After three washes with phosphate-buffered saline (PBS) (supplemented

with.15 M NaCl, 0.03 M phosphate, 0.02% sodium azide, pH 7.2), 0.05% Tween 20. The proteins bound to the cells were eluted by incubation with 0.1 M glycine-HCl, pH 2.0, for 15 min. Cells were removed by centrifugation at 14,000 × g for 20 min at 4°C, and supernatants were then analysed by Western blotting. Protease degradation assay To characterize protease-susceptibility of CFH and FHL-1 binding proteins of B. garinii ST4 PBi, cells were treated with two different proteases as described previously [34]. Briefly, spirochetes were grown to mid-log phase, sedimented by centrifugation at 5,000 × g for 30 min, washed twice with cold PBS containing 5 mM MgCl2 (PBS-Mg), and resuspended in 100 μl PBS-Mg. To the Borrelia cell suspension (at a concentration of 108 in a final volume of 0.5 ml), proteinase K

or trypsin selleckchem was added to a final concentration of 12,5 μg/ml to 100 μg/ml. Following incubation for 1 hour at room temperature, proteolytic degradation with proteinase K or trypsin was terminated by the SCH727965 addition of 5 μl of phenylmethylsulfonyl fluoride or by the addition of 5 μl of phenylmethylsulfonyl fluoride and 5 μl of 4-(2-aminoethyl)-benzenesulfonyl fluoride, respectively. Borrelia were then gently washed twice with PBS-Mg, resuspended in 20 μl PBS-Mg, and lysed by sonication five times using a Branson B-12 sonifier (Heinemann, Schwäbisch Gmünd, Germany). Equal volumes of Borrelia lysates were subjected to Tris/Tricine SDS-PAGE, and proteins were transferred to nitrocellulose membranes as described previously [16]. Susceptibility of proteins to proteolytic degradation was assessed by Western or ligand affinity blotting with the appropriate monoclonal or polyclonal antibodies, learn more followed by incubation with a horseradish peroxidase-conjugated IgG antibody, and then visualized by the addition of 3, 3′, 5, 5′-tetramethylbenzidine.

PCR cloning, expression and purification of recombinant CspA orthologous proteins Sequences of genes encoding for CspA B31 and orthologs from B. garinii ST4 PBi were obtained from genbank (NC_006129 and NC_001857). Primers were designed using primer3 (MIT) and listed in table 2. Amplification reactions were performed in a 50 μl final volume, containing 25 μl IQ Supermix (Bio-Rad, Veenendaal, The Netherlands), 15 pmol forward primer, 15 pmol reverse primer, and 10 μl of a DNA isolate of cultured B31 or PBi. Following an enzyme activation step for 3 min at 95°C, amplification comprised 50 cycles of 30 s at 95°C, 30 s at 55°C and 30 s at 72°C. Genes lacking their leader sequences were ligated in frame into the pGEX-5X3 vector (Amersham Bioscience, Freiburg, Germany). The ligation mixtures were used to transform Escherichia coli MC1061.

SiRNAs were procured through Ambion. SiRNA transfection reagent was purchased from Bio-Rad (USA). Cell Line Nucleofector Kit V was purchased from Amaxa Inc. USA. Cell culture The THP-1 human macrophage-like cell line was

acquired from the American Type Culture Collection, USA and cultured in RPMI-1640 medium containing 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, supplemented with 10% heat inactivated fetal calf serum and 0.05 mM β-mercaptoethanol at 37°C, 5% CO2. Cells were treated with 30 nM PMA for 24 h before using for the experiments. The J774A.1 murine macrophage cell line was maintained at 37°C, 5% CO2 MLN0128 in vivo in DMEM containing 10% fetal calf serum, 2 mM glutamine and essential amino acids. Mycobacteria and macrophage Infection Mycobacterium tuberculosis H37Rv (Rv), Mycobacterium tuberculosis H37Ra (Ra), Mycobacterium bovis BCG (BCG) and Mycobacterium smegmatis MC2 155 (MS) were grown in Middlebrook (MB) 7H9

medium supplemented with 0.5% glycerol, buy Dabrafenib ADC supplement, 0.5% BSA, fraction V, 0.2% dextrose, 0.85% NaCl and 0.05% Tween 80. Cultures were incubated at 37°C. Mycobacteria grown in mid-log phase were used for infecting THP-1 cells. The bacterial suspension was washed and resuspended in RPMI-1640 containing 10% FCS. Bacterial clumps were disaggregated by vortexing five times (each cycle~2 min) else with 3-mm sterile glass beads, and then passed through 26 gauge needle 10 times to disaggregate any remaining clumps. The total number of bacilli per milliliter of suspension was ascertained by measuring OD at 650

nm and by further counting for cfu on MB7H10 agar plates. Infection and preparation of cell lysates for western blotting THP-1 cells were seeded at 2 × 106 cells/well in 6 well plates and were subsequently incubated with 20, mycobacteria/macrophage, for 4 h and lysed in phosphorylation buffer as described previously [18]. Alternatively, 2 × 106 peritoneal macrophages from BALB/c mouse were also infected with MS and Rv. Total 20 μg protein sample was analyzed by 10% SDS-PAGE and electroblotted as described previously [18]. Briefly, after blocking, the membranes were incubated overnight at 4°C with antibodies (anti PKC-α and anti PKCδ, 1:1000, anti pPKC-α and anti pPKCδ, 1:1000, anti tubulin, 1:5000, anti PknG, 1:1000) in 0.1% TBST containing 3% BSA, with gentle shaking. After four washes with 0.05% TBST, the membrane was incubated with goat anti-rabbit (anti-mouse when detecting tubulin) polyclonal antibodies conjugated to horseradish peroxidase (1:50000) in 0.1%TBST containing 3% BSA for 1 h at room temperature. After four washes with 0.05% TBST, the blots were developed using ECL reagents and were analyzed on Chemi-Doc XRS system (Bio-Rad Laboratories, Hercules, CA) using Quantity One program.

The scale contains 11 dichotomous items, representing short-term effects of a day of work. All items were recoded in such a way that higher scores indicate ‘more complaints’, i.e. a higher need for recovery. The recoded scores are presented C646 cost in a range from 0 to 100. The Cronbach’s alpha of the scale is 0.78 (Jansen et al.

2002). Examples of items in the scale are ‘I find it hard to relax at the end of a working day’ and ‘Because of my job, at the end of the working day, I feel rather exhausted’ (Van Veldhoven and Broersen 2003). In the present study, the upper quartile was used to define a contrast between employees with a high versus low-medium need for recovery, which corresponds with a cut-off point of 6 on the 11-item scale as recommended by Broersen et al. (Broersen Paclitaxel manufacturer et al. 2004). The level of need for recovery was determined in each questionnaire (T0, T1, T2, T3, T4, T5, T6). Demographic and health factors Employees provided information on gender, age, educational level and the presence of a long-term illness through self-report in the questionnaires. Employees were divided into five age groups, that is, 18–25, 26–35, 36–45, 46–55 and 56–65 years. Smoking

status was assessed by a single dichotomous item (“Do you smoke every day?”). Characteristics of the private situation Living situation was operationalized as living alone (yes/no). Work–family conflict was measured by one dichotomous item asking employees whether they were able to adequately combine work and family life. Work characteristics Regarding working hours, employees were amongst others asked for their working hours per week, categorized as >40, 36–40, 26–35, 16–25 and <16 h per week. Also, information on overtime was collected using an item on frequent overtime

(yes/no). A Dutch version of the Job Content Questionnaire was used to measure psychological job demands and decision latitude (Karasek 1985). Psychological job demands were assessed by the sum of five items (Chronbach’s alpha 0.69). Decision latitude (Chronbach’s alpha 0.81) was measured by the sum of two subscales: skill discretion and decision authority. The response options BCKDHA varied from “strongly disagree” to “strongly agree” on a four-point scale. The total score was then divided into tertiles, resulting in low, medium and high levels of psychological job demands or decision latitude. To assess whether employees perceived their work as physically demanding, one item of the Dutch questionnaire on Work and Health (VAG; Gründemann et al. 1993) was used. Statistical analysis Because the distribution of need for recovery was skewed to the left, Poisson regression analyses were conducted to test differences in mean levels of need for recovery in the cross-sectional analyses.

Baseline total body weight was not significantly different (p = 0.326) between FEN and PL groups. There were no total body weight changes over the 8 week time course of the study between or within groups (p > 0.05). A significant main effect for time (p = 0.004) for lean body mass was observed, and further pair-wise comparisons revealed a significant increase in lean body mass for FEN at week 4 (p < 0.001) and week 8 (p < 0.001) compared with baseline. No such changes were seen in the PLA group (p > 0.005). A significant interaction effect (p < 0.001) and main effect for time (p < 0.001)

occurred between groups for body fat percentage. Additional pair-wise comparisons displayed significant improvements in body fat percentage at week

4 (p < 0.001) and week 8 (p < 0.001) in FEN compared to baseline, www.selleckchem.com/products/MLN-2238.html while no such changes were noticed in PLA (p > 0.005). Table 3 Body composition changes within and between groups Variable Group Baseline (T1) Week 4 (T2) Week 8 (T3) Between Group Body Weight FEN 90.2 ± 18.2 89.9 ± 18.2 90.4 ± 17.7 G = 0.305 (kg) PLA 85.7 ± 12.7 85.0 ± 13.9 85.8 ± 12.4 T = 0.244           G × T = 0.803 Lean Mass FEN 157.7 ± 23.9 160.2 ± 23.8‡ 162.6 ± 22.9‡ G = 0.640 (kg) PLA 157.2 ± 19.5 156.4 ± 22.4 158.2 ± 19.5 T = 0.004†           G × T = 0.057 Body Fat Obeticholic Acid chemical structure % FEN 19.4 ± 8.4 17.8 ± 8.4 ‡ 17.1 ± 8.6 ‡ G = 0.298   PLA 16.3 ± 4.8 16.0 ± 4.8 15.9 ± 4.5 T < 0.001†           G × T < 0.001† Abbreviations: FEN = fenugreek supplement group, PLA = placebo group Symbols: † = Significant between group difference (p < 0.05), ‡ = Within group difference from baseline (T1), p < 0.05 Training Adaptations Table 4 exhibits all training adaptation data. A significant group × time interaction (p = 0.008) and main effect Lepirudin for time (p < 0.001) was observed between FEN and PLA groups for bench press 1-RM, however pair-wise comparisons revealed no significant differences between FEN and PLA bench press 1-RM's at any time point.

Pair-wise comparisons also showed significant increases in bench press 1-RM at week 4 (p < 0.001) and week 8 (p < 0.001) in comparison with baseline and from week 4 to week 8 (p = 0.002) in FEN. PLA experienced significant increases in bench press 1-RM at week 4 (p = 0.008) and week 8 (p = 0.004) when compared to baseline. A significant group × time interaction (p < 0.001) and main effect for time (p < 0.001) was observed between FEN and PLA groups for leg press 1-RM, as further pair-wise comparisons indicated a significant difference in FEN compared to PLA at week 8 (p = 0.019). Pair-wise comparisons also revealed significant increases in leg press 1-RM at week 4 (FEN: p < 0.001, PLA: p < 0.001) and week 8 (FEN: p < 0.001, PLA: p < 0.001) in comparison with baseline. No significant interactions or main effects (p > 0.005) were noted for muscular endurance repetitions on the bench press or leg press. A significant main effect for time (p = 0.

Local reports [27–29], as well as a national study [30] did

not provide clinical details on chronic illness. The population-based study by Acosta et al. [32] documented only occurrence of diabetes and chronic hypertension among live birth PASS hospitalizations. Bauer et al. [33] reported a broader but still selective range of chronic comorbidities, with the most common being congestive heart failure (6%), systemic lupus (1.5%), and chronic liver disease (0.7%). However, the investigators provided no data on the overall frequency of any chronic comorbidity (of those examined) among PASS hospitalizations, limiting the inference on the overall burden of chronic illness from their findings. Risk factors for the development of PASS were examined in several reports. Reported risk factors included maternal MK2206 age ≥35 years [30, 33], low income [30], black race [32, 33], Medicaid insurance [33] or public insurance/no RG-7204 insurance [32], tobacco use [28] congestive heart failure [33], diabetes [32], hypertension [32], chronic liver disease [33], chronic kidney disease [33], systemic

lupus [33], human immunodeficiency viral infection [33], preeclampsia [28, 32], induced labor [29, 30], cesarean section [28–30], premature rupture of membranes [30, 33], and retained products of conception [33]. Of note, obesity was not an independent risk factor for PASS in the study by Bauer et al. [33], possibly due to its underreporting (1.8%) in their population. The aforementioned predictors identify subsets of obstetric patients requiring extra vigilance for

prevention, early recognition and intervention for PASS. However, as noted by others, the risk factors for maternal sepsis are not well-understood [36]. Clinical Manifestations of Pregnancy-Associated Severe Sepsis The most common sites of infection among patients with PASS in local studies were described variably as involving the genital (39%) [27] and urinary (37%) [35] tracts. Kramer et al. [30] reported in their national study that genital tract infections were the most common, noted in 56% of their patients. No data on sites of infection were reported on PASS hospitalizations in the study by Acosta et al. [32]. Finally, in the national population Forskolin study by Bauer et al. [33], the genital tract was the most common reported site of infection (56.7%) among PASS hospitalizations. Of note, pneumonia was reported in 29.7% of PASS hospitalizations [33]. Although SIRS has been considered part of the bedside definition of sepsis in the general population, it was not validated in obstetric patients pre- or post-delivery and multiple investigators have raised concerns about the appropriateness of its cutoff values, which are often observed among otherwise healthy pregnant women [25]. The clinical findings of PASS include those related to a specific site of infection. Nevertheless, the site of infection is often not readily apparent in these patients.

McCarthy ND, Colles FM, Dingle KE, Bagnall MC, Manning G, Maiden MCJ, Falush D: Host-associated genetic

import in Campylobacter jejuni. Emerg Infect Dis 2007, 13:267–272.PubMedCrossRef 19. de Haan CPA, Kivistö R, Hakkinen M, Rautelin H, Hänninen ML: Decreasing trend of overlapping multilocus sequence types between human and chicken Campylobacter jejuni isolates over a decade in Finland. Appl Environ Microbiol 2010, 76:5228–5236.PubMedCrossRef 20. Kwan PSL, Birtles A, Bolton FJ, French NP, Robinson SE, Newbold LS, Upton M, Fox AJ: Longitudinal study of the molecular epidemiology of Campylobacter jejuni in cattle on dairy farms. Appl Environ Microbiol 2008, 74:3626–3633.PubMedCrossRef 21. Pittenger LG, Frye JG, McNerney V, Reeves J, Haro J, Fedorka-Cray PJ, Harrison MA, Englen MD: Analysis of Campylobacter jejuni whole-genome microarrays: significance of prophage and hypervariable selleck kinase inhibitor regions for discriminating isolates. Foodborne Pathog Dis 2012, 9:473–479.PubMedCrossRef 22. Clark CG, Bryden L, Cuff W, Johnson PL, Jamieson F, Ciebin B, Wang G: Use of the Oxford multilocus sequence typing protocol and sequencing of the flagellin short variable region to characterize isolates from a large outbreak of waterborne Campylobacter sp. strains in Walkerton, Ontario, Canada. J Clin Microbiol 2005, 43:2080–2091.PubMedCrossRef 23. Clark CG, Price L, Ahmed R, Woodward DL, Melito PL, Rogers

FG, Jamieson F, Ciebin B, Li A, Ellis A: Characterization of waterborne outbreak-associated Campylobacter Selleckchem NVP-AUY922 jejuni, Walkerton, Ontario. Edoxaban Emerg Infect Dis 2003, 9:1232–1241.PubMedCrossRef 24. Andrysiak AK, Olson AB, Tracz DM, Doré K, Irwin R, Ng L-K, Gilmour MW: and the Canadian Integrated Program for Antimicrobial Resistance Surveillance Collaborative: Genetic characterization of clinical and agri-food isolates of multi-drug resistantSalmonella entericaserovar Heidelberg from Canada. BMC Microbiol 2008, 8:89.PubMedCrossRef 25. Taboada EN, Acedillo RR, Carrillo CD, Findlay WA, Medeiros

DT, Mykytczuk OL, Roberts MJ, Valencia CA, Farber JM, Nash JHE: Large-scale comparative genomics meta-analysis of Campylobacter jejuni isolates reveals low level of genome plasticity. J Clin Microbiol 2004, 42:4566–4576.PubMedCrossRef 26. Malik-Kale P, Raphael BH, Parker CT, Joens LA, Klena JD, Quiñones B, Keech AM, Konkel ME: Characterization of genetically matched isolates of Campylobacter jejuni reveals that mutations in genes involved in flagellar biosynthesis alter the organism’s virulence potential. Appl Environ Microbiol 2007, 73:3123–3136.PubMedCrossRef Competing interests The authors declare no competing financial interests. Authors’ contributions Conceived and designed the work: CGC, MWG. Performed the laboratory experiments: CGC, CCRG, JM, DMT. Performed the analysis, including statistical analysis: CGC. Wrote the manuscript: CGC. Collected, collated, and provided patient and source data associated with the sentinel site: FP, BM.

Figure  5a shows the SEM image of the PbTe prepared with CTAB, which indicates the formation of mostly cube-shaped nanoparticles with size in the range of 65 to 145 nm. The sample synthesized with

SDS (Figure  5b) shows fewer nanocubes and more irregular nanoparticles compared to the nanoparticles Selleck AZD5363 synthesized with CTAB; the size of nanoparticles ranges from 70 to 230 nm. The synthesis of the PbTe sample with Triton (Figure  5c) yields fine particles with the size in the range of 40 to 120 nm. From the SEM images, it can be concluded that the PbTe nanoparticles synthesized at 140°C for 24 h with a water/glycerol solution with the addition of different surfactants (Figure  5) are more uniform in shape and size compared to the nanoparticles synthesized without surfactants (Figure  4e). This can be attributed to the presence of surfactant as a shape-directing agent which is expected to control the size and shape of the particles. PbTe nanoparticles synthesized with CTAB and Triton are smaller in size, while nanoparticles

synthesized in SDS are bigger in size which are comparable to the nanoparticles synthesized without surfactants. Zhu et al. [18] reported the synthesis of three-dimensional hierarchical structure of PbTe by Talazoparib in vitro a hydrothermal method with or without surfactants using different molar concentrations of NaOH and concluded that the morphology of the PbTe crystals depends on the synthesis temperature, time, and most importantly on the concentration of NaOH. This work also reported the synthesis of PbTe nanoparticles without any hierarchical structure, similar to our PbTe nanostructures, with or without 1 M NaOH at 160°C, and without the use of any surfactants. Figure 5 Effect of use of surfactants on the formation of undoped PbTe. SEM images of undoped Sitaxentan PbTe synthesized with (a) CTAB, (b) SDS, and (c) Triton, respectively,

as surfactants in water/glycerol (3:1 volume ratio) solution at 140°C for 24 h. The structure of the as-prepared PbTe sample synthesized at 140°C for 24 h with a water/glycerol solution (i.e., sample PbTe-2, its corresponding SEM image is Figure  4e) was analyzed by TEM, HRTEM, SAED, and EDS. Figure  6a is the low-magnification TEM image of the PbTe nanoparticles with various sizes of 75 to 220 nm. The high-magnification TEM image of the PbTe sample (Figure  6b) indicates that the nanoparticles have cube-like shape. Poudel et al. [26] also reported the cube-like PbTe nano- and microparticles synthesized hydrothermally at 100°C and 160°C, respectively, for 10 h without surfactant. However, with surfactants, various morphologies of PbTe crystals including hierarchical structures were obtained. Recently, PbTe microcubes were prepared using a composite-hydroxide-mediated approach [27].

Finally, the original alignment was analyzed by maximum likelihood using dnaml but instead of searching for the best tree, the sequences were fitted to the consensus tree. In the resulting tree, the branching was derived from the bootstrap analysis, and the branch lengths from the maximum likelihood analysis. Nucleotide sequence accession find more numbers The partial 16S rRNA gene sequences obtained in this study are available in GenBank under accession numbers JQ062987 and HM639782 to HM639862. T-RFLP analysis

Sludge sample collection and DNA extraction was carried out as described above. Archaeal 16S rRNA genes were amplified as described above but with the forward primer Arch18F labeled with the fluorescent dye 6 – carboxyfluorescein. Three PCR reactions were prepared from each sludge sample. The PCR products were purified using the Agencourt AMPure system (Beckman Coulter) and digested with 10 units of restriction enzyme at 37°C for at least 16 hours. Restriction enzymes AluI and RsaI were used in separate reactions. The restriction digests were purified and analyzed by capillary gel electrophoresis (3730 DNA Analyzer, Applied Biosystems). The size standard LIZ1200 (Applied Biosystems) was used for fragment size determination. The software Genemapper (Applied Biosystems) was used to quantify the electropherogram data and to generate

the TRF profiles. Peaks from fragments of size 50-1020 bases with a height PF-02341066 research buy above 50 fluorescent units were analyzed. The total fluorescence of a sample was defined as the sum of the heights of all the peaks in the profile and was interpreted as a measure of the amount of DNA that was loaded on the capillary gel. Only samples with at least two of the three TRF profiles with a total fluorescence above 500 fluorescent

units were considered for further analysis. The two profiles with the highest total fluorescence were chosen from each sample. The TRFs of the TCL two profiles were aligned using a moving average procedure [67] and then checked manually for errors. The two profiles were then normalized as described by Dunbar et al [68] and combined to a single consensus profile by taking the average size, height and areas of the fragments present in both. Consensus profiles with a low total fluorescence, i.e. where low amounts of DNA had been loaded on the gel, were excluded from the subsequent analysis to avoid excessive normalization. 32 and 33 consensus TRF profiles, for the RsaI and AluI analysis, respectively, were normalized and aligned as described above. The TRFs that were removed by normalization constituted only a minor part of the TRF profiles, on average 2 ± 3% and 1 ± 2% of the total fluorescence in the AluI and RsaI profiles, respectively. The dynamics of the Archaea community were evaluated by pair-wise comparisons of TRF profiles using the Bray-Curtis distance coefficient (described in e.g. [69]).

A double-layered lamella was positioned between the layer of microtubules and a deeper layer of mitochondrion-derived organelles (Figures 4A-B, 4D). The mitochondrion-derived Raf activation organelles were discoidal in shape, were bounded by two membranes and lacked mitochondrial cristae or inclusions such as kinetoplasts (Figures 4A-B, 4E). Moreover, we did not observe any evidence

of euglenid-like pellicle features, such as the presence of S-shape proteinaceous strips or discontinuities in the layer of microtubules. Nucleus, Vestibulum and Associated Pockets An anterior nucleus was positioned near the ventral side of the cell and contained a prominent nucleolus and condensed chromosomes (Figures 3A, 3C-D). The vestibulum was positioned directly above the nucleus as this space passed from the ventral, subapical opening toward the dorsal side of the cell (Figure 3C). The vestibulum then extended posteriorly along the dorsal side of the cell and branched into three distinct pockets: (1) a novel “”extrusomal pocket”", (2) a flagellar pocket and (3) a feeding pocket (Figures 3A, 3C; described in more detail below). A battery of longitudinally arranged extrusomes was connected to the base of the extrusomal pocket and was nested within a notch on the dorsal side of the ventral nucleus (Figures 1B, 3A, 3C). Each extrusome was about 160 nm in diam. (Figure 3G). The battery of extrusomes

was indistinguishable from the feeding rods of euglenids when viewed with the light microscope, and discharged as a single unit through the anterior opening (Figures 1B, 1H). The flagellar pocket was located selleck products on the dorsal side of the cell and contained two flagella that inserted at the bottom of the pocket (Figures 6, 7; described in more detail below). The feeding pocket was located to the right of the flagellar pocket and extended horizontally before tapering posteriorly toward the ventral side of the cell (Figures 8, 9; described in more detail

below). Florfenicol Figure 6 Transmission electron micrographs (TEM) showing paraxonemal rods in the flagella, the flagellar transition zone and the basal bodies of Calkinsia aureus. A. Longitudinal section of the dorsal flagellum (DF) showing the flagellar transition zone and the dorsal basal body (DB) (bar = 500 nm). B-J. Non-consecutive serial sections through the DF (B), the flagellar transition zone (C-G), and the DB (H-J) as viewed from anterior end (images at same scale, bar = 200 nm). B. Section showing the 9+2 configuration of axonemal microtubules and the tubular paraxonemal rod (arrow) in the DF. C. Section showing termination of central microtubules and the 9+0 configuration of axonemal microtubules. D. Section showing the transition zone through an outer concentric ring associated with nine electron dense globules inside of each doublet and faint spokes that extend inward from the each globule (see L for a diagram of this micrograph). E.

( 1998 ), Melendo et al. (2003), and Flora Iberica (2009) Ophioglossum vulgatum Ophioglossaceae L S S           Blanca et al. ( 1998 ) and Muller (2000) Papaver lapeyrousianum Papaveraceae L S S Perennial         Blanca et

al. ( 1998 ), Baudet et al. (2004) and Flora Iberica (2009) Pedicularis furbishiae Scrophulariaceae S S   Perennial Biotic     Sexual Gawler et al. ( 1987 ) Petrocoptis grandiflora Caryophyllaceae S S S Perennial Biotic Abiotic Wind Sexual Guitian and Sanchez ( 1992 ) and Navarro and Guitian (2003) Petrocoptis viscosa Caryophyllaceae S S S Perennial Biotic Abiotic Ballistic Mixed Navarro and Guitian ( 2002 ) Phyllitis scolopendrium var. americana Aspleniaceae S S S           Kuehn and Leopold Roxadustat in vitro ( 1992 ) Primula elatior subsp. lofthousei Primulaceae S S S Perennial Biotic Abiotic Ballistic Sexual Blanca et al. ( 1998 ) and Taylor and Woodell (2008) Rhizophora mangle Rhizophoraceae L S D Perennial   Abiotic Water Mixed Rabinowitz ( 1981

), Krauss and Allen (2003) and Proffitt et al. (2006) Rothmaleria granatensis Asteraceae S S S   Biotic Abiotic Wind   Blanca et al. ( 1998 ) and Melendo et al. (2003) Sagittaria isoetiformis Alismataceae S S D Perennial Biotic Abiotic Ballistic Mixed Edwards and Sharitz ( 2000 ) Sagittaria teres Alismataceae S S D Perennial Biotic Abiotic Ballistic Mixed Edwards and Sharitz ( 2000 ) Salix caprea Salicaceae L G S Perennial         Blanca et al. ( 1998 ) and Falinski (1998) Salix hastata subsp. sierrae nevadae Salicaceae S S S Perennial Biotic Abiotic Wind Mixed Blanca et al. ( 1998 ), buy JQ1 Melendo et al. (2003), and USDA PLANTS Database (2009) Scabiosa pulsatilloides subsp. pulsatilloides Dipsacaceae S S S Perennial Biotic Abiotic Wind Mixed Blanca et al. ( 1998 ) and Melendo et al. (2003) Scrophularia valdesii Scrophulariaceae S S S Perennial Biotic Abiotic Ballistic   Bernardos et al. ( 2006 ) Senecio elodes Asteraceae S S S Perennial Biotic

Abiotic Wind Asexual Blanca et al. ( 1998 ), Melendo et al. (2003), and Baudet et al. (2004) Senecio nevadensis Asteraceae S G S Perennial Biotic Abiotic Ballistic   Blanca et al. ( 1998 ) and Melendo et al. (2003) Setaria geniculata Poaceae L G S Perennial Abiotic     Mixed Rabinowitz and Rapp ( 1985 ) and Dekker Resminostat (2003) Shortia galacifolia Diapensiaceae S S D Perennial   Abiotic   Mixed Vivian (1967) and Rabinowitz ( 1981 ) Silene douglasii var. oraria Caryophyllaceae S S S Perennial   Abiotic Ballistic Asexual Kephart and Paladino ( 1997 ) Sorbus hybrida Rosaceae L S S Perennial       Mixed Blanca et al. ( 1998 ), USDA PLANTS Database (2009), and Flora Iberica (2009) Sphenopholis obtusata Poaceae L G S   Abiotic       Rabinowitz and Rapp ( 1985 ) and USDA PLANTS Database (2009) Spiranthes aestivalis Orchidaceae L G   Perennial         Blanca et al. ( 1998 ), and Flora Iberica (2009) Stylidium chiddarcoopingense Stylidaceae S S   Perennial Biotic     Sexual Coates et al.