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International audience; Branchial chambers constitute the main osmoregulatory site in almost all decapod crustaceans. However, few studies have been devoted to elucidate the cellular function of specific cells in every osmoregulatory structure of the branchial chambers. In decapod crustaceans, it is well-known that the osmoregulatory function is localized in specific structures that progressively specialize from early developmental stages while specific molecular mechanisms occur. In this study, we found that although the structures developed progressively during the larval and postlarval stages, before reaching juvenile or adult morphology, the osmoregulatory capabilities of Litopenaeus vannamei were gradually established only during the development of branchiostegites and epipodites, but not gills. The cellular structures of the branchial chambers observed during the larval phase do not present the typical ultrastructure of ionocytes, neither N a + /K +-ATPase expression, likely indicating that pleura, branchiostegites, or bud gills do not participate in osmoregulation. During early postlarval stages, the lack of N a + / K +-ATPase immunoreactivity of the ionocytes from the branchiostegites and epipodites suggests that they are immature ionocytes (ionocytes type I). It could be inferred from IIF and TEM results that epipodites and branchiostegites are involved in iono-osmoregulation from PL15, while gills and pleura do not participate in this function.

Calcium oxalate (CaOx) crystals in plants are formed in crystal idioblasts cells and have specific geometric shapes. Their proposed functions include calcium homeostasis and carbon source, among others. Amaranth is a plant that presents high tolerance to abiotic stresses and accumulates considerable amounts of CaOx crystals; however, few studies have focused on characterizing the crystals ultrastructure and none is related to identifying proteins bound to them. This information is of great interest to understand the mechanisms related to CaOx crystal formation and to support their proposed functions. Thus, this work aimed to characterize CaOx crystals in amaranth leaves. Crystals were purified and the proteins bound to them were isolated and identified by nLC-MS/MS. Leaf sections were analyzed by light and electron microscopy. The identified proteins were related to the chloroplast such as ATPb synthase, RuBisCO large subunit, and cell wall-related proteins, which were validated by immunohistochemistry and immunogold labeling. In addition, it was observed that CaOx crystal idioblasts were formed from parenchyma cells associated with mesophyll and veins, in which the thylakoid membranes of degraded chloroplasts turned into crystal chambers. These results significantly advance our understanding of the mechanisms of CaOx crystal formation and the potential function as an alternative carbon source in leaves.

Branchial chambers constitute the main osmoregulatory site in almost all decapod crustaceans. However, few studies have been devoted to elucidate the cellular function of specific cells in every osmoregulatory structure of the branchial chambers. In decapod crustaceans, it is well-known that the osmoregulatory function is localized in specific structures that progressively specialize from early developmental stages while specific molecular mechanisms occur. In this study, we found that although the structures developed progressively during the larval and postlarval stages, before reaching juvenile or adult morphology, the osmoregulatory capabilities of Litopenaeus vannamei were gradually established only during the development of branchiostegites and epipodites, but not gills. The cellular structures of the branchial chambers observed during the larval phase do not present the typical ultrastructure of ionocytes, neither Na

Toxoplasmosis is a zoonotic disease caused by the apicomplexa protozoan parasiteToxoplasma gondii. This disease is a health burden, mainly in pregnant women and immunocompromised individuals, in whom they can cause death. Despite advances in the medical area, nowadays there are no new drugs to treat toxoplasmosis. The standard therapy to toxoplasmosis has not had progress for last seven decades; it is a combination of sulfadiazine-pyrimethamine (S-P); which is co-administered with folic acid due to the adverse effects of the drug. Several studies have shown that the conventional treatment has limited effectiveness and severe adverse effects. Thus, the search of better treatments with greater efficacy and without the adverse effects becomes relevant. In the current work we demonstrate for the first time the parasiticidal effect of dehydroepiandrosterone (DHEA), a steroid hormone produced by many mammals, on extracellular tachyzoites (the infective stage ofT. gondii). In vitro treatment with DHEA reduces the viability of extracellular tachyzoites, and both the active and passive invasion processes. The ultrastructural analysis of treated parasites showed that DHEA alters the cytoskeleton structures, leading in the lost of the organelle structure and organization, as well as, the lost of the cellular shape. On a molecular level, we observed an important reduction of the expression of several proteins that are essential for the motility and virulence of parasites when they were exposed to DHEA. These results suggest that DHEA could be used as an alternative treatment against toxoplasmosis.

Emission of bioaerosols from biofilters during the treatment of toluene vapours was studied. A non-culture-dependent technique, known as epifluorescence microscopy (EM), with several fluorochromes was used to characterize and quantify bioaerosols. The bioaerosol emitted concentrations were between 6.4 × 105 and 1.3 × 108 cells m−3air compared with the bioaerosol concentration in ambient air, which was 3.0 × 107±7 × 106 cells m−3air. EM allowed for a better estimation of bioaerosol concentrations than culture-dependent techniques. Bioaerosol emission was dependent on the packing material. Perlite was a better packing material in terms of removal efficiency (RE; RE of 60%), with a lower bioaerosol emission (7 × 107 cells m−3air) than Tezontle (RE = 40%; 1.3 × 108 cells m−3air). The main drawback of perlite was acidification of the bed. Bioaerosols in biofilters A and B were composed of Gram-negative bacteria (45% and 40%, respectively), a similar percentage of Gram-positive bacteria (28%) and fungi (27% and 32%, respectively). After the shutdown periods, Gram-positive bacteria were predominant (∼60%). The biomass concentrations in leachates were twice those in the air flow and were mainly composed of fungi. Overall, the EM technique is a valuable tool to characterize and quantify bioaerosols in biofilters without under evaluation. This is the first estimation of bioaerosol emissions by biofilters inoculated with a microbial consortium using a noncultivable technique.

One-dimensional titanate nanotubes (H2Ti2O5·H2O) functionalized with silver nanoparticles (AgNPs) exhibited unique properties for the effective inactivation of the Gram-negative Escherichia coli within 45 minutes under irradiation using a 65 W halogen lamp. The pathway of the photo-assisted catalytic inactivation was examined by SEM and TEM using a reproducible biological protocol for sample preparations. The membrane integrity of the bacteria was damaged due to the oxidative stress caused by the reactive oxygen species, the bacteriostatic effect of the highly-dispersed-surface AgNPs (∼5 nm) and the sharp nanotube penetration that induced the cell death.

Acidithiobacillus thiooxidans is an acidophilic chemolithoautotrophic bacterium widely used in the mining industry due to its metabolic sulfur-oxidizing capability. The biooxidation of sulfide minerals is enhanced through the attachment of At. thiooxidans cells to the mineral surface. The Type IV pili (TfP) of At. thiooxidans may play an important role in the bacteria attachment since TfP play a key adhesive role in the attachment and colonization of different surfaces. In this work, we report for the first time the mRNA sequence of three TfP proteins from At. thiooxidans, the adhesin protein PilY1 and the TfP pilins PilW and PilV. The nucleotide sequences of these TfP proteins show changes in some nucleotide positions with respect to the corresponding annotated sequences. The bioinformatic analyses and 3D-modeling of protein structures sustain their classification as TfP proteins, as structural homologs of the corresponding proteins of Ps. aeruginosa, results that sustain the role of PilY1, PilW and PilV in pili assembly. Also, that PilY1 comprises the conserved Neisseria-PilC (superfamily) domain of the tip-associated adhesin, while PilW of the superfamily of putative TfP assembly proteins and PilV belongs to the superfamily of TfP assembly protein. In addition, the analyses suggested the presence of specific functional domains involved in adhesion, energy transduction and signaling functions. The phylogenetic analysis indicated that the PilY1 of Acidithiobacillus genus forms a cohesive group linked with iron- and/or sulfur-oxidizing microorganisms from acid mine drainage or mine tailings.

To our knowledge, there are no studies about the structure and ecological function of extrafloral nectaries (EFNs) in Opuntia robusta. This is the first description of EFNs in O. robusta, where young spines have an interesting structure and a secreting function, which are different from EFNs described in other Cactaceae species. We used light, scanning-electron, and transmission-electron microscopy to examine morphology, anatomy, and ultrastructure of the secretory spines in areoles in female and hermaphrodite individuals of O. robusta. Young cladodes develop areoles with modified and secretory spines as EFNs only active during the early growth phase. EFNs are non-vascularized structures, with no stomata, that consist of a basal meristematic tissue, a middle elongation region, and an apical secretory cone formed by large globular epidermal cells, containing nectar and medullar elongated cells. We observed the presence of Golgi apparatus, vesicles and plastids in the medullar and sup-epidermal cells of the spine. We propose that the nectar is stored in the globular cells at the apex of the spine and secreted by breaking through the globular cells or by pores. We recorded a more frequent presence of ants on younger cladode sprouts producing young secreting spines: this result is parallel with the predictions of Optimal Defense Hypothesis, which states that younger plant organs should be better defended than older ones because their loss produces a higher fitness impairment. Although Diaz-Castelazo's hypothesis states that a more complex structure of EFNs correlates with their lower among-organs dispersion, comparing to less complex EFNs, non-vascularized structure of EFNs in O. robusta is not associated with their higher among-organs dispersion likened to O. stricta, which produces vascularized EFNs. We provide evidence that this characteristic is not a good taxonomic feature of Opuntia genus. Moreover, the comparison of EFNs of O. robusta and O. stricta suggests that the hypothesis of Diaz-Castelazo should be revised: it is rather a rule but not a law.

Acidithiobacillus thiooxidans is an acidophilic chemolithoautotrophic bacterium widely used in the mining industry due to its metabolic sulfur-oxidizing capability. The biooxidation of sulfide minerals is enhanced through the attachment of A. thiooxidans cells to the mineral surface. The Type IV pili (TfP) of At. thiooxidans may play an important role in the bacteria attachment, since among other functions, TfP play a key adhesive role in the attachment to and colonization of different surfaces. In this work, we reported for the first time the confirmed mRNA sequences of three TfP proteins from At. thiooxidans, the protein PilY1 and the TfP pilins PilW and PilV. The nucleotide sequences of these TfP proteins show changes of some nucleotide positions with respect to the corresponding annotated sequences. The bioinformatic analyses and 3D-modeling of protein structures sustain their classification as TfP proteins, as structural homologs of the corresponding proteins of P. aeruginosa, results that sustain the role of PilY1, PilW and PilV in pili assembly. Also, that PilY1 comprises the conserved Neisseria-PilC (superfamily) domain of the tip-associated adhesin, while PilW of the superfamily of putative TfP assembly proteins and PilV belongs to the superfamily of TfP assembly protein. Also, the analyses suggested the presence of specific functional domains involved in adhesion, energy transduction and signaling functions. The phylogenetic analysis indicated that the PilY1 of Acidithiobacillus genus forms a cohesive group linked with iron- and/or sulfur-oxidizing microorganisms from acid mine drainage or mine tailings. This work enriches knowledge regarding colonization, adhesion and biooxidation of inorganic sulfurs by A. thiooxidans.

The role of the renin-angiotensin system (RAS) in blood pressure regulation in MS during aging is unknown. It participates in metabolic syndrome (MS) and aging regulating vascular tone and remodeling. RAS might participate in a compensatory mechanism decreasing blood pressure and allowing MS rats to reach 18 months of age and it might form part of therapeutical procedures to ameliorate MS. We studied histological changes and distribution of RAS receptors in aortas of MS aged rats. Electron microscopy images showed premature aging in MS since the increased fibrosis, enlarged endothelium, and invasion of this layer by muscle cells that was present in control 18-month-old aortas were also found in 6-month-old aortas from MS rats. AT1, AT2, and Mas receptors mediate the effects of Ang II and Ang 1-7, respectively. Fluorescence from AT2 decreased with age in control and MS aortas, while fluorescence of AT1 increased in aortas from MS rats at 6 months and diminished during aging. Mas expression increased in MS rats and remained unchanged in control rats. In conclusion, there is premature aging in the aortas from MS rats and the elevated expression of Mas receptor might contribute to decrease blood pressure during aging in MS.

Phloem loading is a critical process in plant physiology. The potential of regulating the translocation of photoassimilates from source to sink tissues represents an opportunity to increase crop yield. Pyrophosphate homeostasis is crucial for normal phloem function in apoplasmic loaders. The involvement of Arabidopsis (Arabidopsis thaliana) type I proton-pumping pyrophosphatase (AVP1) in phloem loading was analyzed at genetic, histochemical, and physiological levels. A transcriptional AVP1 promoter::GUS fusion revealed phloem activity in source leaves. Ubiquitous AVP1 overexpression (35S::AVP1 cassette) enhanced shoot biomass, photoassimilate production and transport, rhizosphere acidification, and expression of sugar-induced root ion transporter genes (POTASSIUM TRANSPORTER2 [KUP2], NITRATE TRANSPORTER2.1 [NRT2.1], NRT2.4, and PHOSPHATE TRANSPORTER1.4 [PHT1.4]). Phloem-specific AVP1 overexpression (Commelina Yellow Mottle Virus promoter [pCOYMV]::AVP1) elicited similar phenotypes. By contrast, phloem-specific AVP1 knockdown (pCoYMV::RNAiAVP1) resulted in stunted seedlings in sucrose-deprived medium. We also present a promoter mutant avp1-2 (SALK046492) with a 70% reduction of expression that did not show severe growth impairment. Interestingly, AVP1 protein in this mutant is prominent in the phloem. Moreover, expression of an Escherichia coli-soluble pyrophosphatase in the phloem (pCoYMV::pyrophosphatase) of avp1-2 plants resulted in severe dwarf phenotype and abnormal leaf morphology. We conclude that the Proton-Pumping Pyrophosphatase AVP1 localized at the plasma membrane of the sieve element-companion cell complexes functions as a synthase, and that this activity is critical for the maintenance of pyrophosphate homeostasis required for phloem function.

A bioelectrochemical study of charge transfer in the biofilm–chalcopyrite interface was performed to investigate the effect of surficial reduced sulfur species (RSS), in the form of non-stochiometric compounds or polysulfides (Sn2−) and elemental sulfur (S0) on a biofilm structure, during the earliest stages (1, 12 and 24 h) of chalcopyrite biooxidation by Acidithiobacillus thiooxidans alone and adding Leptospirillum sp. The surface of massive chalcopyrite electrodes was exposed to the bacteria, which were analyzed electrochemically, spectroscopically, and microscopically. At the studied earlier times, charge transfer and significant differences in the biofilm structure were detected, depending on the presence of Leptospirillum sp. acting on A. thiooxidans biofilms. Such differences were a consequence of a continuous chalcopyrite pitting and promoting changes in biofilm hydrophobicity. A. thiooxidans modifies the reactive properties of RSS and favors an acidic dissolution, which shifts into ferric dissolution when Leptospirillum sp. is present. A. thiooxidans allows H+ and Fe3+ diffusion, and Leptospirillum sp. enables to surpass the charge transfer (reactivity) barrier between the mineral interface and the ions. The observed changes of hydrophobicity on the interface are associated to ions and electrons activity and transfer. Finally, a model of S0 biooxidation by A. thiooxidans alone or with Leptospirillum sp. is proposed.

A bioelectrochemical study of charge transfer in the biofilm/chalcopyrite interface was performed to investigate the effect of surficial sulfur reduced species (SRS), as non-stochiometric compounds or polysulfides (Sn2-), and elemental sulfur (S0) on a biofilm structure during the earliest stages (1, 12 and 24 h) of chalcopyrite biooxidation by A. thiooxidans alone and adding Leptospirillum sp. The surface of massive chalcopyrite electrodes was exposed to the bacteria, which were analyzed electrochemically, spectroscopically, and microscopically. At the studied earlier times, charge transfer and significant differences in the biofilm structure were detected, depending on the presence of Leptospirillum sp. acting on A. thiooxidans biofilms. Such differences were a consequence of a continuous chalcopyrite pitting and promoting changes in biofilm hydropathy. A. thiooxidans modifies the reactive properties of SRS and favors an acidic dissolution, which shifts into ferric when Leptospirillum sp. is present. A. thiooxidans allows H+ and Fe3+ diffusion, and Leptospirillum sp. allows surpassing the charge transfer (reactivity) barrier between the mineral interface and the ions. The observed changes of hydropathy on the interface are associated to ions and electrons activity and transfer. Finally, a model of S0 biooxidation by A. thiooxidans alone or with Leptospirillum sp., is proposed.

Previous literature has shown the presence of a plasma membrane (PM) localized type I H+-PPase in sieve elements of Ricinus communis. Unfortunately, the physiological relevance of these findings remains obscure due to the lack of genetic and molecular reagents to study R. communis. The availability of H+-PPase gain and loss-of-function mutants in Arabidopsis thaliana makes this plant an attractive genetic model to address the question, but data on the PM localization of this H+-PPase in A. thaliana are limited to two proteomic approaches. Here we present the first report on the localization of the type I H+-PPase AVP1 in sieve element–companion cell complexes (SE–CCc) from A. thaliana. Double epifluorescence and immunogold labeling experiments are consistent with the co-localization of AVP1 and PIP1 (a bona fide PM maker) in PM of SE–CCc from A. thaliana.

Mating in yeast is initiated by binding of pheromone to G-protein-coupled receptors expressed in haploid cells. We analysed the role of KlSte2p and KlSte3p receptors in the Kluyveromyces lactis mating pathway. By sequence analysis, KlSte2p and KlSte3p are the homologues of the Saccharomyces cerevisiae α-pheromone and a-pheromone receptors, respectively. However, by expression experiments, we determined that KlSTE2 gene is expressed in the cells typified as MATα and therefore is the receptor for the K. lactis a-pheromone and KlSTE3 gene is expressed in the MATa cells and binds the α-pheromone. The KlSTE2 gene is silent in MATa cells, while it is highly expressed in MATα cells, and conversely the KlSTE3 gene is expressed in MATa cells and repressed in MATα cells. Disruption mutants of both genes were found to be sterile, and this defect is reversed by plasmidic copies of each gene. The cytoplasmic C-terminus of KlSte3p interacts strongly with the KlGpa1p (Gα) subunit, which is involved in the transduction of the pheromone stimulus to induce mating. Remarkably, this same domain does not interact with a constitutive active allele of the Gα subunit, indicating that the C-terminus is able to discriminate between the active (GTP-bound) and inactive (GDP-bound) forms of the Gα subunit. Copyright © 2007 John Wiley & Sons, Ltd.

Candida glabrata is a haploid yeast considered the second most common of the Candida species found in nosocomial infections, accounting for approximately 18% of candidemias worldwide. Even though molecular biology methods are easily adapted to study this organism, there are not enough vectors that will allow probing the transcriptional and translational activity of any gene of interest in C. glabrata. In this work we have generated a set of expression vectors to systematically tag any gene of interest at the carboxy-terminus with three different fluorophores (CFP, YFP and mCherry) or three epitopes (HA, FLAG or cMyc) independently. This system offers the possibility to generate translational fusions in three versions: under the gene’s own promoter integrated in its native locus in genome, on a replicative plasmid under its own promoter, or on a replicative plasmid under a strong promoter to overexpress the fusions. The expression of these translational fusions will allow determining the transcriptional and translational activity of the gene of interest as well as the intracellular localization of the protein. We have tested these expression vectors with two biosynthetic genes, HIS3 and TRP1. We detected fluorescence under the microscope and we were able to immunodetect the fusions using the three different versions of the system. These vectors permit coexpression of several different fusions simultaneously in the same cell, which will allow determining protein–protein and protein–DNA interactions. This set of vectors adds a new toolbox to study expression and protein interactions in the fungal pathogen C. glabrata.

Candida glabrata (CG) is an opportunistic fungal pathogen that initiates infection by binding to host cells via specific lectin-like adhesin proteins. We have previously shown the importance of lectin-oligosaccharide binding in cardiac responses to flow and agonists. Because of the lectinic-oligosaccharide nature of CG binding, we tested the ability of CG to alter the agonist- and flow-induced changes in cardiac function in isolated perfused guinea pig hearts. Both transmission and scanning electron microscopy showed strong attachment of CG to the coronary endothelium, even after extensive washing. CG shifted the coronary flow vs. auricular-ventricular (AV) delay relationship upward, indicating that greater flow was required to achieve the same AV delay. This effect was completely reversed with mannose, partially reversed with galactose and N-acetylgalactosamine, but hyaluronan had no effect. Western blot analysis was used to determine binding of CG to isolated coronary endothelial luminal membrane (CELM) receptors, and the results indicate that flow-sensitive CELM receptors, ANG II type I, α-adrenergic 1A receptor, endothelin-2, and VCAM-1 bind to CG. In addition, CG inhibited agonist-induced effects of bradykinin, angiotensin, and phenylephrine on AV delay, coronary perfusion pressure, and left ventricular pressure. Mannose reversed the inhibitory effects of CG on the agonist responses. These results suggest that CG directly binds to flow-sensitive CELM receptors via lectinic-oligosaccharide interactions with mannose and disrupts the lectin-oligosaccharide binding necessary for flow-induced cardiac responses.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.