages that are linked by dispersal, but strong empirical tests of the theory are rare. We analyzed the metacommunity dynamics of Florida rosemary scrub, a patchily distributed pyrogenic community, to test predictions about turnover rates, community nestedness, and responses to patch size, arrangement, and quality. We collected occurrence data for 45 plant species from 88 rosemary scrub patches in 1989 and 2005 and used growth form, mechanism of regeneration after fire, and degree of habitat specialization to categorize species by life history. We tested whether patch size, fire history, and structural connectivity (a measure of proximity and size of surrounding patches) could be used to predict apparent extinctions and colonizations. In addition, we tested the accuracy of incidence-function models built with the patch survey data from 1989. After fire local extinction rates were higher for herbs than woody plants, higher for species that regenerated only from seed than species able to resprout, and higher for generalist than specialist species. Fewer rosemary specialists and a higher proportion of habitat generalists were extirpated on recently burned patches than on patches not burned between 1989 and 2005. Nestedness was highest for specialists among all life-history groups. Estimated model parameters from 1989 predicted the observed (1989-2005) extinction rates and the number of patches with persistent populations of individual species. These results indicate that species with different life-history strategies within the same metacommunity can have substantially different responses to patch configuration and quality. Real metacommunities may not conform to certain assumptions of simple models, but incidence-function models that consider only patch size, configuration, and quality can have significant predictive accuracy.

Physical symptoms and psychosocial correlates of somatization in pediatric primary care.

A group of children, aged 8 to 13 years, presenting to their pediatricians with multiple medically unexplained physical symptoms (MUPS) were compared with a control group of children from the identical age range who were, according to their pediatricians, free of unexplained physical symptoms. The groups were compared on both self-reported and parented-rated scales assessing physical symptoms and psychosocial functioning. The multiple MUPS group, relative to controls, exhibited significantly higher levels of parent-reported emotional/behavioral symptoms and a trend toward higher patient-reported anxiety than controls. Parents' and child's reports of symptomatology were modestly correlated. Symptom patterns characteristic of pediatric somatization differed as a function of whether child or parent reports were analyzed. Methodological issues in research on pediatric somatization were addressed and some directions for future research emerged.

Lipid corrections in carbon and nitrogen stable isotope analyses: comparison of chemical extraction and modelling methods.

1. Lipids have more negative delta(13)C values relative to other major biochemical compounds in plant and animal tissues. Although variable lipid content in biological tissues alters results and conclusions of delta(13)C analyses in aquatic food web and migration studies, no standard correction protocol exists. 2. We compared chemical extraction and mathematical correction methods for freshwater and marine fishes and aquatic invertebrates to better understand impacts of correction approaches on carbon (delta(13)C) and nitrogen (delta(15)N) stable isotope data. 3. Fish and aquatic invertebrate tissue delta(13)C values increased significantly following extraction for almost all species and tissue types relative to nonextracted samples. In contrast, delta(15)N was affected for muscle and whole body samples from only a few freshwater and marine species and had a limited effect for the entire data set. 4. Lipid normalization models, using C : N as a proxy for lipid content, predicted lipid-corrected delta(13)C for paired data sets more closely with parameters specific to the tissue type and species to which they were applied. 5. We present species- and tissue-specific models based on bulk C : N as a reliable alternative to chemical extraction corrections. By analysing a subset of samples before and after lipid extraction, models can be applied to the species and tissues of interest that will improve estimates of dietary sources using stable isotopes.

A finite-state continuous-time approach for inferring regional migration and mortality rates from archival tagging and conventional tag-recovery experiments.

Spatially structured population dynamics models are important management tools for harvested, highly mobile species and although conventional tag recovery experiments remain useful for estimation of various demographic parameters of these models, archival tagging experiments are becoming an important data source for analyzing migratory behavior of mobile marine species. We provide a likelihood-based approach for estimating the regional migration and mortality rate parameters intrinsic to these models that may use information obtained from conventional tag recovery and archival tagging experiments. Specifically, we assume that the regional location and survival of animals through time is a finite-state continuous-time stochastic process. The stochastic process is the basis of probability models for observations provided by the different types of tags. Results from application to simulated tagging experiments for western Atlantic bluefin tuna show that maximum likelihood estimators based on archival tagging observations and corresponding confidence intervals perform similar to conventional tagging observations for a given number of tag releases and releasing tags in each region can improve the behavior of maximum likelihood estimators regardless of tag type. We provide an example application with Atlantic bluefin tuna released with conventional tags in 1990-1992.

FXYD5 modulates Na+ absorption and is increased in cystic fibrosis airway epithelia.

FXYD5, also known as dysadherin, belongs to a family of tissue-specific regulators of the Na(+)-K(+)-ATPase. We determined the kinetic effects of FXYD5 on Na(+)-K(+)-ATPase pump activity in stably transfected Madin-Darby canine kidney cells. FXYD5 significantly increased the apparent affinity for Na(+) twofold and decreased the apparent affinity for K(+) by 60% with a twofold increase in V(max) of K(+), a pattern that would increase activity and Na(+) removal from the cell. To test the effect of increased Na(+) uptake on FXYD5 expression, we analyzed Madin-Darby canine kidney cells stably transfected with an inducible vector expressing all three subunits of the epithelial Na(+) channel (ENaC). Na(+)-K(+)-ATPase activity increased sixfold after 48-h ENaC induction, but FXYD5 expression decreased 75%. FXYD5 expression was also decreased in lung epithelia from mice that overexpress ENaC, suggesting that chronic Na(+) absorption by itself downregulates epithelial FXYD5 expression. Patients with cystic fibrosis (CF) display ENaC-mediated hyperabsorption of Na(+) in the airways, accompanied by increased Na(+)-K(+)-ATPase activity. However, FXYD5 was significantly increased in the lungs and nasal epithelium of CF mice as assessed by RT-PCR, immunohistochemistry, and immunoblot analysis (P < 0.001). FXYD5 was also upregulated in nasal scrapings from human CF patients compared with controls (P < 0.02). Treatment of human tracheal epithelial cells with a CFTR inhibitor (I-172) confirmed that loss of CFTR function correlated with increased FXYD5 expression (P < 0.001), which was abrogated by an inhibitor of NF-kappaB. Thus FXYD5 is upregulated in CF epithelia, and this change may exacerbate the Na(+) hyperabsorption and surface liquid dehydration observed in CF airway epithelia.

S163 is critical for FXYD5 modulation of wound healing in airway epithelial cells.

The FXYD family, which contains seven members, are tissue specific regulators of the Na,K-ATPase. Increased expression of FXYD5, a cancer-cell-associated membrane glycoprotein, has been associated with increased cell motility and metastatic potential. To better understand how FXYD5 may modulate cell motility, we analyzed S163, a conserved residue in all FXYD family members located in the C-terminus. Ectopic expression of human FXYD5 S163 mutants in HEK 293 cells showed that negative charge at S163 (S163D) decreased membrane localization, assessed by immunofluorescence. Coimmunoprecipitation studies revealed decreased FXYD5/Na,K-ATPase interaction for S163D compared with wild-type or S163A mutants. Interestingly, FXYD5 overexpression induced expression of vimentin, a marker of epithelial-mesenchymal transition, in murine airway epithelial cells. Because Na,K-ATPase expression is decreased in some forms of cancer and is critical for establishing cell polarity and suppressing cell motility, we analyzed S163 mutants in an epithelial cell scratch-wound model as a measure of cell migration. Wild-type FXYD5 overexpression increased reepithelialization (p<0.0001), which was further increased in S163D mutants (p<0.005). However, S163A mutants inhibited epithelial cell migration compared with wild-type FXYD5 overexpression (p<0.0001). We conclude that negative charge at S163 regulates FXYD5/Na,K-ATPase interaction and that this interaction modulates cell migration across a wound in airway epithelial cells.