Our results suggest that (1) the composition of stream bacterial communities change in response to watershed urbanization and (2) such changes may have important consequences for critical ecosystem functions such as denitrification.Watershed urbanization leads to dramatic changes in draining streams, with urban streams receiving a high frequency of scouring flows, together with the nutrient, contaminant, and thermal pollution associated with urbanization. These changes are known to cause significant losses of sensitive insect and fish species from urban streams, yet little is known about how these changes affect the composition and function of stream microbial communities. Over the course of two years, we repeatedly sampled sediments from eight central North Carolina streams affected to varying degrees by watershed urbanization. For each stream and sampling date, we characterized both overall and denitrifying bacterial communities and measured denitrification potentials. Denitrification is an ecologically important process, mediated by denitrifying bacteria that use nitrate and organic carbon as substrates. Differences in overall and denitrifying bacterial community composition were strongly associated with the gradient in urbanization. Denitrification potentials, which varied widely, were not significantly associated with substrate supply. By incorporating information on the community composition of denitrifying bacteria together with substrate supply in a linear mixed-effects model, we explained 45% of the variation in denitrification potential ( Streams occupy low lying points in landscapes and are thus strongly affected by the detrimental impacts of watershed urbanization  and nutrient inputs It is less clear how urbanization might affect the composition and function of bacterial functional groups . One particularly important functional group and function in streams is denitrifying bacteria (denitrifiers) and denitrification. Denitrification is the transformation of nitrate to nitrogen gas and is one of the few ways to permanently remove nitrate from surface waters. The global supply of nitrate has more than doubled over the last century Most denitrifiers are facultative anaerobes, using oxygen as the electron acceptor for organic matter oxidation whenever available and nitrate as the next best alternative electron acceptor under hypoxic or anoxic conditions. Denitrification is thus expected to be highest in habitats with low dissolved oxygen and high nitrate and organic carbon substrate supply. Urban streams tend to have low oxygen and high substrate concentrations We propose two mechanisms by which watershed urbanization could affect stream denitrification. The first mechanism focuses on direct effects of urbanization on denitrification through changes to nitrate and organic carbon concentrations. The second mechanism focuses on indirect effects of urbanization on denitrification through changes to denitrifier community composition . To compWe surveyed microbial communities and measured denitrification potentials in eight study streams located in the Raleigh-Durham area in the Piedmont region of North Carolina (USA) . We sele3 was collected. Sediment cores ranged from 1 to 12 inches in vertical depth, depending on the depth of the stream bedrock. Sediment cores were sieved (2 mm opening) and composited in the field, resulting in a single composited sample from each site on each date. Sediment subsamples for molecular analyses and denitrification potentials were kept on ice for transport to the laboratory and then stored at −80°C and 4°C, respectively. Denitrification potentials were measured within 48 hours of sample collection. It is worth noting that streamwater temperatures are nearly always above 4°C in these streams, so denitrification potentials may have been negatively affected by the sudden temperature change associated with transport and storage conditions. Although not ideal in some respects, it was important to keep samples cool to minimize changes to bacterial abundances following sampling.We collected sediments from streams on four sediment sampling dates – sampling date 1 (June 2008), 2 (December 2008), 3 (June 2009), and 4 (July 2009). All streams were open to public access and did not require permits for collecting sediments. Mud Creek and Lower Mud were dry on sampling date 4, so sediments were not collected from those streams on that date. To collect sediments, we randomly selected five points along each ∼100 m study reach. We then demarcated a 2 m segment of streambed  upstream and downstream of each selected point and used PVC corers (6.35 cm diameter) to take multiple sediment cores in each sampling area until a total volume of at least 4,024 cmWe collected streamwater samples from each site on the same day of sediment sampling and at least once per month from June 2008 to July 2009. Samples were field filtered through Whatman GF/F filters  and kept on ice for transport to the laboratory. All samples were stored at 4°C and analyzed for nitrate and total organic carbon (TOC). We measured nitrate with an ion chromatograph equipped with an AS18 anion column and KOH eluent generator . We measured TOC as non-purgeable organic carbon with a TOC analyzer . For all statistical analyses of nitrate and TOC concentrations, we used all available measurements from the month of each sediment sampling date to calculate a mean value for that particular site and sediment sampling date.While pharmaceuticals, herbicides, and other toxic chemicals can also contaminate urban streams, we chose to focus primarily on heavy metals as an indicator of overall contamination intensity, because previous work has documented denitrifier community shifts in response to heavy metal contamination To measure heavy metal concentrations, sediments were re-sieved (1 mm opening), dried at 60°C for 48 hours, and then weighed out into three replicate 1 g subsamples per site per date for digestion (EPA method 3050B) To correct for differences in organic carbon content among sediment samples, we standardized all heavy metal concentrations using ash-free dry mass (AFDM). AFDM is the amount of dry mass of a sediment sample that is organic and can, therefore, be combusted. To determine AFDM values, we weighed out three replicate 5 g subsamples per site per date, dried at 60°C for 48 hours, weighed to get dry mass, combusted at 400°C for 4 hours, and weighed again to get combusted mass In addition to the concentrations of individual heavy metals, we also calculated an additional metric  to represent the cumulative heavy metal load in sediments by categorizing each heavy metal concentration into a quintile category  and then summing quintile values across all nine measured heavy metals for each site. Quintile values give equal weight to all metals, as opposed to a simple sum of concentrations, which would weigh metals with the highest concentrations (that may not be the most toxic) most heavily.The focus of this study was on assessing variability in community composition among sites, rather than within each site. We therefore extracted DNA from sediment subsamples taken from field composited (per site and sampling date) sediment cores. Extractions were done using PowerSoil kits , according to manufacturer instructions. Samples collected on sampling dates 1 and 2 were extracted in triplicate, while those collected on sampling dates 3 and 4 were extracted in duplicate. We used extractions from sampling dates 3 and 4 to characterize overall bacterial communities and extractions from sampling dates 1, 2, 3, and 4 to characterize denitrifier communities.5′-AGAGTTTGATCCTGGCTCAG, HEX labeled) 5′- ACGGGCGGTGTGTACAAG) We amplified bacterial 16S rRNA genes with the bacterial primer set 8F (nirK and nosZ. The nirK primer set was nirK1F (5′- GG(A/C)ATGGT(G/T)CC(C/G)TGGCA, FAM labeled) and nirK5R (5′-GCCTCGATCAG(A/G)TT(A/G)TGG) nosZ primer set was nosZ-F (5′- CG(C/T)TGTTC(A/C)TCGACAGCCAG, FAM labeled) and nosZ1622R (5′- CGC(G/A)A(C/G)GGCAA(G/C)AAGGT(G/C)CG) nirK and nosZ denitrifier PCRs.We amplified denitrifier DNA with two functional gene primer sets – nirK is a functional gene that encodes the copper containing form of nitrite reductase, which catalyzes the first step in the denitrification pathway. Each of 33 nirK PCR cycles consisted of 30 seconds at 95°C, 30 seconds at 46°C, and 45 seconds at 72°C. Primers were also tested for the amplification of nirS, which encodes an alternate form of nitrite reductase , but repeated amplification difficulties with the primer set (nirS1F and nirS6R) nosZ is a functional gene that encodes for nitrous oxide reductase, which catalyzes the last step in the denitrification pathway. Each of 35 nosZ PCR cycles consisted of 30 s at 94°C, 60 s at 53°C, and 60 s at 72°C. Each DNA extraction (two to three per site by sampling date) was amplified in triplicate PCRs for both denitrifier primer sets.nirK products, or Mn lI for nosZ products. Endonucleases were chosen based on test runs with different endonucleases; we used endonucleases that yielded the largest number of fragments, as visualized on 1% agarose gels, for that particular PCR product type. All endonucleases were from New England Biolabs . Subsequent electrophoresis runs were done with an ABI Prism 3100 genetic analyzer  using a 1000 ROX size standard (Applied Biosystems).Resulting PCR products were composited , cleaned with Qiaquick PCR purification kits , checked for appropriate sizes by agarose gel electrophoresis, and then used to generate terminal restriction fragment length polymorphism (TRFLP) profiles with either endonuclease Msp I for 16S rRNA products, Hae III for To process TRFLP data, we used T-REX software The TRFLP method is a cost-effective tool that has been accepted as an appropriate and efficient means of assessing overall dissimilarity among microbial community composition We used denitrification enzyme activity (DEA) assays to measure denitrification potentials 2) flushing. The incubation was initiated by adding 10 mL of acetylene (C2H2) gas to each flask. C2H2 inhibits nitrous oxide (N2O) reduction (the last step of the denitrification pathway), allowing N2O to accumulate in the flask headspace. Flasks were continuously shaken (125 rpm) on reciprocal shakers during the 90 minute incubation. Gas samples were taken at the start of the incubation and every 30 minutes thereafter. Chloramphenicol inhibits the synthesis of new enzymes and bacterial communities are unlikely to change significantly during the short incubation period After adding media, flasks were topped with butyl rubber stoppers  to achieve airtight conditions and made anaerobic by three successive cycles of evacuation and nitrogen gas  the concentration of denitrification enzymes, which reflects stream conditions at the time of sampling, and 2) the structure of the denitrifier community, which reflects a legacy of stream conditions over a period of time leading up to the time of sampling. DEA assays are widely used as a valid means of comparing denitrification rates among sites 2O concentrations using a Teledyne Tekmar 7000 headspace autosampler  to inject samples into a Shimadzu GC-17A ver.3 gas chromatograph with a Porapak Q column and electron capture detector . We used Bunsen coefficients to determine N2O concentrations in each sample and calculated rates of N2O production as the average rate observed over any 30 minute interval. N2O production rates were then divided by the dry mass of sediments in the flask to calculate denitrification potentials (ng N/g sediment/hr). Statistical analyses of denitrification potentials were based on averaged potentials across all five DEA assay replicates done for each site on each sampling date.We measured NTo explore relationships among measured variables, we used R 2.11.1 (R Core Development Team) software to conduct simple linear regressions. To equalize variances and normalize residuals, denitrification potential, nitrate, TOC, and heavy metal values were natural log transformed, while watershed % IC and % development were arcsine square root (arcsq) transformed prior to this and all subsequent data analyses.a priori into three groups: low , intermediate , and high  impervious cover streams  with watershed impervious cover as a factor. That is, we categorized streams  streams .F-statistic by comparing the total variance explained by sample identity  to that explained by random permutations of sample identities. To avoid pseudoreplication, permutations  were constrained by sampling date. Calculations were based on presence-absence matrices and Jaccard distance measures.To test the null hypothesis of no difference in community composition among streams, we conducted permutational multivariate analysis of variance (perMANOVA) To visualize differences in community composition among sites, we created non-metric multidimensional scaling (NMS) ordinations in R using the nmds function in ecodist Following the ordination, we analyzed potential correlations between bacterial community composition and urbanization intensity by regressing mean ordination scores for each primer set against arcsq-transformed % IC nirK and nosZ ordination scores as fixed effects and sampling date, nested within site as a random effect. We tested auto-correlation of residuals with a simple autoregressive model of order 1 2 based on a likelihood ratio test We built a linear mixed-effects (LME) model of log-denitrification to compare the explanatory power of substrate concentration versus denitrifier community composition variables. We used LME because the method can account for non-independence of errors  by differentiating between fixed versus random effects nirK ordination axes scores, and all three nosZ ordination axes scores. Substrate concentrations were means of all available measurements from the month of each sediment sampling date. We simplified the complete model by sequentially removing the least significant term and using a likelihood ratio test to compare the deviance of the simpler versus more complex model We started with a complete model that included log-nitrate, log-TOC, all three We built a second LME model with interaction terms to explore potential interactions between substrate concentration and denitrifier community composition. We used the same random effects structure and model simplification approach as for the first model. For the second LME model, the complete model included log-nitrate, log-TOC, and the two best performing (based on results of the first LME model) community composition parameters as single terms, along with all possible two-way interaction terms. We did not include more single terms and interactions because of the relatively small dataset size (n = 30).3-N L−1) and TOC  concentrations across sites. Study streams also had a wide range of heavy metal concentrations (Study streams varied widely in terms of watershed impervious cover (0.5 to 39.4%), and watershed development (4.4 to 100%) . Monthlytrations . Total mEphemeroptera, Plecoptera, and Trichoptera (EPT) Macroinvertebrate surveys conducted within these same study streams revealed substantial declines in the diversity of sensitive macroinvertebrate species within the families nirK denitrifier (n = 119 OTUs), and 20 for nosZ denitrifier (n = 139 OTUs) communities. For reference, the mean relative fluorescence units for OTUs retained after data processing was 371 for overall bacterial, 301 for nirK denitrifier, and 1054 for nosZ denitrifier communities. Microbial community composition was significantly affected by watershed urbanization. Across these eight streams, the composition of overall bacterial, nirK denitrifier, and nosZ denitrifier communities clustered into the three groups we defined based on % IC: low, intermediate, and high impervious cover streams .The mean relative fluorescence units for OTUs that were present in less than 5 percent of samples and therefore eliminated was 107 for overall bacterial (n = 101 OTUs), 17 for nirK ordination, and axis 3 for the nosZ ordination  . The linear regression of nosZ NMS axis 3 against arcsq-transformed % IC was also highly significant .The linear regression of overall bacterial NMS axis 1 against arcsq-transformed % IC was highly significant (F= 0.005) . The sam−1 hr−1, with a mean DEA of 195 ng N g sediment−1 hr−1 . Log-denitrification potentials were also not significantly different across sampling dates .Denitrification potentials ranged between 41 and 561 ng N g sedimentt−1 hr−1 . WatershnirK ordination scores were removed from the final, best fitting model , the final model (AIC = 61.21) had lower deviance .There was no evidence of autocorrelation of observations within groups, suggesting that errors were normally distributed within groups . In the first LME model without interaction terms, log-nitrate, log-TOC, and all ng model . The finnosZ ordination axis 1, were deleted from the final, best fitting model .In the second LME model with two-way interaction terms, the starting model included log-nitrate, log-TOC, and nosZ ordination axes 1 and 2, along with all two-way interactions. We used the ordination axes 1 and 2 because they explain a larger proportion of the variation in composition than any other combination of axes. Log-nitrate, log-TOC, and all two-way interactions, except that between log-TOC and ng model . The finnirK) or nitrous oxide (nosZ) reductase primers were used to characterize communities. Knowledge of denitrifier community composition, in turn, greatly improved our ability to capture observed variations in denitrification potential.Watershed urbanization imposes numerous stressors on stream inhabitants, including increased contaminant concentrations, stream water temperatures, and hydrologic disturbance Our study streams encompassed chemical , physical (stream water temperatures) and hydrologic (flashiness) gradients that were generally positively associated, though not always significantly, with watershed % IC. While several factors are likely to regulate bacterial community composition, we found that a single variable, watershed % IC, was strongly and significantly correlated with overall bacterial and denitrifier community composition across our study streams. Moreover, communities from low, intermediate, and high % IC streams were significantly different from one another.nirK denitrifiers, and nosZ denitrifiers) appeared to sort along the urbanization gradient identified in this study.Clearly, watershed land cover itself is not directly driving differences in bacterial community composition. Rather, % IC provides an integrative measure of the intensity with which watershed urbanization may be altering numerous different measured and unmeasured aspects of stream conditions, which are, in turn, regulating bacterial community composition. Given the limited number of sites in this study, we cannot identify the specific urban impacts primarily responsible for observed patterns in bacterial community composition. We can, however, conclude that watershed % IC is a good predictor of bacterial community composition in these streams and that watershed urbanization has the potential to strongly affect bacterial community composition, albeit through as yet unidentified pathways. Further support for these conclusions can be found in the consistency with which the composition of different groups of bacteria  that may play some role in driving observed differences in bacteria community structure in these study streams. Thus, further studies, using both observational and experimental approaches, are needed to definitively describe the relationships between watershed urbanization and bacterial community composition in receiving streams. Depsite this limitation, we feel this study provides intial insights into the potential for stream bacterial communities to respond to urban impacts in a similar manner as larger fish and insect communities.Model results indicate that denitrifier community composition may be more important than substrate supply in driving denitrification rates in our study streams. The first LME model captured an estimated 38% of the variation in denitrification potential among streams and included three composition parameters, but no substrate parameters. The second LME model captured an estimated 45% of the variation in denitrification potential among streams and included two composition parameters and an interaction term between composition and organic carbon. The interaction suggests that the capacity of communities to utilize available carbon substrates depends, in part, on which denitrifier taxa are present.These results suggest that one important pathway through which watershed urbanization may alter stream ecosystem functioning is by changing bacterial community composition in streams. That is, community composition can directly influence rates of functioning, independent of environmental factors. It remains unclear whether the observed links between urbanization-driven shifts in denitrifier community composition and denitrification potential extend to other microbe-mediated ecosystem processes in streams, such as decomposition and carbon cycling. Future studies could address this research gap by characterizing different functional groups of bacteria and measuring their function rates in streams with varying degrees of watershed urbanization.nosZ denitrifiers) in relation to urbanization intensity. Our findings suggest that the composition of nosZ denitrifier communities may be strongly affected by urbanization, which may speculatively help explain recent observations indicating that urban streams may release more nitrous oxide to the atmosphere than non-urban streams Study findings also provide novel insight into the potential impacts of watershed urbanization on the production of nitrous oxide gas from incomplete denitrification in streams. Nitrous oxide is a potent greenhouse gas that contributes to climate change and stratospheric ozone depletion. Although we did not measure nitrous oxide emissions, we did examine patterns of distribution for nitrous oxide reducing denitrifiers Click here for additional data file.Table S2Stream sediment mean water and organic carbon content.(DOC)Click here for additional data file.Table S3r) between watershed metrics and measured stream characteristics across all sites and dates.Correlations (Pearson's (DOC)Click here for additional data file.Table S4Denitrification potential.(DOC)Click here for additional data file.Table S5Non-significant terms deleted from the complete version of the first linear mixed-effects model of log-denitrification (without interaction terms).(DOC)Click here for additional data file.Table S6Non-significant terms deleted from the complete version of the first linear mixed-effects model of log-denitrification (with interaction terms).(DOC)Click here for additional data file.


Disturbance can both initiate and shape patterns of secondary succession by affecting processes of community assembly. Thus, understanding assembly rules is a key element of predicting ecological responses to changing disturbance regimes. We measured the composition and trait characteristics of plant communities early after widespread wildfires in Alaska to assess how variations in disturbance characteristics influenced the relative success of different plant regeneration strategies. We compared patterns of post-fire community composition and abundance of regeneration traits across a range of fire severities within a single pre-fire forest type– black spruce forests of Interior Alaska. Patterns of community composition, as captured by multivariate ordination with nonmetric multidimensional scaling, were primarily related to gradients in fire severity  and secondarily to gradients in soil pH and regional climate. This pattern was apparent in both the full dataset (n = 87 sites) and for a reduced subset of sites (n = 49) that minimized the correlation between site moisture and fire severity. Changes in community composition across the fire-severity gradient in Alaska were strongly correlated to variations in plant regeneration strategy and rooting depth. The tight coupling of fire severity with regeneration traits and vegetation composition after fire supports the hypothesis that disturbance characteristics influence patterns of community assembly by affecting the relative success of different regeneration strategies. This study further demonstrated that variations in disturbance characteristics can dominate over environmental constraints in determining early patterns of community assembly. By affecting the success of regeneration traits, changes in fire regime directly shape the outcomes of community assembly, and thus may override the effects of slower environmental change on boreal forest composition. A focus on plant traits can provide additional insights into community assembly by indicating why certain species are arrayed along observed gradients The structure and composition of plant communities is governed by a hierarchy of abiotic and biotic filters that act on the regional species pool to control which species and traits are present at a site Variation in the distribution and severity of fire disturbance influences landscape and stand-level vegetation patterns e.g. , in partFire is the dominant disturbance in the North American boreal forest, as reflected in regional and local species pools and landscape-level vegetation patterns th-century, boreal Alaska has warmed twice as rapidly as the global average, with warming since the 1950s apparently unprecedented in at least the last 400 years During the 20We measured the composition of early post-fire vegetation communities following the 2004 fire season, the largest annual area burned in Alaska’s 58-year fire record Interior Alaska is a region of extensive but discontinuous permafrost, with a continental climate and temperature extremes ranging from −70°C to +35°C Widespread fires in 2004 burned 2.7 million ha of forest in Interior Alaska Following an initial reconnaissance of the burned areas, we selectively identified 90 sites (∼30 sites in each burn complex) that encompassed the range of fire severities, site moisture, topographic positions, and geographic dispersion that we encountered in burned black spruce forests 2 sub-sample area at each site In 2005 and 2006 we measured environmental conditions, pre-fire stand composition, and fire characteristics at each site, as described in detail elsewhere http://www.tropicos.org). Nomenclature for lichens follows Salix spp., the relative proportion of individuals arising from each regeneration mode was estimated based on plant morphology and size.Measurements of post-fire plant community composition were made at each site in July 2006 to quantify the initial responses of the plant community to the 2004 wildfires. We conducted a relevé at each site following the methods of To estimate the potential of plant species to survive a fire via underground organs, we documented the rooting depth and root biomass distribution of species in eight burned and four unburned plots (hereafter “rooting plots”). Plots were selected to span a range of topographic positions representative of black spruce forests, and were located in the Caribou Poker Creeks Research Watershed (CPCRW) about 45 km northeast of Fairbanks . CPCRW iCorydalis sempervirens, Equisetum sylvaticum, and Luzula rufescens) not found in our unburned rooting plots, we made similar measurements in the eight burned plots within 1 km of our unburned rooting plots (http://www.lter.uaf.edu/data_b.cfm).We measured belowground distribution of root and rhizome biomass of all vascular species found along a randomly-oriented 30 m×5 m transect in each of the four unburned plots . At 5 m intervals, we measured the closest individual of all vascular species until 5 individuals of each species were obtained in each plot (n = 5/species/rooting plot). Belowground measurements were taken by carefully digging up the rooting structure of the individual until the lowest depth of secondary roots and rhizomes was found. For each individual we collected all belowground biomass of the rooting structure and separated it by the soil layer in which it was found. Soil layers were categorized as moss (living moss), upper duff (organic matter that is still identifiable), lower duff (unidentifiable and well-decomposed organic matter), and mineral soil. Belowground biomass was then dried and weighed in the laboratory. For three obligate post-fire species  were characterized based on our qualitative observations of the frequency with which we encountered them on different substrates across all 87 sites. For example, it was extremely rare to observe Ceratodon purpureus on any other substrate besides mineral soil, whereas Polytrichum spp. were observed to be anchored on both lower duff and mineral soil. Finally, all lichens observed after fire were residual lichens anchored in the unburned organic layer and were categorized as “rooting” in the moss substrate. Vascular species for which we did not have data on belowground biomass distribution (22 of the 81 understory species) were given zero values for all rooting substrates, so that these species provided no information on the distribution of this trait.We calculated the frequency of occurrence of dominant regeneration modes  between pair-wise distances of objects in ordination space and distances in the original distance metric We performed Nonmetric Multidimensional Scaling (NMDS) ordinations Our initial ordination results indicated that plant responses to moisture and fire severity across all 87 sites were complicated by a confounding association between pre-fire and post-fire organic layer depths that reflected a broad constraint of site moisture on fire severity field measurements).To evaluate the influence of fire on the relative distribution of plant regeneration traits, we compared the frequency of traits between the post-fire species pool and the observed trait distribution at each site We calculated the % frequency of regeneration strategy and rooting depth across species as well as within plant growth forms  and between fire severity classes. Finally, to determine the likelihood of a given regeneration strategy occurring on a given substrate, we closely examined species that exhibited multiple regeneration strategies across sites. We built a multinomial logistic regression model to test whether likelihood of a particular regeneration strategy was correlated to % of a site covered in organics as well % of the site covered in <3 cm organic depth . The 3 cm threshold in organic depth is an experimentally determined threshold for germination of deciduous tree species Sphagnum spp.), and 15 lichens (6 families). Of the 101 vascular plants, there were 4 trees (out of 6 tree species found in Interior Alaska), 43 forbs, 17 graminoids, 18 deciduous shrubs, 12 evergreen shrubs, and 7 seedless vascular plants. This is approximately 50% fewer plant species than found in mature black spruce stands (30% fewer vascular and 75% fewer nonvascular species) 2), alpha diversity (average species richness/site) was 19.93±0.51, beta diversity was 6.08 , and gamma diversity was 134 . Species richness varied significantly among fire severity classes , with richness declining as fire severity increased. Two years after burning, low-severity sites had an average richness of 25.34±1.09 species per site (mean ± SE), significantly greater than sites with moderate severity  or high severity .Across all 87 post-fire sites, we encountered 134 plant and lichen species distributed across 50 families . We obseOrdination of sites in species-space (Matrix A) captured three dominant gradients (axes) in species composition . The NMDAll three axes of the NMDS ordination were strongly correlated with gradients in measured environmental factors . The strPopulus tremuloides and forb Chamerion angustifolium were most commonly found in severely burned sites, while shrubs Betula nana and Rubus chamaemorus were positively associated with sites that burned at low severity. Among the species that showed strong correlations with Axis 2 (Equisetum arvense and Equisetum scirpoides) have been characterized as diagnostic species for nonacidic mature black spruce communities and a third species (Rhododendron tomentosum) is diagnostic of acidic black spruce communities Equisetum sylvaticum) in our study showed a strong correlation (r2>0.2) with axis 3 (climate).We assessed the correlation of ordination axes with the main species (A) matrix and a plant trait matrix (AS’). Many species that are indicators of a high or low severity fire were significantly correlated with Axis 1 , Table 1h Axis 2 , two  matrix. Associations with different regeneration modes and rooting substrates were predominantly expressed along the fire severity gradient represented by Axis 1 , Fig. 2d2 = 8.99, p = 0.0112, df = 2). It is therefore difficult to know if the correlation of plant traits with axis 1 is a consequence of site moisture or fire severity. To better disentangle the effects of site moisture and fire severity on post-fire community composition, we analyzed patterns of species composition across a subset of sites with minimized correlation between pre-fire and post-fire organic depth. In this subset of sites, observed volumetric soil moisture explained 8% of the variance in site-level CBI , a substantially weaker association than observed between observed volumetric moisture and CBI in the full dataset . We found very similar ordination patterns with these sites as we did with the full number of study sites  also exhibited this strategy, as they are able to resprout from points along the stem below the capitulum Salix spp. that recruited from seed and are a particularly important component of the colonizer group because they often persist through succession to mature stands Over 40% of the post-fire species displayed the “resprouter” strategy, making it the most common regeneration mode in the post-fire species pool . This asThe effect of fire severity as a filter on the distribution of plant regeneration traits can be visualized by comparing the observed, abundance-weighted distribution of regeneration traits in different severity classes  versus tRhododendron tomentosum and Vaccinium vitis-idaea) exhibited both resprouter and residual strategies, three deciduous trees/shrubs and two forbs  exhibited both resprouter and colonizer strategies, and one moss (Polytrichum commune) exhibited all three regeneration strategies. However, multinomial logistic regressions predicting the likelihood of a given regeneration strategy for a given species from site substrate characteristics were not significant (p>0.1).Although most post-fire species encountered were characterized by a single regeneration strategy across all sites, eight of the species exhibited site-dependent regeneration strategies. Two evergreen shrubs , community composition and structure in unburned, mature black spruce has been strongly linked to site moisture, mineral soil pH and elevation Nevertheless, variations in fire characteristics are strongly linked to environmental gradients, and we found significant associations between fire severity and site moisture across our study sites. Paleo-records suggest that this relationship has existed in boreal Alaska for as long as black spruce has dominated the landscape Observational results suggested that a few species had multiple regeneration strategies and therefore were able to regenerate across a broader range of fire severities. We were interested in whether we could use species with multiple regeneration strategies to test the importance of site substrate characteristics to a given regeneration strategy, or whether a species may exhibit preferential use of a regeneration strategy depending on substrate characteristics. Our inability to statistically predict regeneration strategy of a species at a given site was likely due to the low number of observations , however, poor prediction of regeneration strategy from substrate characteristics could also suggest that species with multiple regeneration strategies are responding to other environmental factors that act as filters on boreal forest composition Corydalis sempervirens) that is an obligate seed-bank species. Nevertheless, this species was positively associated with increases in fire severity, suggesting that the positive effects of improved seedbed quality outweigh negative effects on seedbank consumption even in severely burned sites, at least within organic-rich black spruce forests.Regeneration modes that were important in Alaska apply to many fire-prone ecosystems Betula nana, Rubus chamaemorus, Oxycoccus microcarpus, Eriophorum vaginatum, and Rhododendron tomentosum), as well as many lichens and mosses that currently define and dominate boreal Alaska Populus tremuloides, Chamerion angustifolium, and Equisetum arvense) and would likely increase with more severe fires. These responses to increasing fire severity could cause biogeographic shifts to occur more rapidly than would be expected from species’ direct responses to climate. Similarly, the higher productivity of species favored by high severity fire (e.g. those listed above) could amplify the expected stimulation of decomposition and nutrient turnover expected with climate warming Our results have important biogeographic and functional implications for Alaska’s boreal forest. The most striking differences in community composition occurred between low-severity burns and moderate-to-high-severity burns, mimicking post-fire colonization patterns of tree species Table S1Site/Trait Matrix (S×T) derived by multiplying Species/Site matrix (A×S and Species/Trait matrix (A×T) then relativizing data by site so that each set of traits summed to 1. A×T was derived by multiplying the species abundance by its plant trait dominance as measured in the field for each trait. Sites are sorted by fire severity.(XLSX)Click here for additional data file.Table S2Complete vascular and nonvascular species list across all study sites. Vascular species were verified in the University of Alaska herbarium and nonvascular species were verified from voucher species in the BNZ LTER working herbarium.(XLSX)Click here for additional data file.


Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and are located in a highly dynamic microenvironment called “niche” that influences all aspects of stem cell function, including homing, self-renewal and differentiation. Several studies have recently identified specific proteins that regulate the fate of SSCs. These studies also aimed at identifying surface markers that would facilitate the isolation of these cells in different vertebrate species. The present study is the first to investigate SSC physiology and niche in stallions and to offer a comparative evaluation of undifferentiated type A spermatogonia (Aund) markers  in three different domestic equid species . Aund were first characterized according to their morphology and expression of the GFRA1 receptor. Our findings strongly suggest that in stallions these cells were preferentially located in the areas facing the interstitium, particularly those nearby blood vessels. This distribution is similar to what has been observed in other vertebrate species. In addition, all three Aund markers were expressed in the equid species evaluated in this study. These markers have been well characterized in other mammalian species, which suggests that the molecular mechanisms that maintain the niche and Aund/SSCs physiology are conserved among mammals. We hope that our findings will help future studies needing isolation and cryopreservation of equids SSCs. In addition, our data will be very useful for studies that aim at preserving the germplasm of valuable animals, and involve germ cell transplantation or xenografts of equids testis fragments/germ cells suspensions. Spermatogonial stem cells (SSCs) are the undifferentiated male germ cells committed to the establishment and maintenance of spermatogenesis In addition to transmitting genetic information to the next generation, and being capable to repopulate the germ cell-depleted testis through the germ cell transplantation technique Dolichos biflorus agglutinin) Three SSC markers involved in the regulation of self-renewal and themaintenance of the SSC pool in mice have gained special attention. One is a transcription factor known as PLZF (promyelocytic leukaemia zinc finger) and the others are membrane receptors named GFRA1  and CSF1R (Colony stimulating factor 1 receptor) As reviewed by Oatley and Brinster (2012) Few reports in the literature have investigated the biology of equid spermatogonia All surgical procedures were performed by a veterinarian and followed approved guidelines for the ethical treatment of animals. The protocol was approved by the Committee on Animal Experimentation-CETEA, Federal University of Minas Gerais (Permit Number: 056/11). All surgery was performed under anesthesia and analgesia, and all efforts were made to minimize suffering. The animals were anaesthetized with acepromazine and xylazine intravenously followed 15 minutes later by ketamine. Following xylazine administration, lidocaine was administered subcutaneously along the incision line and by intrafunicular injection. Prophylactic and postoperative antibiotic and anti-inflammatory therapy was performed using benzylpenicillin and phenylbutazone respectively.Fourteen adult stallions (n = 14), two adult donkeys (n = 2) and four adult mules (n = 4) were used in this study. The animals were obtained from farms located in the Southeast region of Brazil (Minas Gerais province). For stallions, testes sampling was performed by orchiectomy during the breeding season (n = 7) (September to March in the South Hemisphere) and during the non-breeding season (n = 7). Testis from donkeys and mules were obtained during the breeding season.After orchiectomy, the testes were separated from the epididymides and weighed, then cut longitudinally with a razor blade into small fragments. Testes from eight stallions were fixed by immersion in 4% buffered glutaraldehyde for 12 hours. Tissue samples measuring 2 to 3 mm in thickness were routinely processed and embedded in glycol methacrylate  for histological and morphometric analyses. In order to characterize the different spermatogonial types using high resolution microscopy, samples measuring 1–2 mm in thickness were embedded in epoxy Stages of the seminiferous epithelium cycle (SEC) were characterized in stallions based on the development of the acrosomic system, morphology of the developing spermatid nucleus and in the overall germ cells associations We characterized the morphology of different spermatogonial types in stallions by analyzing images obtained from each stage of the SEC To qualitatively evaluate the presence of specific markers for SSCs , immunoblots were performed using total protein lysates from stallion (during the breeding and non-breeding seasons), donkey and mule testes stored at −80°C. For this evaluation, 300 mg of testis parenchyma from each individual investigated were placed in 0.9% NaCl containing protease inhibitors , and the tissues were homogenized and sonicated. After that, the lysates were centrifuged at 14,000×g for 20 minutes. Supernatants were collected, and then frozen at −80°C. Protein samples were diluted to 1∶2 in a solution of 10% sodium dodecyl sulfate , 2% glycerol, 0.2% bromophenol blue and, when reducing conditions were required, 1% beta-mercaptoethanol was added in 0.5 M TRIS buffer pH 6.8. The samples were boiled for 5 minutes. Denaturing 12% SDS polyacrylamide mini-gels were prepared and 25 µL samples were loaded into the wells. High-molecular-weight markers (Sigma Aldrich) were run parallel to the samples. Gels were run under a 90 mA current and the separated proteins were transblotted to nitrocellulose membrane for 75 minutes using a 100 mA current. The strips were blocked with 3% BSA (Sigma Aldrich) in TBS for 1 hour at room temperature. Proteins were detected through 90 minutes incubation, at room temperature, with one of the following primary antibodies diluted in TBS 1% BSA: polyclonal goat anti-GFRA1 , monoclonal mouse anti-PLZF  and polyclonal rabbit anti-CSF1R . Subsequently, the strips were rinsed three times with TBS 0.05% Triton-X buffer and then incubated for 1 hour with biotinylated anti-goat , anti-mouse  or anti-rabbit  IgG antibodies at room temperature. Before exposing the strips to streptavidin solution  for 15 minutes at room temperature, they were rinsed three times with TBS 0.05% Triton-X buffer. Prior developing the reaction, the samples were then rinsed twice with TBS 0.05% Triton-X and once with pure TBS buffer. The strips were exposed to DAB solution (Sigma Aldrich) in TBS containing chloronaphthol (Sigma Aldrich), methanol and hydrogen peroxide (Sigma Aldrich) during approximately 1 minute and then washed in water. Finally, the strips were scanned with an Epson Perfection 4990 photo scanner.in situ expression of proteins analyzed by Western blotting, we performed immunostaining using the immunoperoxidase method. Slides were analyzed by light microscopy. The tissue samples were fixed in Bouin and embedded in paraplast (Sigma Aldrich). Five micrometer sections were immunostained using protocols specifically developed for each antigen and with antibody dilutions previously tested. After dewaxing and rehydration, antigen retrieval was performed in citrate buffer (pH 6.0) for 5 minutes after boiling in a microwave oven . For immunohistochemistry, endogenous peroxidase was quenched for 30 minutes with 3% H2O2 (Sigma Aldrich) in PBS at room temperature. Non-specific binding was blocked with Ultra-V-Block (Thermo Scientific) for 30 minutes at room temperature. Primary antibodies GFRA1 , PLZF , CSF1R  and Cleaved Caspase-3  were applied and the slides were incubated overnight at 4°C. The slides were exposed to biotinylated rabbit anti-goat , horse anti-mouse  and goat anti-rabbit  immunoglobulin G (IgG) antibodies during 60 minutes at room temperature. Detection of the signal was performed by incubating the sections in streptavidin for 10 minutes at room temperature, followed by the reaction with peroxidase substrate 3,3′-diaminobenzidine  and counterstaining with hematoxylin (Merck). Following dehydration, sections were mounted and analyzed.In order to evaluate the In order to quantify the percentage of GFRA1(+), PLZF(+) and CSF1R(+) in type A undifferentiated spermatogonia (Aund) , 200 Aund cells were randomly evaluated for each stallion (n = 6), donkey (n = 2) and mule (n = 4). Only for stallions, qualitative double staining immunofluorescence was also performed in the present study. Non-specific background was blocked for 30 minutes with Ultra-V-Block (Thermo scientific) incubation. The proteins were detected using primary antibodies at the following dilutions: GFRA1 (1∶50), PLZF (1∶50) or CSF1R (1∶100). After the incubation with Alexa Fluor secondary antibodies  for 1 hour at room temperature, the confocal images were obtained using 40X and 60X oil immersion objectives in a 510 META Laser Scanning Confocal Microscope , equipped with 488 and 543 nm lasers. Dual channel images were obtained by sequential scanning.http://rsb.info.nih.gov/ij/). Fifteen randomly chosen fields/images (8100 points) were scored per testis for each animal (n = 8) at 200× magnification. In order to investigate the influence of seasonality in the testicular components, these stallions were divided separated in two groups of four animals.The volume densities of the testicular tissue components in stallions were determined by images captured by light microscopy, using a 540-intersection grid from ImageJ software  evaluations were considered. In this regard, images from 10 seminiferous tubules cross-sections of each stage of the SEC previously characterized were obtained for each animal and the seminiferous tubules cross-sections were subdivided into four regions, as follows: I) adjacent to another tubule (Tubule-Tubule contact or TT); II) adjacent to the interstitial compartment without blood vessels (Tubule-Interstitium without blood vessels or TI−BV); III) adjacent to the interstitial compartment containing blood vessels (Tubule-Interstitium with blood vessels or TI+BV); and IV) adjacent to the interstitial compartment containing connective tissue (TIC). Considering that the tubular circumference corresponds to 360 degrees, the numbers of Aund cells obtained in the four different regions evaluated were expressed per degree Parametric data were analyzed by ANOVA and differences were compared by Tukey’s test, whereas the Student’s “t” test was used for two-parameter analysis. Non-parametric data were compared by Chi-square test. All analyses were performed using the GraphPad Prism 5 . All data were expressed as mean ± SEM (standard error of the mean) and the significance level considered was p<0.05.Based on the development of the acrosomal system in spermatids, twelve stages of the SEC were characterized in stallions ; I–XII. Based on the criteria previously described in our laboratory for donkeys Contrary to Aund cells, which were present in all stages, differentiating spermatogonia were only observed in well-defined stages of the SEC . In thisUsing immunohistochemistry, we detected expression of GFRA1, PLZF and CSF1R by Aund spermatogonia in all three equid species investigated. Aund cells could be visualized as single, pairs and chains of spermatogonia , and 5A.As explained above, Aund include A single (As), A paired (Apr), and A aligned  spermatogonia. In the immunolabeling quantitative analysis for horses, we observed that approximately 90 and 80% of Aund cells expressed GFRA1 and PLZF respectively  and 4C. We measured the volume density of components of the testicular parenchyma in stallions during the breeding ; 1 and nAccording to the four seminiferous tubules regions considered, and based on morphological and GFRA1 immunolabeling evaluations , GFRA1+. Except To our knowledge, the present study in the first that investigates the SSC niche in stallions. This study also offers a comparative evaluation of Aund markers in three different domestic equid species . In order to better understand the topology of cellular compartments within the testis, we first characterized the stages of the SEC in stallions according to acrosome development in spermatids. We also characterized the different types of spermatogonia in this species and investigated their distribution in four different regions of seminiferous tubules cross-sections, using morphological and Aund markers criteria. Based on the distribution of GFRA1-positive spermatogonia, our findings strongly suggest that, similar to the other vertebrate species investigated in this respect, Aund cells in stallions were preferentially located in the areas facing the interstitium, particularly those nearby blood vessels. Also, all three Aund markers investigated  were expressed in stallions, donkeys and mules, which was confirmed by Western blot analysis. These results suggest that the molecular mechanisms driving SSCs physiology and maintenance of the niche are conserved among mammals.The GFRA1 membrane receptor is considered one of the most useful markers of Aund Kit and Crtc1 gene expression, whose products are both involved in spermatogonial differentiation Although slightly less frequent, expression of the transcription factor PLZF was observed in ∼80% of Aund in equids. This result, also confirmed by Western blotting, is similar to findings already described in rabbits In the present study, we also show that like in mice In comparison to the data obtained for rodents To our knowledge, the present study is the first to investigate the distribution of GFRA1 positive spermatogonia in equids. Our data strongly suggest that the interstitium, particularly the vascular network, may play an important role in the regulation of equid SSCs, like in other species Due to drastic alterations in the intertubular compartment volume density during the sexually inactive period, an evident niche was not observed in golden hamsters In the present investigation, we defined twelve stages of spermatogenesis in equids according to overall germ cells composition and acrosome development. A similar situation was previously described in donkeys The different types of stallion spermatogonia exhibited morphological characteristics already described in donkeys In summary, our findings suggest that the molecular mechanisms driving SSCs physiology and niche maintenance are conserved among mammals. Also, the characterization of Aund spermatogonia performed in the present study provides for the first time a foundation for future studies of SSCs self-renewal and differentiation in equids. Our data will facilitate isolation and cryopreservation of these cells. In addition, our findings will be very useful for studies involving germ cells transplantation and xenografts of equid testis fragments/germ cells suspensions to preserve the germplasm of valuable animals.


Aging is associated with increased oxidative stress levels and impaired neovascularization following ischemia. CuZnSOD has an important role to limit oxidative stress in the vasculature. Here we investigated the role of CuZnSOD for the modulation of ischemia-induced neovascularisation during aging.−/− mice. We found that blood flow recovery after ischemia and vascular density in ischemic muscles were significantly reduced in older compared to young WT mice. Both in young and older mice, CuZnSOD deficiency led to a further reduction of neovascularization. Accordingly, the resulting neovascularisation potential in a young CuZnSOD−/− mouse was similar to that of an older WT mouse. Oxidative stress levels were also increased to similar levels in the ischemic muscles of young CuZnSOD−/− and older WT mice. To identify potential mechanisms involved, we investigated the effect of aging and CuZnSOD deficiency on the number and the function of endothelial progenitor cells (EPCs). Both aging and CuZnSOD deficiency were associated with reduced number of bone marrow and peripheral EPCs. The effect of moderate aging alone on specific functional activities of EPCs  was modest. However, CuZnSOD deficiency was associated with severe age-dependent defects in EPC functional activities.Hindlimb ischemia was surgically induced in young (2- month-old) or older (8-month-old) wild type (WT) and CuZnSODCuZnSOD deficiency is associated with accelerated vascular aging and impaired ischemia-induced neovascularization. Our results suggest that in the context of aging, CuZnSOD has an essential role to protect against excessive oxidative stress in ischemic tissues and preserve the function of EPCs. In patients with cardiovascular diseases, the capacity of the organism to develop new blood vessels (neovascularization) constitutes an important adaptive mechanism against ischemia Advanced age is a major risk factor for coronary and peripheral artery disease. In addition, one of the consequences of aging is a decline in the ability of the organism to respond to different stresses, including ischemia. For instance, advanced age is associated with a defect in neovessel formation following arterial occlusion in different animal models −/−) mice. Moreover, the functional importance of CuZnSOD for the age-dependent modulation of ischemia-induced neovascularization is currently unknown.A loss of the adaptive response to oxidative stress with the passage of time is one of the major characteristic of aging Here we used a mouse model of hindlimb ischemia to study the effect of CuZnSOD deficiency on oxidative stress levels and reparative ischemia-induced neovascularization in the context of aging. We found that CuZnSOD-deficient mice exhibit accelerated vascular aging and impaired neovascularization in response to ischemia. We also demonstrate that CuZnSOD has an essential role to maintain the functional activities of EPCs in older animals.tm1Leb/J) mice obtained from Jackson Laboratory . Four groups of mice were studied: young (2-month-old) and older (8-month-old) homozygous CuZnSOD-deficient (CuZnSOD−/−) mice were compared to young and older wild-type (CuZnSOD+/+) littermates. Mice were maintained in 12 hours light-dark cycle and fed ad libitum. Genotyping of each mouse was assessed by polymerase chain reaction of DNA isolated from tail biopsy samples as described on the Jackson laboratory Web site.Mice used for this study were derived from breeding pairs of heterozygous CuZnSOD-deficient  was approved by the Comité Institutionnel de Protection des Animaux (CIPA) of the Centre Hospitalier de l'Université de Montréal (CHUM). Unilateral hindlimb ischemia was surgically induced in mice as previously described Hindlimb perfusion was measured with a Laser Doppler Perfusion Imager (LDPI) system . After anesthesia with a ketamine-midazolam solution , consecutive measurements were obtained after scanning of the same region of interest (leg and foot) with the LDPI. The perfusion signal was split into six different intervals, each displayed in a separate color. Low or no perfusion was displayed in dark blue, whereas the highest perfusion interval was displayed in red. Color photographs were recorded and analyses were performed by calculating the average perfusion of the ischemic and non-ischemic hindlimb. To account for variables such as ambient light and temperature, the results are expressed as the ratio of perfusion in the left (ischemic) vs. right (non-ischemic) hindlimb. The mice were killed at predetermined arbitrary time points after surgery with an overdose of sodium pentobarbital.2). Serial sections were cut at three different levels, and representative fields were analyzed by counting the number of capillaries in each field. Arterioles were identified using a Modified Verhoeff Van Gieson Elastic Stain Kit . Serial sections were cut at three different levels, and positive vessels identified by the presence of a continuous internal elastic laminae and muscle spindle were analyzed for the entire section under a 100× magnification. To characterize premature vascular senescence, goat polyclonal antibodies against mouse p16 and p53  were used. To investigate local oxidative stress levels in ischemic muscles, an antibody against nitrotyrosine  was used. Nitrotyrosine immunostaining is a reflection of peroxinitrite produced locally in tissues. Intensities of fluorescence were measured and analyzed using computer-based analysis (Metamorph) with the same threshold for all sections under a 200× magnification. The specificity of the test was confirmed by pre-incubating the antibody with 10 mM nitrotyrosine (data not shown). To evaluate superoxide production in ischemic muscles, dihydroethidium (DHE) fluorescence labeling was performed. After bones had been carefully removed at the level of the gastrocnemius muscle, ischemic muscles were put in 25% sucrose solution for 10 minutes. Muscles were frozen in eppendorf tubes at −80°C for 24 hours, mold in OCT and kept at −20°C. 3 µm frozen sections were made at three different levels in the ischemic muscles. Sections were labeled with 10 µM DHE  for 30 minutes. Intensities of fluorescence were measured and analyzed using a computer-based software (Metamorph) with the same threshold for all sections under a 100× magnification. The specificity of the test was confirmed by pre-incubating the section with superoxide dismutase polyethyleneglycol (PEG-SOD) 500 U/ml for 1 hour (data not shown).For immunohistochemistry, whole ischemic hindlimbs were immediately fixed in tissue-fix overnight. After bones had been carefully removed, 3-µm thick tissue transverse sections of the hindlimbs were cut at the level of the gastrocnemius muscle and paraffin-embedded so that the whole leg could be analyzed on each section. Identification of endothelial cells was performed by immunostaining for platelet endothelial cells adhesion molecule-1 (PECAM-1 or CD31) with a rat monoclonal antibody directed against mouse CD31 . Capillaries, identified by positive staining of CD31 and appropriate morphology, were counted by a single observer blinded to the treatment regimen under a 200× magnification to determine the capillary density  of protein extract was separated in nonreducing 15% polyacrylamide gel and electroblotted on nitrocellulose membranes. The membranes were probed with 1∶500 CuZnSOD antibody . Specific proteins were detected by chemiluminescent reaction . Results are expressed as density values normalized to Ponceau red staining.Progenitor cells contained in the total viable population derived from the spleen were analysed by FACs , using fluorescence-coupled antibodies against the following markers: CD34-FITC, VEGFR2 (Flk-1)-PE and CD117 (c-kit)-APC . Corresponding isotype-matched immunoglobulins for the three antibodies gave similar low levels of fluorescence compared to unstained cells confirming that the primary antibody binding is specific and not a result of non-specific Fc receptor binding or other protein interactions. The results are expressed as the percentage of triple-marked cells from the initial population. Cell phenotypes were determined by the analysis of 300 000 events.Seven days following ischemia, mononuclear cells were mechanically isolated from the femora, tibiae and humeri by flushing the bone marrow cavities using culture medium. After red blood cell lysis and washing, mononuclear cells were plated on 0.05% fibronectin (Sigma) and cultured in complete Medium 200 containing 20% FBS. After 4 days in culture, non-adherent cells were removed by thorough washing with PBS. Adherent cells were stained with 1,1′-dictadecyl-3,3,3′,3′ tetramethyllindocarbocyanine perchlorate-acetylated low-density lipoprotein  and FITC-labeled lectin BS-1 . Spindle-shaped cells were observed, and the vast majority of adherent cells (95%) were found to be double-positive for the uptake of DiI-acLDL and binding of FITC-labeled lectin. FACS analyses revealed that 83% of adherent cells express CD45, 57% CD14, 18% CXCR4, 9% CD31 and 8% Sca-1. These cells were shown to migrate in response to VEGF stimulation and were capable of incorporation into a network of tubular-like structures when cocultured with mature endothelial cells. On the basis of these morphological and functional characteristics, these cells were defined as early outgrowth EPCs.2−, DHE is oxidized to ethidium bromide, which binds to DNA. EPCs were plated at a density of 15000/well in a 96-well plate in complete Medium 200, 20% FBS for 24 hours. The fluorescence reaction was carried out by incubating EPCs with DHE (2 µmol/L) for 30 minutes. DHE fluorescence was detected with a fluometer  using a 585 nm filter. The specificity of the test was confirmed using 50 ng/ml TNF-alpha and 500 U/ml PEG-SOD as positive and negative controls, respectively (data not shown).Dihydroethidium (DHE) was used to evaluate the presence of superoxide anion oxygen radicals. In the presence of OCell migration was assessed using a modified Boyden chamber assay. Polyvinylpyrrolidine-free polycarbonate filter Transwell inserts  were coated with 0.1% gelatin. Inserts were placed in a 24-well plate containing DMEM with 50 ng/ml of VEGF. EPCs (15 000) were added to the upper chamber of the inserts in DMEM, 0.1% fetal bovine serum. Cells were allowed to migrate from the upper to the lower chamber for 6 h at 37°C. Non-migratory cells were removed from the upper chamber by wiping the upper surface with an absorbent tip. Cells that had migrated to the lower side of the transwell insert were fixed for 10 min with 3.7% formaldehyde and stained with hematoxilin. The number of cells that had migrated was counted in six different representative high power (200×) fields per insert (2 inserts/condition). The data are presented as number of migrated cells ± standard error from the mean.5 cells (passage 2–5) in each well of 24 wells plate. HUVECs were pretreated for 12 hours with tumor necrosis factor-α  (1 ng/ml). Cells were stained with DAPI and fixed with 2% paraformaldehyde. EPCs were labeled with DiI-acLDL and 11 000 EPCs were added to each well and incubated for 3 hours at 37°C. Non-attached cells were gently removed with PBS and adherent EPCs were fixed with 2% paraformaldehyde and counted in three random fields. The data are presented as a ratio of the number of adherent EPCs/HUVECs per field ± standard error of the mean.A monolayer of human umbilical vein endothelial cells (HUVECs) was prepared 48 hours before the assay by plating 2×10EPC (4000) labeled 1 hour with DiI-acLDL were co-plated with HUVECs (14 000) in 96-well plates that had been precoated with 50 µl of growth factor reduced Matrigel Matrix (BD Biosciences) and cultured at 37°C for 6 h with 50 ng/ml of VEGF. Tubular-like structures were photographed and the number of incorporated EPCs in tubules was determined in 6 random fields. A tube was defined as a straight cellular segment connecting two cell masses (nodes). The data are presented as number of incorporated EPCs/tube ± standard error of the mean.All results are expressed as mean ± SEM. Statistical significance was evaluated using a one-way ANOVA followed by Newman-Keuls post-hoc test for multiple group comparison. A value of p<0.05 was interpreted to denote statistical significance.−/− mice was similar to that of older WT mice . At the microvascular level, capillary density was significantly reduced in CuZnSOD-deficient mice (−/− mice that were similar to that of older WT mice (In control wild type (WT) mice, we found that aging is associated with a significant decrease of CuZnSOD expression in hindlimb muscles . To charent mice . Moreove WT mice .−/− and WT mice. Figure S2).Because excessive oxidative stress has been associated with impaired angiogenesis in different situations, we compared oxidative stress levels in ischemic muscles of young and older CuZnSODTo identify potential mechanisms involved in the detrimental effect of aging and CuZnSOD deficiency on ischemia-induced neovascularization, we quantified the number of EPCs in the different animal groups at day 7 after hindlimb ischemia. Early outgrowth bone marrow EPCs were isolated in culture and shown to endocytose acLDL and bind BS-1 lectin . We founSuperoxide generation was measured in EPCs isolated from the bone marrow using DHE immunostaining. A small non-significant increase of superoxide generation was seen in EPCs isolated from older vs. young wild-type animals. However, CuZnSOD deficiency was associated with a very important increase of EPC superoxide levels in older animals . We also−/− mice have been shown to exhibit acceleration of several age-related diseases including skeletal muscle atrophy +/− mice −/− mice −/− mice are relatively modest, and the effect of aging in these conditions is unknown. Here we studied the response to ischemia in both young and older homozygous CuZnSOD-deficient mice in order to specifically define the role of CuZnSOD and aging for the modulation of neovascularization.To our knowledge, the present study is the first one to investigate the role of CuZnSOD for postnatal ischemia-induced neovascularization in the context of aging. Since oxidative stress is thought to contribute to the pathophysiology of aging −/− mice exhibit neovascularisation levels that are similar to that of older control mice, both at the macrovascular (Laser Doppler) and microvascular (vascular density) levels. In older CuZnSOD−/− mice, surgically-induced hindlimb ischemia resulted in severe necrosis and auto-amputation in the second week after surgery (data not shown) and the current study was therefore limited to a one-week follow-up. Although impaired ischemia-induced neovascularisation has previously been shown with aging Our results indicate that CuZnSOD expression in hindlimb muscles is decreased with aging. We also demonstrate that CuZnSOD deficiency accelerates vascular aging in the context of tissue ischemia. In fact, young CuZnSODThe mechanisms by which CuZnSOD modulates neovascularization in the context of aging are potentially diverse. Ischemic tissues are characterized by high levels of inflammatory cytokines that can activate ROS production In the current study, we propose that another important factor involved in the accelerated vascular aging of CuZnSOD-deficient mice is endothelial progenitor cells (EPCs) dysfunction. The importance of EPCs for the development of neovessels in different physiological and pathological situations has recently been recognized Age-dependent endothelial dysfunction has been shown to correlate with impairment in the functional activity of EPCs In summary, our study demonstrates that CuZnSOD deficiency leads to accelerated vascular aging and impaired post-natal neovascularization after ischemia. We propose that in the context of aging, CuZnSOD has an essential role to protect against excessive oxidative stress in ischemic tissues and preserve the number and the functional activities of EPCs. The finding that CuZnSOD is essential to maintain EPC functions might have important clinical implications. In ‘healthy’ aging, stable CuZnSOD expression could limit oxidative stress accumulation and help maintaining EPC function and vascular integrity. However, it has been shown that clinical conditions such as diabetes and chronic heart failure are associated with defective levels of antioxidant enzymes, including CuZnSOD Figure S1+/+) mice (n = 2–4/group).CuZnSOD expression in hindlimb muscles of young (Y) and older (O) wild type Click here for additional data file.Figure S2+/+ and CuZnSOD−/− mice.Histo-immunostaining showing expression of p53 (A) and p16 (B) in ischemic hindlimb muscles of young (Y) and older (O) CuZnSOD Representative results are shown.(TIF)Click here for additional data file.


Cerebrospinal fluid shunt infection can be recalcitrant. Recurrence is common despite appropriate therapy for the pathogens identified by culture. Improved diagnostic and therapeutic approaches are required, and culture-independent molecular approaches to cerebrospinal fluid shunt infections have not been described.To identify the bacteria and fungi present in cerebrospinal fluid from children with cerebrospinal fluid shunt infection using a high-throughput sequencing approach, and to compare those results to those from negative controls and conventional culture.This descriptive study included eight children ≤18 years old undergoing treatment for culture-identified cerebrospinal fluid shunt infection. After routine aerobic culture of each cerebrospinal fluid sample, deoxyribonucleic acid (DNA) extraction was followed by amplification of the bacterial 16S rRNA gene and the fungal ITS DNA region tag-encoded FLX-Titanium amplicon pyrosequencing and microbial phylogenetic analysis.The microbiota analyses for the initial cerebrospinal fluid samples from all eight infections identified a variety of bacteria and fungi, many of which did not grow in conventional culture. Detection by conventional culture did not predict the relative abundance of an organism by pyrosequencing, but in all cases, at least one bacterial taxon was detected by both conventional culture and pyrosequencing. Individual bacterial species fluctuated in relative abundance but remained above the limits of detection during infection treatment.Numerous bacterial and fungal organisms were detected in these cerebrospinal fluid shunt infections, even during and after treatment, indicating diverse and recalcitrant shunt microbiota. In evaluating cerebrospinal fluid shunt infection, fungal and anaerobic bacterial cultures should be considered in addition to aerobic bacterial cultures, and culture-independent approaches offer a promising alternative diagnostic approach. More effective treatment of cerebrospinal fluid shunt infections is needed to reduce unacceptably high rates of reinfection, and this work suggests that one effective strategy may be reduction of the diverse microbiota present in infection. Cerebrospinal fluid (CSF) shunt placement has been the mainstay of treatment for hydrocephalus for over 50 years. For children with first CSF shunt infection, prognosis for clearance is poor, with re-infection rates ranging from 12–26 percent. in vitro exist in a largely dormant, antibiotic-resistant mode, This recalcitrance of CSF shunt infections to treatment may be related to complex, adherent assemblages of microbes, known as biofilms, on the shunt catheter surface. Given the recalcitrant nature of CSF shunt infection, the emerging evidence of a role for biofilms, and the increased application of culture-independent molecular approaches to medical microbiology, we sought to better characterize the diversity and natural history of the microbial communities  in CSF shunt infections using molecular approaches. The study received Institutional Review Board approval from the Seattle Children’s Research Institute and the University of Utah, as well as approval from the Primary Children’s Medical Center (PCMC) Privacy Board. For all study subjects, except those from PCMC prior to March 18, 2010, consent was obtained for additional CSF to be collected on each occasion that regular CSF samples were obtained during treatment for CSF shunt infection. Prior to March 18, 2010 at PCMC, we used CSF remaining for after routine processing and testing in the PCMC Microbiology Laboratory.Children ≤18 years old undergoing treatment for conventional culture-confirmed CSF shunt infection at either Seattle Children’s Hospital (SCH) or PCMC were eligible for enrollment in this study. A CSF shunt infection was defined as identification of organisms on microbiological culture of CSF fluid obtained from a partial or complete CSF shunt system. CSF shunt system(s) included ventriculoperitoneal, ventriculoatrial, ventriculopleural, arachnoid cyst shunts, subdural shunts, and lumboperitoneal shunts; temporary devices only such as external ventricular drain(s), Ommaya reservoir(s), ventricular access devices (reservoirs) and subgaleal shunts were excluded. Negative controls included phosphate buffered saline (PBS) and five sets of both stock CSF from donors without CSF shunts. Donors included adult research participants with brain tumors, and commercially available sterile CSF from adults. Negative controls underwent concurrent deoxyribonucleic (DNA) extraction along with either one or two infection episode samples, followed by subsequent testing as described below.Sterile conditions were standard practice throughout recovery and storage of CSF. The first CSF sample for diagnosis of infection was usually obtained from needle aspiration of the shunt reservoir under sterile conditions outside the operating room in a bedside “shunt tap”. The initial CSF sample analyzed in this study either was leftover from this first diagnostic sample or was obtained in the operating room under sterile conditions from the system being removed during the first surgery to treat infection. Subsequent CSF samples, including those at the end of the infection, were generally obtained under sterile bedside conditions through a sampling port within sterile extension tubing attached to the external ventricular drain.After CSF was obtained for the study, samples were stored at 4°C for up to a week. CSF was then aliquoted into vials of ∼100 µl for the study and stored at −70°C; PCMC samples were shipped overnight to Seattle on dry ice for analysis. After December 9, 2011, 200 µl of CSF was stored in 450 µl MO-BIO CB1 solution at the time of sample collection.All CSF samples were tested using routine aerobic culture techniques in hospital-certified laboratories at both SCH and PCMC. Conventional cultures are the traditional diagnostic approach used to detect typical pathogens in infectious diseases and were performed in a clinical microbiology laboratory following Clinical and Laboratory Standards Institute guidelines; however they do not detect all bacteria present in human disease For each CSF sample, bacterial DNA was extracted from either 100 µl of CSF or 200 µl of 650 µl of CSF/buffer using the BiOstic Bacteremia DNA isolation kit (MO-BIO). DNA was eluted in 100 µl of buffer. At a minimum, the initial CSF sample from the infection episode was analyzed, and in one instance the entire infection course was also analyzed.Partial ribosomal amplification was performed as previously described.  CCC TGT GTG CCT TGG CAG TCT CAG TCC TCC GCT TAT TGA TAT GC -3′).The fungal primers ITS1F, 5′- CTT GGT CAT TTA GAG GAA GTA A-3′ and ITS4R, 5′- TCC TCC GCT TAT TGA TAT GC-3′ were used for amplifying a 500 bp region of the fungal internal transcribed spacer (ITS) DNA sequence. The primer sets used for FLX-Titanium amplicon pyrosequencing included the addition of linker A and 8 base pair subject-specific barcode sequence at the 5′ end of forward primer ITS1F-A (5′-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG-barcode- CTT GGT CAT TTA GAG GAA GTA A-3′) and the biotin and linker B sequence at the 5′ end of reverse primer ITS4R-B  was used for polymerase chain reaction (PCR) under the following conditions: 95°C for 5 min followed by 35 cycles of 95°C for 30 s; 54°C for 40 s and 72°C for 1 min, a final elongation step at 72°C for 10 min was also included. The PCR products were cleaned by using Diffinity Rapid Tip  and pooled. The small fragments were removed by Agencourt Ampure Beads .Tag-encoded FLX-Titanium amplicon pyrosequencing (TEFAP) was performed as described previously. Pseudomonas aeruginosa PAO1 (ATCC number: BAA-47) was grown in tryptic soy broth  medium at 37°C with shaking for 16 hours. An aliquot was serially diluted in TSB broth. The diluted bacteria were plated out by using a Whitley automatic spiral plater , which was used to count colony-forming units (cfu). The bacteria broth was further diluted from 1010 to 104 cfu ml−1. Total DNA extractions were performed using 1 ml of each diluted samples.5′-CCA TGA AGT CGG AAT CGC TAG-3′; 16S-R, 5′-GCT TGA CGG GCG GTG T-3′; 16S-Probe, 5′-6-FAM-TAC AAG GCC CGG GAA CGT ATT CAC CG-3′. Roche LightCycler 480 Probes Master was also used for the assay. The PCR reactions were as follows: 50°C for 2 min, 95°C for 10 min, and 35 cycles of 95°C for 15 sec and 60°C for 1 min. The DNAs extracted from the series dilutions were used to set up a standard curve, the Ct values of the quantitative real-time PCR assay (qPCR) assay are compared with the standard curve. The qPCR assay using probe and primers showed that a concentration as low as 106 cfu/ml can be detected on the DNA samples.The CSF samples’ DNAs were detected using a Roche LightCycler 480. The specific primers and probe for bacteria 16S rRNA gene were as follows: 16S-F, The proportion of individual bacterial species detected in each initial CSF sample was summarized descriptively using graphical displays. Bacterial species that comprised less than 1% of reads within a sample were grouped together as “other”. Individual species or taxa identified by sequencing were compared with findings from bacterial culture. Changes in proportion of bacterial species for a single subject over the course of CSF shunt infection treatment were tabulated for species or taxa that comprised over 2% of sequenced reads; this threshold was selected for ease of interpretation.Paenibacillus species, Bacillus species, and Methylobacterium jeotgali. bTEFAP of several of the samples  identified a large variety of additional bacterial species, each generally at relatively low abundance, and many of which are not considered typical CSF shunt infection pathogens.Bacterial species identified by 16S rRNA bTEFAP in the initial samples from the CSF shunt infections in eight different children are shown in Bacterial culture results are compared to 16S bTEFAP findings in Fungi were also identified by ITS fTEFAP at the time of initial CSF samples for the same eight CSF shunt infections Table 2.S. epidermidis, which was identified by bTEFAP along with a substantial proportion of Paenibacillus species. Bacterial quantitation by qPCR of the samples detected 1.15×107 CFU/ml equivalents on day 2, followed by no detectable bacterial DNA above the assay’s limits of detection  thereafter. After day 2, bTEFAP identified a large variety of other species at relatively low abundances throughout the course of infection treatment. bTEFAP did not identify any 16S reads among CSF and PBS negative controls. The proportions of some bacteria fluctuated, such as Streptococcus species; others, such as Bacillus and Pseudomonas species, which are not targets of antimicrobial therapy, even rose over the course of infection treatment. Intravenous antibiotics that were used in this infection and that may have driven the observed microbial abundance changes are shown in Finally, the proportion of bacterial species detected by bTEFAP over the course of CSF shunt infection A is shown in To our knowledge, this is the first study that describes the microbiota present in CSF shunt infection. Using 16S and ITS TEFAP, we have demonstrated that numerous bacterial species, including anaerobes, as well as fungal organisms were detected in the CSF of children with CSF shunt infections. With the exception of fungal counts in two samples, these organisms were detected at TEFAP counts well above those in negative controls. The organisms recovered by TEFAP, including many beyond those recovered by standard aerobic bacterial culture, suggest heterogeneous microbiota. The composition of the microbiota changed over the course of infection treatment, probably in response to antibiotic use and surgical removal of the shunt. Future studies of a larger patient population will be needed to determine the relationships between the diverse CSF microbiota and clinical outcomes, particularly the response to treatment and occurrence of reinfection.Paenibacillus species, Bacillus species, and Methylobacterium jeotgali. Paenibacillus species are particularly intriguing in this context, as they have been reported to have biomedical applications. Bacterial organisms that comprised substantial proportions of the 16S TEFAP findings included Staphylococcus aureus; however, the proportion or burden of S. aureus at the time of shunt replacement is unknown. Study of additional, longitudinal samples from children who have developed serial CSF shunt infections is needed to determine if bacterial species identified at the first infection are related to those seen at the second infection. Further investigations such as these will hopefully inform more effective CSF shunt infection treatment.16S bTEFAP analysis suggested that CSF bacterial microbiota changed over the course of infection treatment. The results in There are several limitations to this work. The selection of appropriate negative controls is difficult. Since the negative controls we selected were from adults, they may not be physiologically comparable to the infection samples. While we considered the use of CSF from children with shunt obstruction without clinical evidence of infection, we were concerned about the potential role of microbial biofilms in this clinical scenario and felt it warrants a separate, independent line of inquiry. CSF from children with functioning shunts may be an ideal negative control, but challenging to obtain. Moreover, while the ideal negative control would be CSF from uninfected children whose shunts have been in-situ for a similar period to the infected cases, this would not be ethically feasible since sampling of CSF from the shunt introduces risk of infection. We anticipate future work examining bacteria and fungal sequences will need to incorporate negative controls from several sources, including but not limited to: donor CSF, children undergoing shunt revision surgery for shunt failure, and children undergoing initial CSF shunt placement.In addition, there are several opportunities for contamination of CSF samples during recovery, storage, and experimentation. However, sterile conditions were sought throughout recovery and storage of CSF samples, and contamination during experimentation is improbable given low recovery from negative controls that were handled identically to and concurrently with the CSF samples. Therefore, contamination is unlikely to have a major contribution to the bacterial and fungal diversity observed. The low numbers of infection episodes analyzed renders this study preliminary in nature. Testing from the hospital-certified laboratories was limited to routine aerobic cultures, practices that are subject to individual laboratory practices, including policies affecting identification of all organisms on a plate, incubation for extended periods of time, and enrichment for slow growing and fastidious organisms. We did not have information about variability or density of colonies on the original plates. In addition, fungal and anaerobic cultures were not obtained. Finally, detection of microbial DNA, which could be from either viable or nonviable cells, by PCR does not guarantee that living bacteria or fungi were present at the time of sample collection. However, the differences observed between infection and control samples indicates that these microbes were present at some point up to collection.While the clinical importance of these CSF microbiota remains to be determined, the current findings provide additional support to the emerging evidence of a role for biofilms in CSF shunt infection. Biofilms are often comprised of multiple microbial taxa held together by an extracellular matrix, Numerous bacterial and fungal organisms appear to be present in the microbiota that comprises CSF shunt infection. In evaluating CSF shunt infection, fungal and anaerobic bacterial cultures should be considered in addition to aerobic bacterial cultures, and culture-independent approaches may prove to be useful as a primary or adjunctive diagnostic modality. Furthermore, better understanding of the diverse microbial environment in active CSF shunt infection is needed. The persistence of these microbiota despite antibiotics may suggest the presence of biofilm. More effective treatment of CSF shunt infection is badly needed to reduce unacceptably high rates of reinfection, and this early work suggests that one effective target may be the reduction of the diverse microbiota present in infection. Future work must focus on defining the relationships between the diverse CSF microbiota found in this study with clinical outcomes, including response to treatment and reinfection; as well as on attainment of optimal negative control subjects.


Alzheimer’s disease (AD), the most common cause of dementia in the elderly, is characterized by the presence of Aβ plaques, which results in a progressive neuronal degeneration and subsequent cognitive decline. Dendrites and axons in the region surrounding the plaques may become damaged and swollen, forming what are known as dystrophic neurites (DNs). However, the mechanisms by which DNs affect cognitive function in AD patients are not understood.While characterizing the reticulon (RTN)/Nogo family of proteins, which have been shown to negatively regulate BACE1 activity, we found that RTN3, which is primarily neuronally expressed, accumulates in a distinct population of DNs called RTN3 Immunoreactive Dystrophic Neurites (RIDNs) in the brains of AD patients and in APP transgenic mice, an animal model for AD. More importantly, transgenic mice overexpressing RTN3 (Tg-RTN3) produce RIDNs in the hippocampus at a young age, while wild-type mice produce similar RIDNs only at older ages.The importance of RIDNs in relation to cognitive decline is further evidenced by the finding that Tg-RTN3 mice where RIDNs are present show impaired learning and spatial memory, reduced spine density and synaptic plasticity. Molecular characterization of RIDNs indicates that RIDNs contain RTN3 aggregates and inducible expression of RTN3 in a mouse model reveals that RTN3 aggregates are formed irreversibly.Together, our results suggest that inhibition of RTN3 aggregation is a promising approach for improving cognitive dysfunction associated with Alzheimer’s pathology.