Hargreaves-Allen et al [71] suggest that MPAs are unlikely to be

Hargreaves-Allen et al. [71] suggest that MPAs are unlikely to be successful if there are high levels of conflict, numerous uncontrollable external stressors, or alternative forms of development and livelihoods

are not possible Governance is the structural, institutional, ideological, and procedural umbrella under which development programs and management practices operate. Natural resource governance can be defined as “the interactions among structures, processes and traditions that determine how power and responsibilities are exercised, how decisions are taken, and how citizens or other stakeholders have their say” [110]. Governance determines how and whether the interactions of structures, processes, and institutions Selleck PD0332991 coalesce to solve societal and environmental problems [111] and [112]. Effective governance requires the design of institutions that are instrumental in “encourag[ing] people to choose to behave in a manner that provides for certain strategic policy outcomes, particularly biodiversity conservation objectives, to be fulfilled” [37] and [113].

Governance can be evaluated based on whether it effectively supports the achievement of MPA outcomes and also whether it engages with the principles of “good” governance—including legitimacy, transparency, accountability, inclusiveness, fairness, integration, capability, and adaptability [102] and [110]. OSI-744 mouse The importance of these guiding principles is generally supported by the recent literature on MPA governance, management, and development. The following section will explore three aspects of governance that are required to establish a solid base for management and development and the achievement of beneficial socio-economic and environmental outcomes from MPAs: (1) the creation

of an enabling institutional and organizational environment; (2) the process of implementation and design of MPAs; and, (3) the choice of management structures and MPA design (i.e., strict no-take, multiple use, multiple use with no-take zone). The concept of institutions often refers to both “soft” and “hard” institutions BCKDHA such as norms, rules, policies, and laws after [114]. Institutions are manifest in formalized organizations (e.g., governmental, non-governmental, and community based organizations) and structures (e.g., co-management and MPA format) and the interactions amongst these bodies. Institutions and organizations can act as drivers, constraints, or supports for effective MPA management and local development depending on the level of institutional linkage, congruence, coordination, and cooperation across scales [73], [100] and [115]. The harmonization of legal frameworks and mandates, policies at various levels, local rules and regulations, cultural norms and individual attitudes is both a challenge and an imperative for enabling effective management and development. As Camargo et al.

DSC results are presented in Table 4 and Fig  1 and Fig  2 All t

DSC results are presented in Table 4 and Fig. 1 and Fig. 2. All the flour samples exhibited at least two endothermic peaks at different temperatures, with the exception of severe extrusion flour. They are referred to hereafter

as transitions 1, 2 and 3 (Tp1, Tp2, Tp3). The first Tp for whole and defatted native amaranth flour were similar (76 °C) and coincided with the paste temperature obtained by RVA. Some authors (Baker & Rayas-Duarte, 1998) have reported that the gelatinization temperature of amaranth starch was higher than wheat or rice starches. They have suggested there are more organized regions in amaranth as higher temperatures were needed to record a melting transition. learn more These Tp and their respective δH could indicate starch gelatinization whereas the other small peaks could be attributable

to protein denaturation. In fact, Baker and Rayas-Duarte (1998) reported a Tp for amaranth starch of around 70 °C and Kong et al. (2009) observed Tp for fifteen selleck kinase inhibitor cultivars of amaranth which ranged from 68 °C to 78 °C. Martínez and Añón (1996) reported different temperatures for amaranth protein denaturation. Albumin and glutelin presented Tp of 64 °C and 70 °C, respectively, which indicate lower thermal stability. It was also observed a higher Tp (in excess of 90 °C), corresponding to globulin, albumin-2 and glutelin subfraction that are more thermostable. However, it is worth noting that these comparisons to the present work are not straightforward because in this case all amaranth fractions must be considered and also distinct water:starch proportions were used. Initially, it was thought that the small endothermic peak observed for whole native flour could be attributed to an amylose–lipid complex. However, this peak still occurred after defatting at the same temperature (defatted native flour),

indicating that it was not related to the lipid content of SPTLC1 the flour. In addition, lipid–amylose complexes start to melt only at temperatures approaching 110 °C (Doublier, Paton, & Llamas, 1987) and the waxy characteristic of amaranth flour starch did not confirm this hypothesis, again suggesting denaturation of thermostable protein, as outlined earlier. It is noteworthy that Okechukwu and Rao (1997) also reported two DSC peaks in a study with cowpea protein plus starch (cowpea and corn) gels, the first peak being due to starch gelatinization and second to protein denaturation. The absence of an endothermic peak at around 70 °C for extruded flours could indicate total degradation of starch that occurred prior to the extrusion process. Indeed, these results agree with those discussed previously in that the extruded flours also showed a very small peak and low final viscosity compared to native flours. González, Carrarra et al. (2007) reported similar values of enthalpy for an extruded amaranth starch-rich fraction to those observed in this study.

Commercial carriers Kaldnes™ K1 (Anoxkaldnes™, Sweden), made of h

Commercial carriers Kaldnes™ K1 (Anoxkaldnes™, Sweden), made of high density polyethylene (density 950 kg/m3; surface area 500 m2/m3), were used as inert supports. The carriers were autoclaved at 121 °C for 20 min until used. Sunflower (Helianthus annuus) seeds were obtained from a local market Afatinib and the shells were collected after normal human consumption of the seeds. The sunflower seed shells (SS) were autoclaved at 121 °C for 20 min before use. The chemical

composition of the SS according to Gullón et al. [6] is 23 ± 0.15% glucan, 29.4 ± 0.0016% klason lignin, 25.8 ± 0.07% hemicelluloses, 5.40 ± 0.03% extractives and 4 ± 0.15% ash. Cultivation was carried out in cotton-plugged Erlenmeyer flasks (250 mL) containing 3 g of Kaldnes™ K1 carriers or 1.5 g SS, according to the experiment, and 20 mL of culture medium. The culture medium composition was the same as that of the medium M1 described in Rodriguez-Couto [16] and consisted of 10 g/L glucose, 20 g/L yeast extract, 0.9 g/L

(NH4)2SO4, 2 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 0.1 g/L CaCl2·2H2O, 0.5 g/L KCl and 0.5 g/L thiamine hydrochloride in 0.05 M citrate–phosphate buffer (pH 4.5). To boost laccase production 0.5 mM Cu+2 was added to the cultures on the 3rd cultivation day [18]. Inoculation was carried out directly in the Erlenmeyer flasks. Three agar plugs (diameter, 7 mm), from a 7-day grown fungus on PDA, per Erlenmeyer were used as inoculum. The Erlenmeyer flasks were incubated statically under an air atmosphere at 30 °C and in complete darkness. Glucose consumption, Bak apoptosis measured as reducing sugars, was determined with the dinitrosalicylic acid reagent (DNS) using d-glucose as a standard according to the method described by Miller [11]. Laccase activity

was spectrophotometrically determined as described by Niku-Paavola et al. [12] with 2,2′-azino-di-[3-ethyl-benzo-thiazolin-sulphonate] (ABTS) as a substrate. One activity unit (U) was defined as the amount of enzyme very that oxidised 1 μmol of ABTS per min. The activities were expressed in U/L. Manganese-dependent peroxidase activity was spectrophotometrically assayed at 468 nm by the method of Kuwahara et al. [9]. The reaction was started by adding 0.4 mM H2O2. One activity unit (U) was defined as 1 μmol of 2,6-dimethoxyphenol oxidised per minute and the activities were expressed in U/L. Lignin peroxidase activity was spectrophotometrically determined at 310 nm according to Tien and Kirk [22]. The reaction was starting by adding 0.4 mM H2O2. One activity unit (U) was defined as 1 μmol of veratryl alcohol oxidised in 1 min and the activities were reported as U/L. The dyes used were the textile dyes Bemaplex Navy M-T (CI Acid Blue 193), an acid chromium-complex dye and Bezaktiv Blue BA (CI Reactive Blue 235), a reactive copper-complex dye. They were a kind gift of CTH R. Beilich GmbH (Barcelona, Spain). They were used as received, without further purification.

In nonlifting cases, because of underlying fibrosis endoscopic su

In nonlifting cases, because of underlying fibrosis endoscopic submucosal dissection may be necessary for complete resection.14 Figure options Download full-size image Download high-quality image (240 K) Download as PowerPoint slide Fig. 48. Ensuring complete resection. Close endoscopic visualization of the surroundings of the resection area to ensure complete resection cannot be overemphasized. In this case, indigo carmine is applied EPZ5676 to delineate its borders. EMR is performed, showing significant fibrosis. However, close inspection of the defect borders shows residual lesion

(arrows). Repeat snare of the site is immediately performed to achieve complete resection. Argon plasma coagulation is then used to coagulate the base and edges of the resection. Figure options Download full-size image Download high-quality image (284 K) Download as PowerPoint slide Fig. 49. Evaluation of the surroundings is critical. Following resection, close inspection of the resection defect borders should be performed, and any residual neoplasia removed. In addition, the mucosa

around the site should be biopsied to exclude the presence of invisible dysplasia. Figure options Download full-size image Download high-quality image (315 K) Download as PowerPoint slide Fig. 50. Multiple nonpolypoid neoplasms can be endoscopically resected during a single procedure. A 62-year-old patient with long-standing Crohn’s colitis underwent surveillance colonoscopy Vincristine mw that showed multiple neoplasms distributed throughout the colon. (1A to 1C) and (2A to 2E) illustrate details of diagnosis Progesterone and resection of the lesions. Chromoendoscopy using indigo carmine 0.4% was used for delineation of the borders and examination of the epithelial surface. En bloc EMR resections were performed (1C, 2E). Histopathology showed LGD within chronic colitis. Figure options Download full-size image Download

high-quality image (543 K) Download as PowerPoint slide Fig. 51. Endoscopic resection in patients with Crohn or ulcerative colitis can be very difficult because of underlying thickened mucosa and fibrosis. Multiple biopsies for removal of such lesions must be avoided. EMR is usually the most appropriate endoscopic therapy, noting still the high level of difficulty and risk in endoscopic resection of IBD lesions. Endoscopic submucosal dissection may be necessary for complete resection in some cases, such as shown here. Following injection of the submucosa, there is minimal lifting. Thus, a dual knife is used to make a circumferential incision around the lesion border and dissect the fibrosis submucosally, after which a snare is used to remove the lesion in one piece. Figure options Download full-size image Download high-quality image (195 K) Download as PowerPoint slide Fig. 52. Severe fibrosis in Crohn’s or ulcerative colitis can make endoscopic removal technically difficult.

When exposed

When exposed AC220 to repeated levels of disturbance throughout the day, the net effect is an altered activity budget, in which killer whales spend less time feeding in the presence of boats than during no-boat, control conditions

(Lusseau et al., 2009 and Williams et al., 2006). A number of studies have demonstrated effects of noise from large ships on a variety of cetacean species, including Cuvier’s beaked whale (Aguilar Soto et al., 2006), North Atlantic right whale (Nowacek et al., 2004 and Rolland et al., 2012), beluga (Erbe and Farmer, 1998 and Erbe and Farmer, 2000) and fin whales (Castellote et al., 2012). These studies provide a hint that ship noise can reduce a whale’s foraging efficiency (Aguilar Soto et al., 2006); elevate the risk of ship strikes (Nowacek et al., 2004); and cause physiological stress that is detectable in hormone levels (Rolland et al., 2012). A combination of captive experiments and computer models (Erbe and Farmer, 2000) enabled researchers to estimate that icebreaker noise is audible to belugas and capable of eliciting behavioral responses and causing http://www.selleckchem.com/products/PTC124.html communication masking at ranges to 62 km. A temporary hearing shift was modeled to occur if a beluga stayed within 1–4 km of the icebreaker

for at least 20 min. Whales have evolved in an ocean environment that becomes naturally noisy during storms and surf zones, and they have evolved some mechanisms to compensate for noise. Fin whales change their song characteristics to try to maintain communication in high levels

of shipping noise (Castellote et al., 2012). There is some evidence to suggest that killer whales can compensate for increases in ambient noise by lengthening their calls (Foote et al., 2004) or increasing the source level of social calls (Holt et al., 2008). There is no evidence that killer whales can adjust their echolocation patterns to compensate for masked signals used in foraging, and no information on the upper limit to Selleckchem Fludarabine the whales’ compensatory mechanisms. In many behavioral response studies, the received levels that trigger responses are rarely known (but see (Williams et al., 2002a)). A recurring theme in the literature describing marine mammals and noise is that the most rigorous behavioral studies rarely report information on the acoustic stimulus, and the best acoustic studies often have very small sample size for inferring behavioral responses (Nowacek et al., 2007). No studies have yet examined the responses of killer whales to presence and activities of large ships. Such studies are needed (Wright, 2008). Global shipping represents a large and growing contributor to ocean ambient soundscapes (Hildebrand, 2009), and creative solutions are needed to quantify and mitigate impacts of chronic ocean noise on sensitive marine mammals (Wright et al., 2011).

Thus, the amaranth flour film should meet some requirements, rega

Thus, the amaranth flour film should meet some requirements, regarding mechanical strength, flexibility, and permeability to water vapor and gases, in order to ensure food preservation during storage. Therefore, the aim of this work was to examine the effect of the drying conditions find more on the mechanical, solubility, barrier properties, and drying time of amaranth flour films plasticized with glycerol or sorbitol and optimize the drying process by using a response surface methodology and multi-response analyses, targeting the production

of films with low solubility and good mechanical properties. The Amaranthus cruentus BRS Alegria seeds were grown in the state of Santa Catarina (Brazil) at 18.8–22 °C, soil pH of 5.5. The seeds were harvested in early October, transported to Campinas (Brazil), cleaned, and stored at 10 °C. The amaranth flour was obtained by using a modification to the alkaline wet milling method of Perez, Bahnassey, and Breene (1993), as proposed by Tapia-Blácido et al. (2005a). The composition of amaranth flour is: moisture content 8.3 ± 0.4 g/100 g, ashes 2.1 ± 0.0 g/100 g, lipids 7.9 ± 0.2 g/100 g,

protein 14.1 ± 0.3 g/100 g, and starch 75.7 ± 0.3 g/100 g (11.9 ± 0.3 g amylose/100 g flour) (db). All the reagents were analytical PI3K Inhibitor Library chemical structure grade. Sorbitol and NaOH were purchased from Synth (São Paulo, Brazil). All the solutions were prepared with deionized water. The films were produced by the casting method. Amaranth flour films were prepared by using the methodology proposed by Tapia-Blácido et al. (2005a). A suspension ifoxetine of flour in water (4 g/100 g) was homogenized in a mixer for 25 min, and the pH was regulated to 10.7 with 0.1 mol equi/L NaOH, to dissolve the protein. This suspension was then heated at 75 °C for 15 min, followed by addition of the plasticizer (29.6 g sorbitol/100 g flour or 20.02 g glycerol/100 g flour). For each film, 85 ± 3 g of the film-forming solution was poured onto acrylic plates (18 × 21 cm), in order to obtain a constant thickness

of 80 ± 5 μm. The films were dried under different drying conditions by using an oven with air circulation and controlled temperature (model MA 415UR, Marconi, Piracicaba, Brazil). The studied drying conditions were 30 °C, 40% RH; 30 °C, 70% RH; 50 °C, 40% RH; 50 °C, 70% RH; 25.9 °C, 55% RH; 54.1 °C, 55% RH; 40 °C, 33.8% RH; 40 °C, 76.2% RH; and 40 °C, 55% RH, defined according to the experimental design that was being used (Tables 1 and 2). The drying kinetics curves of the amaranth flour films were determined for all the studied conditions. Prior to characterization, all the films were preconditioned for at least 48 h in desiccators containing a saturated NaBr solution (25 ± 3 °C, 58 ± 2% RH). The thickness of the films was measured with a digital micrometer Fowler (average of 8 measurements).

To have a representative set of the

liver contigs of B m

To have a representative set of the

liver contigs of B. microlepidotus, reads of each individual were mapped back to the assembled transcriptome using the alignment program TMAP (http://github.com/iontorrent/TMAP/tarball/tmap.0.3.7) (for more details see Supplementary methods) and contigs showing expression in the three individuals were chosen. In total 13,724 contigs (Supplementary information 1) with an average length of 836.8 bp were retained for the functional annotation ( Table 1; Fig. 1A). The raw sequence data is accessioned in the NCBI Sequence Read Archive (SRA accession SRP046041). The Blastx function was performed with a minimum E-value score of 1.0E− 06 and the gene ontology (GO) terms of molecular function, cellular component, and biological process were GKT137831 price assigned to the 13,724 retained contigs using the Blast2GO software (Conesa et al., 2005). A total of 2803 sequences presented Blast results and 7938 (57.8%) sequences were successfully annotated (Fig. S1, Supplementary information 2). As expected, the species distribution of the Blast hits showed that most hits correspond to fish species (Fig. S2, Supplementary information 2). A total of 40,814 annotations (Supplementary information 3) for the 13,724 contigs were obtained; the biological processes class was the most highly represented (44.2%), followed

by molecular function (35%) and cellular component (20.8%) (Fig. 1B). These proportions were similar to these described for Oncorchynchus mykiss ( Fox et al.,

2014). For B. microlepidotus, the biological see more processes involved mainly the diversity of gene expression, with Mannose-binding protein-associated serine protease predominance of cellular, metabolic and single-organism processes ( Fig. 2A), while the GO annotations for molecular functions were mostly represented by binding and catalytic activity ( Fig. 2B). The cellular component class was mainly composed of cell, organelle, membrane and macromolecular complex components ( Fig. 2C). See Supplementary methods for details regarding functional annotation. The following are the supplementary data related to this article. Supplementary methods We thank C Quezada-Romegialli, JP Oyanedel and P Muñoz-Rojas for support during field work and Dr. Arne Nolte for support during the analyses. The authors thank R Espejo and Omics-Solutions Chile for sequencing. DV thanks Basal Grant PFB 023, ICM P05-002 and Nucleo Milenio NC120030; CVR thanks Conicyt Doctoral Fellowship 21090188 and doctoral thesis fellowship 24121005. All analyses were conducted in Chile and complied with its existing laws (Resolución Exenta No. 3329 Subsecretaria de Pesca). “
“Brine shrimp (Artemia franciscana) are small crustaceans found worldwide, mainly in hypersaline environments. This zooplanktonic organism has been extensively used in fish aquaculture as larval feed for over 85% of cultured species ( Kayim et al., 2010). Besides this role in aquaculture, Artemias spp.

One of the advantages of our study was the large number of partic

One of the advantages of our study was the large number of participants in the study compared to previous researches, 84 patients with MS and 115 healthy controls. Most of the participants in our study were RRMS and SPMS, with a small percentage of PPMS. We recommend future studies to include other types of MS in the evaluation to check for differences between all types of the disease. As there is controversy between different studies assessing CCSVI criteria in MS patients and above-mentioned reports about IJV resection consequences, reconsidering the criteria may be an option. Another reason for these controversies might be differences

in techniques, instruments, anatomical site and patient’s position when performing sonography, which can be decreased by using the same method and mode of sonography. The person who performed sonographic evaluations was not blind to patient’s group in our http://www.selleckchem.com/products/DAPT-GSI-IX.html study. Blinding the assessors also can decrease the bias in the future studies. The authors would like to thank Dr. Jalil Kouhpayezadeh for his confidential

supports in statistical procedures and sample size calculation. Also we would like to appreciate the staff of Firoozgar Clinical Research Development Center (FCRDC) for their technical supports and helps. “
“Optic Neuritis (ONe) is a common feature of Multiple Sclerosis (MS) both in the early phase and during the disease course [1]. Pembrolizumab order MS and ONe are due to demyelination [2], but it has been postulated Urease that vascular mechanisms may have a role in MS and

ONe pathogenesis [3], [4], [5] and [6]. According to a recent hypothesis, cerebrospinal venous system alterations may contribute to the development of the disease and may drive its clinical course [7] and [8]. As a matter of fact, a correlation between the hemodynamic pattern of Chronic Cerebrospinal Venous Insufficiency (CCSVI) and the clinical features in patients with MS has been described [9]. In particular, ONe at onset seems to be associated with Internal Jugular Veins (IJV) and/or of proximal Azygous Vein (AV) high grade stenosis, with consequent reflux in the deep cerebral veins. The blood then flows to the pterygoid plexus, and from there to the facial veins via the deep facial vein, to the cavernous sinus and to the ophthalmic veins. While changes in the hemodynamics of the eye’s arterial system, detected by Doppler ultrasound sonography, have been previously described in MS patients with both acute and chronic ONe [10], [11], [12] and [13], the venous flow has not been studied yet, as far as we know. Taking into account the peculiar environment of the arterial-venous system supplying and draining the Optic Nerve, we have considered it as a representative site for studying the relationship between veins and nervous parenchyma.

Samples were obtained with informed consent A detailed protocol

Samples were obtained with informed consent. A detailed protocol learn more for gastric culture is provided in the Supplementary materials. Briefly, glands were extracted from 1 cm2 of human tissue using EDTA in cold chelation buffer,17 seeded in Matrigel (BD Biosciences), and overlaid with medium containing advanced Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with penicillin/streptomycin, 10 mmol/L HEPES, GlutaMAX, 1 × B27 (all

from Invitrogen), and 1 mmol/L N-acetylcysteine (Sigma-Aldrich). Growth factors were added to the basal medium as indicated in the Figures. The final human stomach culture medium contained the following essential components: 50 ng/mL epidermal growth factor (EGF) (Invitrogen), 10% noggin-conditioned medium, 10% R-spondin1–conditioned medium, 50% Wnt-conditioned medium, 200 ng/mL fibroblast growth factor (FGF)10

(Peprotech), 1 nmol/L gastrin (Tocris), and 2 μmol/L transforming growth factor (TGF)βi (A-83-01; Tocris). The facultative component was 10 mmol/L nicotinamide (Sigma-Aldrich). After seeding, 10 μmol/L RHOKi (Y-27632; Sigma-Aldrich) was added. Additional tested components were as follows: 100 ng/mL insulin-like growth factor (IGF) (Peprotech), 10 μmol/L p38 inhibitor (SB202190; Sigma-Aldrich), 3 μmol/L GSK3β inhibitor (CHIR99021; Axon Medchem), and 500 nmol/L prostaglandin E (PGE)2 (Tocris). Approximately 1 cm2 of cancer tissue was cut into small fragments and washed in cold chelation buffer until the supernatant was clear. Fragments were subjected to enzymatic Ergoloid digestion by 1.5 mg/mL collagenase (Gibco) and VX-809 nmr 20 μg/mL hyaluronidase (Sigma) in 10 mL advanced

DMEM/F12 (Gibco), supplemented with antibiotics (Primocin; Invivogen), for 1 hour at 37°C with shaking. Cells were washed twice in advanced DMEM/F12, seeded into Matrigel, and overlayed with medium containing HEPES, GlutaMAX, penicillin, streptomycin, B27, n-acetylcysteine, EGF, R-spondin1, noggin, Wnt, FGF10, gastrin, TGFβ inhibitor, and RHOK inhibitor as described earlier. Bacterial strains and culture conditions are specified in the Supplementary materials. For infection studies, organoids were seeded in 50 μL Matrigel in 4-well multidishes (Thermo Scientific). Antibiotic-free medium was refreshed every 2–3 days, with a minimum of 3 medium changes before infection to allow removal of antibiotics from the culture. Organoids were microinjected on day 10 after seeding with an approximate multiplicity of infection (MOI) of 50 unless otherwise stated. For calculation of MOI, organoids were disrupted into single cells by EDTA and cells were counted (approximately 4000 cells/organoid). To achieve a final MOI of 50, bacteria were suspended in advanced DMEM/F12 at a density of 1 × 109/mL and organoids were injected with approximately 0.2 μL bacterial suspension using a micromanipulator and microinjector (M-152 and IM-5B; Narishige) under a stereomicroscope (MZ75; Leica) inside a sterile bench (CleanAir).

Cellular

monolayer

Cellular

monolayer Selleck GDC-941 is comprised of midgut epithelial cells and is surrounded on its basal side by a well-established extracellular space. Muscle cells and tracheoles were found adjacent to the extracellular space ( Fig. 1C). Columnar and goblet cells are the most abundant cell types and no specific distribution pattern was observed ( Fig. 1C–E). Both cells define the monolayer height and present luminal-oriented microvilli. They differ by the presence of the goblet cell cavity (GV), a specific luminal space (besides EcS and EnS), rich in microvilli. Vesicles could be detected in the columnar cell, suggesting a trafficking route, perhaps involving multivesicular bodies ( Fig. 1D). Regenerative cells were a less often observed cell type limited to the basal side of the cellular monolayer. As vesicles could be observed inside the epithelial cells cytoplasm, we proceeded towards detecting PolyP stores

using both the modified exopolyphosphatase GSK458 research buy PolyP-binding domain (PPBD) (Saito et al., 2005) and DAPI staining on OCT embedded sections. DAPI has been used as PolyP reporter as its interaction with PolyP yields fluorescence in a different wavelength than the blue emission from DAPI–DNA (Allan and Miller, 1980 and Aschar-Sobbi et al., 2008). Although PolyP stores were present along both columnar and goblet cells, goblet cell cavities and its surroundings were the major regions of accumulation of HAS1 PolyP stores (Fig. 2A and B). To confirm storage of PolyP inside epithelial cells, tissue homogenates were analyzed using a recombinant yeast exopolyphosphatase-based assay (Ruiz et al., 2001b). PolyP strongly concentrated in the posterior midgut of A. gemmatalis but was also detected in the anterior midgut ( Fig. 3A). In that regard, both regions were used in the following experiments. After mechanical lysis and decantation, we could obtain

a fraction rich in PolyP granules as detected by DAPI staining ( Fig. 3B). Under the transmission electron microscope, midgut PolyP granules presented an electron dense morphology ( Fig. 3C, inset) similar to PolyP granules from other models. X-ray microanalysis showed an elemental composition identical to previously found spherite profiles ( Fig. 3C). In that regard, detectable levels of metallic atoms like calcium, magnesium, potassium, sodium, and zinc were present. Phosphorous and chloride were also detected. Manganese, iron and sulfur were less often detected ( Fig. 3D). In our samples, calcium peaks were only observed inside spherites and allowed us to use calcium as a spherite reporter in subsequent experiments. A specific group of polyP-containing organelles from protozoans have been shown to contain bafilomycin A1-sensitive V-ATPases (Docampo et al., 2005 and Scott et al., 1995a) and vanadate-sensitive Ca+2-ATPases (Docampo et al., 1995b) important for metal uptake.