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H.R.5152 - Savings, Accountability, Value, and Efficiency III Act of 2014113th Congress (2013-2014)
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|Rep. Murphy, Patrick [D-FL-18] (Introduced 07/17/2014)
|House - Oversight and Government Reform; Energy and Commerce; Armed Services; Ways and Means; Veterans' Affairs
|Latest Action:
|House - 08/15/2014 Referred to the Subcommittee on Health. (All Actions)
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Cosponsors: H.R.5152 — 113th Congress (2013-2014)All Information (Except Text)
Sponsor: Rep. Murphy, Patrick [D-FL-18] | Cosponsor statistics: 11 current - includes 11 original
* = Original cosponsor
|Date Cosponsored
|Rep. Meadows, Mark [R-NC-11]*


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Youth Red Cross
Youth Red Cross Society has been formed in this College for the purpose of inculcating in the youth of the country the ideals of peace and the practice of service especially in relation to the cause of their own health and that of others. The Youth should cultivate a spirit of friendly helpfulness towards the children and youth of other countries.
The aims of Youth Red Cross Society -(1) Promotion of health;
(2) Service to the sick and suffering;
(3) Development of international friendliness.
Membership to Youth Red Cross is open to girls of all classes who desire to participate in its various activities.
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Antimicrobial peptides play an important role in host defense against microbial pathogens. Their high cationic charge and strong amphipathic structure allow them to bind to the anionic microbial cell membrane and disrupt the membrane bilayer by forming pores or channels. In contrast to the classical pore-forming peptides, studies on histatin-5 (Hst-5) have suggested that the peptide is transported into the cytoplasm of Candida albicans in a non-lytic manner, and cytoplasmic Hst-5 exerts its candicidal activities on various intracellular targets, consistent with its weak amphipathic structure. To understand how Hst-5 is internalized, we investigated the localization of FITC-conjugated Hst-5. We find that Hst-5 is internalized into the vacuole through receptor-mediated endocytosis at low extracellular Hst-5 concentrations, whereas under higher physiological concentrations, Hst-5 is translocated into the cytoplasm through a mechanism that requires a high cationic charge on Hst-5. At intermediate concentrations, two cell populations with distinct Hst-5 localizations were observed. By cell sorting, we show that cells with vacuolar localization of Hst-5 survived, while none of the cells with cytoplasmic Hst-5 formed colonies. Surprisingly, extracellular Hst-5, upon cell surface binding, induces a perturbation on the cell surface, as visualized by an immediate and rapid internalization of Hst-5 and propidium iodide or rhodamine B into the cytoplasm from the site using time-lapse microscopy, and a concurrent rapid expansion of the vacuole. Thus, the formation of a spatially restricted site in the plasma membrane causes the initial injury to C. albicans and offers a mechanism for its internalization into the cytoplasm. Our study suggests that, unlike classical channel-forming antimicrobial peptides, action of Hst-5 requires an energized membrane and causes localized disruptions on the plasma membrane of the yeast. This mechanism of cell membrane disruption may provide species-specific killing with minimal damage to microflora and the host and may be used by many other antimicrobial peptides.
Author Summary
In most healthy individuals, the yeast Candida albicans is found within the oral cavity as part of the normal microflora. Though under immunocompromising conditions, this benign microbe can become an opportunistic pathogen causing oral candidiasis (i.e. thrush). Although antifungal drugs are typically efficacious, the paucity of drugs and their increasing usage has led to rising drug resistance. Thus, researchers have begun to look at alternative therapeutics, such as the candidacidal salivary peptide histatin-5. To date, little is known about the initial binding and subsequent internalization that facilitates Hst-5's killing activity. Thus, our study attempted to determine how Hst-5 is internalized into C. albicans. It was thought that Hst-5 is transported into the cytoplasm without disruption of the plasma membrane. However, our study finds that Hst-5, under physiological concentrations, disrupts the plasma membrane and is rapidly translocated into the cytoplasm, leading to cell death. Interestingly, the internalization of Hst-5 is initiated from a single spatially restricted site on the plasma membrane rather than multiple breaches on the cell surface. This novel mechanism of membrane disruption provides new insights into how Hst-5 and other antimicrobial peptides may be acting on pathogenic microorganisms.
Citation: Mochon AB, Liu H (2008) The Antimicrobial Peptide Histatin-5 Causes a Spatially Restricted Disruption on the Candida albicans Surface, Allowing Rapid Entry of the Peptide into the Cytoplasm. PLoS Pathog 4(10): e1000190. https://doi.org/10.1371/journal.ppat.1000190
Editor: Aaron P. Mitchell, Carnegie Mellon University, United States of America
Received: May 2, 2008; Accepted: September 26, 2008; Published: October 31, 2008
Copyright: © 2008 Mochon, Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work is supported by National Institute of General Medicine and Allergy and Infectious Diseases grants 3R01GM/AI55155 and P01AI061537.
Competing interests: The authors have declared that no competing interests exist.
Oral candidiasis is most commonly associated with individuals infected with the human immunodeficiency virus (HIV), and it is also seen in infants, patients with diabetes mellitus, and those receiving broad-spectrum antibiotics. However, oral candidiasis is relatively uncommon in the general population, despite the fact that Candida albicans can be recovered in the alimentary canal of healthy individuals in over 50% of cases [1]. Antimicrobial peptides, including histatins, play an important role in the innate defense against oral Candida infections.
Histatins are a family of low-molecular weight histidine-rich cationic peptides that are found specifically in human salivary secretions [2]. Histatin-5 (Hst-5), a 24-residue peptide is the most potent member of the family with respect to fungicidal activity against C. albicans. It kills C. albicans in vitro at physiological concentrations (15–30 µM) [3],[4]. Structural studies of Hst-5 have shown that it takes on a random coil structure in aqueous solvents and adopts a largely α-helical conformation in non-aqueous solutions [5],[6]. Many antimicrobial cationic peptides form either α-helical or β-sheet structures that exhibit a strong amphipathic nature. After the initial electrostatic attraction to an anionic microbial surface, these cationic amphipathic peptides can spontaneously insert into cell membranes and form pores/channels, causing lysis of the cell membrane [7],[8]. However, unlike the classical pore-forming peptides, Hst-5 is predicted to lack sufficient amphipathic character to insert spontaneously into microbial membranes [3],[6]. Consistent with this prediction, Hst-5 has little lytic effect in a liposome model system [9].
The activity of Hst-5 against C. albicans is believed to be initiated through cell wall binding, followed by translocation and intracellular targeting. Hst-5 has been shown to localize to the cytoplasm of C. albicans cells, where it associates with the energized mitochondria and inhibits respiration [10],[11]. Cytoplasmic Hst-5 also affects cell membrane functions. Hst-5 induces a sizeable noncytolytic efflux of ATP and potassium and magnesium ions into the extracellular milieu, causing a loss in cell volume and ionic imbalance to the yeast cell [12]–[15]. On the other hand, Hst-5 has also been reported to cause slow depolarization of the cytoplasmic and mitochondrial membranes, indicating a lytic activity towards the membranes [16]. Cells treated with Hst-5 also have elevated cell permeability to the small cationic dye propidium iodide (PI) as shown by FACS analysis [10]. Internalization of Hst-5 by C. albicans cells is tightly linked to killing and is dependent on cellular metabolism [16]–[18]. Oxygen depletion or inhibition of respiration by sodium azide blocks the translocation of Hst-5 into the cell by rigidifying the cell membrane [10],[17]. The initial binding of Hst-5 to the C. albicans cell surface is thought to be initiated by specific binding to Ssa1/2, a heat shock protein present on the cell surface of the yeast [19],[20]. Cells without Ssa2 show an impaired Hst-5 uptake [20]. However, how Hst-5 is internalized is not known. The initial cell surface binding by Hst-5 does not lead to cellular lysis as seen with other antimicrobial peptides [12].
Most polar macromolecules are excluded from the interior of cells due to the impermeability of the plasma membrane. The typical mechanism of entry of extracellular components into yeast cells is dependent on either fluid-phase or receptor-mediated endocytosis [21]–[23]. Recently, cationic cell-penetrating peptides, such as HIV Tat and polyarginine, have been shown to translocate directly from the extracellular surface into the cytoplasm without the need for endocytic vesicles in both yeast and mammalian cells [24],[25]. This novel internalization requires cationic charge and an electrostatic interaction with cell the surface.
This study aimed to determine how Hst-5 is translocated into the cytoplasm of C. albicans. To date, the mode of internalization of Hst-5 is not defined, though it has been suggested that internalization of the antifungal peptide is initiated through a non-lytic manner by an unidentified translocase [26]. Here we report that the internalization event is actually a non-classical lytic event. We visualized an immediate and rapid internalization of Hst-5 and fluorochromatic dyes into the cytoplasm from a spatially restricted site on the plasma membrane, and a concurrent rapid expansion of the vacuole. Cell death is completely correlated with the appearance of cytoplasmic Hst-5. Our study provides the first direct evidence for a breach in the plasma membrane as the initial damage by extracelluar Hst-5 on C. albicans and a mechanism of its internalization into the cytoplasm.
The intracellular localization of histatin-5 is concentration-dependent
Hst-5 at physiological concentration (10–30 µM) has been found distributed throughout the cytoplasm of C. albicans cells [27],[28]. However, by using FITC-conjugated Hst-5 at 10 µM or 20 µM, we observed two populations of cells with distinct Hst-5 localization (Figure 1A). In some cells, Hst-5 was localized strictly to the vacuole, but not in the cytoplasm; in other cells, Hst-5 was localized throughout the cytoplasm. The internalization of the FITC-conjugated Hst-5 produced varying fluorescent intensity in both the vacuolar- and cytoplasmic-associated cells. It seemed that more cells showed vacuolar localization at 10 µM than at 20 µM Hst-5, and the reverse was seen for cells with cytoplasmic localization at these concentrations. To examine a possible effect of Hst-5 concentration on its cellular localization, we examined cells in 5 µM and 50 µM Hst-5. Strikingly, cells exposed to 5 µM Hst-5 showed mostly vacuolar localization and cells in 50 µM showed largely cytoplasmic localization with little vacuolar fluorescence (Figure 1A). Therefore, with increasing Hst-5 concentrations, we observed a shift in cell population from vacuolar to cytoplasmic localization. Because the fluorescent intensity of cells with cytoplasmic Hst-5 was much higher than that of cells with vacuolar Hst-5, we were able to quantify the percentage of cells in each population by flow cytometry (Figure 1B and 1C). Cells with vacuolar Hst-5 are in the peak with low intensity on the left, and cells with cytoplamic Hst-5 are in the high intensity peak on the right. Moreover, with increasing Hst-5 concentrations, more cells are shifted from the low intensity peak to the high intensity peak in flow cytometry (Figure 1B and 1C). Since the majority of cells with cytoplasmic Hst-5 still had enlarged intact vacuoles that excluded Hst-5, the concentration dependent localization of Hst-5 is likely through two distinct pathways of internalization.
C. albicans cells were incubated for 30 minutes with 5, 10, 20, and 50 µM FITC-Hst-5. (A) Fluorescent and DIC images of cells with FITC-Hst-5 at the indicated concentrations and (B) flow cytometry analysis of the same cells. The concentration dependent localization is represented by the markers M1 (vacuole) and M2 (cytoplasm). (C) The bar graph represents the percentage of the population where Hst-5 is localized to the vacuole (M1) and to the cytoplasm (M2) for the varying concentrations.
Vacuolar uptake of histatin-5 is dependent on receptor-mediated endocytosis
The vacuolar uptake of Hst-5 could be through either receptor-mediated or fluid-phase endocytosis. To determine which is responsible for vacuolar Hst-5 uptake, mutants that block receptor-mediated, but not fluid-phase, transport to the vacuole were used. Monoubiquitination of transmembrane receptors at their cytoplasmic domains is required for the sorting of these integral membrane proteins into the luminal vesicles of multivesicular bodies (MVBs). ESCRT (endosomal sorting complex) complexes are essential for the sorting event. Receptors that normally internalize and traffic into MVBs and subsequently degrade in the vacuole are instead accumulated on the membrane of MVBs in the mutants of VPS36 or SNF7, components in the ESCRT complexes [23], [29]–[31]. After incubating C. albicans wild-type, vps36, and snf7 mutant cells in 10 µM Hst-5 and 10 µM FM4-64, a lipophilic membrane dye, for 30 minutes at 30°C, Hst-5 was found to accumulate on the surface of, but excluded from the lumen of vacuole-like membrane structures in vps36 and snf7 mutant cells (Figure 2A). It should be noted that FM4-64 increased the necessary threshold concentration of Hst-5 in order to observe vacuolar or cytoplasmic-associated Hst-5 localization. These results indicate that Hst-5 is internalized to the vacuole by a receptor-mediated endocytic pathway.
(A) C. albicans wild-type, vps36, and snf7 cells were incubated for 30 minutes with 10 µM FITC-Hst-5 and 10 µM FM4-64 at 30°C. (B) C. albicans wild-type and myo5 cells were incubated for 10 minutes with 10 µM of FITC-Hst-5 and 10 µM FM4-64 at 30°C. The populations of plasma membrane and cytoplasmic localization, 80.99% and 19.01% as indicated, were determined by flow cytometry. (C) C. albicans wild-type cells were treated with either 50 µM latrunculin A or 5 µM cytochalasin A for 60 minutes at room temperature. The cells were then exposed to 5 µM FITC-Hst-5 for 30 minutes at 30°C.
Next, we evaluated the effect of blocking the initial internalization step of endocytosis on Hst-5 uptake. The actin cytoskeleton and type I myosins are critical for the initial invagination event at the cell surface [21],[32]. Mutation of the type I myosin gene (MYO5) in C. albicans is also deficient in endocytosis, as the uptake of FM4-64 is impeded at the plasma membrane [33]. When myo5 mutant cells were incubated with 10 µM Hst-5 and 10 µM FM4-64 for 10 minutes at 30°C, both FM4-64 and Hst-5 were found on the plasma membrane, but not in the vacuole in the majority of the cell population (Figure 2B). This observation is consistent with the reported defect of myo5 cells in endocytosis. Surprisingly, a moderate proportion of the population, 19.01%, had Hst-5 localized within the cytoplasm. Similarly, in cells treated with latrunculin A (LatA) or cytochalasin A (CytoA), which prevents actin polymerization, Hst-5 at 5 µM was observed in the cytoplasm in a majority of the cells, especially CytoA-treated cells, but was not observed in the vacuole or on the plasma membrane (Figure 2C). Therefore, cells without functional actin cytoskeleton greatly decreased the threshold necessary to induce cytoplasmic Hst-5 translocation.
The cationic charge of histatin-5 is critical for its translocation into the cytoplasm
The cationic charge of antimicrobial peptides is critical for the initial electrostatic attraction of the peptides to negatively charged cell membranes, and there is a strong correlation between peptide cationicity and antimicrobial activity [34]. For Hst-5, cationic charge is also important for its candicidal activity. A single lysine substitution for a histidine or amidation of the C-terminus of Hst-5 analogs increases the candidacidal activity of the peptide by almost two-fold [35],[36]. Whereas, replacement of lysine-13 with glutamic acid and arginine 22 with glycine, a variant of Hst-5 (m68) reported by Tsai et al., caused a significant reduction in its killing ability [37]. The amino acid substitutions in m68 reduce the cationic charge at pH 7.0 from 6.6 in Hst-5 to 3.6. To determine whether the cationic charge of Hst-5 affects how the antimicrobial peptide is internalized into the fungal cell, we observed the localization of a FITC-conjugated m68 in C. albicans. When cells were exposed to concentrations of 5, 10, 20, 50, and 200 µM FITC-m68 at 30°C for 30 minutes, cytoplasmic m68 was not observed at 5, 10, and 20 µM (data not shown) and was only seen in a small portion of the cell population at 50 and 200 µM m68 (Figure 3A). In contrast to Hst-5 at these concentrations, FITC-m68 was localized to the vacuole in the majority of cells (Figure 3A). The result suggests that a robust cationic charge is necessary for cytoplasmic localization of Hst-5.
(A) C. albicans cells were incubated with 50 µM and 200 µM FITC-Hst-5 m68 for 30 minutes at 30°C. (B) C. albicans yeast cells were treated with 20 µM FITC-Hst-5 for 30 minutes at 30°C under different pH conditions (4.5, 7.0, and 9.5) as indicated.
Since Hst-5 sequence is 29% histidine, which has a pI at around physiological pH, the peptide is expected to have different charges at pHs 4.5, 7.0, and 9.0, which are within the resting pH range of the oral cavity [38]. By incubating C. albicans with 20 µM Hst-5 in varying pHs, we could examine the effect of cationic charge on the peptides cellular localization. In cells exposed to Hst-5 at pH 9.0, giving the peptide a charge of +3.6, the majority of the cells showed either cell surface or vacuolar localization, while no cytoplasmic localization was observed. Conversely, when the cells were incubated with Hst-5 at pH 4.5, which induces a positive net charge of 12.9, a preponderance of cells had Hst-5 localized within the cytoplasm and only a small population had the antimicrobial peptide bound to the cell surface (Figure 3B). This is consistent with the previous observation that a strong cationic charge is important for the cytoplasmic localization of Hst-5. However, extreme pHs may have effects on cell physiology that potentially could influence the uptake of Hst-5. Indeed, endocytosis has been shown to be impaired at a low pH [39], which may account for why vacuolar Hst-5 was not observed at pH 4.5. Therefore, Hst-5 uptake pathway is likely affected by both the pH-dependent cationic charge of Hst-5 and pH effects on cell physiology. Nonetheless, we have shown through both amino acid substitution and the ionization of histidine using the pH range of the oral cavity that a strong cationic charge determines the mode of uptake for Hst-5.
Cytoplasmic localization of histatin-5 is linked to killing, whereas vacuolar histatin-5 is non-cytotoxic
Hst-5 at 10 µM kills with around 50% efficiency in a population of 106 cells/ml. Since this concentration gives a mixed population of cells with either vacuolar or cytoplasmic Hst-5, we were interested in determining whether cells with different Hst-5 localizations had different fates. We have observed that cells with cytoplasmic Hst-5 have higher fluorescence intensity than cells with vacuolar Hst-5, and the difference in fluorescence intensity is sufficient to allow us to separate cells into two populations with different Hst-5 localizations (Figure 1). After incubating C. albicans cells in 10 µM Hst-5 at 30°C for 30 minutes, cell sorting was carried out to give us two distinct populations of Hst-5 localized cells (Figure 4A), which were confirmed by fluorescence microscopy. Approximately two hundred cells from each population were aliquotted onto plates to determine colony-forming units. Cells from a no-peptide control and cells with vacuolar Hst-5 showed 100% survival. Conversely, none of the cytoplasmic-localized cells produced colonies (Figure 4A). These results provide irrefutable evidence that cytoplasmic Hst-5 is linked to killing, whereas vacuolar Hst-5 is non-cytotoxic to the cells and is maintained in the vacuole.
(A) C. albicans cells were incubated for 30 minutes with 10 µM FITC-Hst-5 at 30°C. The cells were sorted by gating the two peaks of the histogram representing vacuolar and cytoplasmic localization. The sorted cells were then plated onto YPD plates and incubated overnight at 30°C (data as a mean±1SD of triplicate cultures). (B) C. albicans, C. glabrata, and S. cerevisiae were incubated with either 50 µM FITC-Hst-5 or 50 µM FITC-Hst-5 and 10 mM NaN3 for 30 minutes at 30°C. The cells treated with and without sodium azide had equal fluorescence exposure times of 900 and 9 milliseconds, respectively.
Resistance of C. glabrata and S. cerevisiae to the peptide correlates with a lack of histatin-5 translocation into the cytoplasm
Previous research has shown that the yeasts C. glabrata and S. cerevisiae show a marked resistance to Hst-5 [40]–[42]. We wanted to determine if their insensitivity to the antimicrobial peptide was due to the lack of cytoplasmic translocation of Hst-5. The yeasts C. albicans, C. glabrata, and S. cerevisiae were exposed to 50 µM of Hst-5 for 30 minutes and examined using fluorescent microscopy with a fixed exposure time (9 ms). Interestingly, in C. glabrata cells, Hst-5 was predominantly localized to the vacuole, whereas in S. cerevisiae, the peptide was found bound to the cell surface (Figure 4B). Thus, the resistance of C. glabrata and S. cerevisiae to Hst-5 may be due to the lack of cytoplasmic translocation of the antifungal peptide. To determine if the lack of Hst-5 uptake was due to a difference in cell surface binding, we exposed Hst-5 to the yeasts in the presence of sodium azide, which has been shown to prevent the internalization of Hst-5 [17]. Hst-5 uniformly bound to the cell surface of C. albicans and S. cerevisiae (Figure 4B). Therefore, the lack of cytoplasmic translocation of Hst-5 in S. cerevisiae was not due to the lack of cell surface binding. The uniform Hst-5 localization was not observed in C. glabrata. Instead, punctate regions were detected, which may correspond to ligand-receptor interactions and receptor-mediated endocytosis. Our data suggest that the resistance of C. glabrata and S. cerevisiae to Hst-5 is dependent on its ability to hinder the mechanism of cytoplasmic translocation.
Histatin-5 induces perturbation at a spatially restricted site on the cell surface, leading to a rapid translocation of the peptide into the cytoplasm
Our previous experiments indicated that the cytoplasmic localization of Hst-5 was achieved in as little as 10 minutes after cells were exposed to the peptide. To further evaluate the internalization event, we used time-lapse confocal microscopy to visualize the process of Hst-5 uptake. 50 µM FITC-Hst-5 was added to C. albicans cells in a thin glass-bottom dish. Image acquisition started prior to the addition of the peptide, and frames were recorded every 9 seconds for a total of 7 minutes and 30 seconds. Hst-5 bound uniformly to the cell surface of the yeast almost immediately after addition of the peptide. Surprisingly, shortly afterwards, a green punctuate site was observed mostly on or near the plasma membrane in many cells (Figure 5A). Only one restricted region of concentrated Hst-5 was seen for each cell. Hst-5 rapidly spread throughout the cytoplasm by diffusion, resulting in a uniform accumulation of Hst-5 in the yeast cell (Figure 5A and Video S1). Concurrent with the internalization of Hst-5, we observed a rapid expansion of the vacuole with a parallel loss in cell volume in less than sixty seconds (Video S2). Eventually, at about 10 minutes, the vacuole collapsed in some cells (Video S2). The disruption in cellular membrane compartments is in agreement with the published electron microscope images of Hst-5 treated cells [43].
(A) 50 µM FITC-Hst-5 was added to the buffer containing PI, and uptake of fluorescence was followed by time-lapse confocal microscopy at room temperature with frames recorded every 9 seconds for 7 minutes and 30 seconds. Six frames recorded at 9, 18, 27, 63, 90, and 297 seconds are shown. (B) 50 µM FITC-Hst-5 was added to the buffer containing RB, and uptake of fluorescence was followed by time-lapse confocal microscopy at room temperature with frames recorded every 8 seconds for a total of 6 minutes and 40 seconds. Six frames recorded at 8, 56, 64, 72, 112, and 128 seconds are shown.
The observation of Hst-5 concentrated in a spatially constricted region is intriguing. One possible explanation is that Hst-5 caused a break on the plasma membrane and the peptide entered into the cytoplasm through the damaged site. If so, the site should also allow the entrance of other molecules. It is known that Hst-5 treatment of cells causes internalization of the cationic molecule propidium iodide (PI). Therefore, we asked whether PI enters cells from the same site as the antifungal peptide. Interestingly, PI was found localized as a single small dot either at or right under the cell surface that also colocalized with Hst-5 (Figure 5A). PI rapidly spread throughout the cytoplasm as was seen with FITC-Hst-5 (Figure 5A and Video S3). The co-localization of PI and Hst-5 at the same spatially restricted site suggests the existence of a breach of the cell surface by Hst-5. No internalization of PI was observed in the absence of Hst-5 (data not shown). Whereas, PI localized to a spatially restricted site on the cell surface when cells were treated with unconjugated Hst-5, confirming that FITC-Hst-5 and native Hst-5 have the same property in membrane perturbation (Figure S1). Among cells in several time-lapse experiments, we never observed a cell with more than one site of PI internalization. This was further supported by the acquisition of a three-dimensional image of the initial uptake of PI into the cytoplasm shortly after C. albicans was treated with FITC-Hst-5 (Video S4). Due to the rapid uptake of the fluorochrome, wide-field microscopy and constrained iterative deconvolution were used in place of confocal microscopy. The initial Hst-5 uptake site on the cell surface could not be observed in this image due to the high fluorescein signal on cell surface. Even myo5 mutant cells, which have a lowered Hst-5 concentration threshold for cytoplasmic translocation, had solitary breaches on their cell surface (data not shown).
To further validate the findings that translocation of Hst-5 into the cytoplasm is mediated by the formation of a spatially restricted site on the cell surface of the yeast, C. albicans was treated with 50 µM FITC-Hst-5 in the presence of 5 µg/ml of rhodamine B (RB). The fluorchrome RB is useful indicator of membrane integrity and viability and is advantageous in this particular assay since it does not bind mitochondria or DNA [44],[45]. As observed with PI, the uncharged fluorochrome RB was internalized from a single regional area that colocalized with FITC-Hst5 (Figure 5B). Taken together the data indicates that Hst-5 induces a spatially localized breach on the cell surface of the C. albicans, leading to a disruption in its membrane integrity and a loss in viability.
Having shown that Hst-5 causes spatially restricted sites on the cell surface, we wanted to determine whether the site correlated with known cell surface markers. To evaluate whether the breach site was in regions where daughter cells had budded off, C. albicans cells were treated first with calcofluor white, and then with PI and biotin-conjugated Hst-5. The site of PI uptake did not correlate with bud scars (Figure 6A). This is consistent with the previous finding that Hst-5 is capable of killing spheroplasts of C. albicans [46]. We then examined whether there was any correlation between the site of the breach and cell polarity, which is linked to the active sites of secretion and endocytosis. Sterol and sphingolipid-rich raft domains are also concentrated at the site of active growth [47]. We performed time-lapse experiments with 50 µM biotin-Hst-5 and 5 µg/ml PI with C. albicans cells carrying GFP-tagged Spa2p, a component of the polarisome that controls cell polarity [48]. To facilitate analysis, hyphal cells with highly polarized growth were used. Spa2p was found to be localized to sites of polarized growth, such as the small bud, the bud neck, and the hyphal tip. However, the site of PI uptake did not correlate with the cellular localization of GFP-Spa2p (Figure 6B). Therefore, the spatially restricted site on the cell surface does not correlate with either of the two tested cell surface landmarks and is either a random manifestation on the cell surface or the uptake of the peptide is dependent on an unknown cellular marker found in association with either the cell wall and/or the plasma membrane of C. albicans.
(A) 50 µM biotin-Hst-5 was added to C. albicans in buffer containing PI and calcofluor white. Uptake of PI was followed by time-lapse fluorescence microscopy at room temperature with frames recorded every 25 seconds for 10 minutes. The frame recorded at 400 seconds is shown. (B) C. albicans was incubated in water with 2% fetal calf serum and PI for 50 minutes at 37°C to induce Spa2-GFP to the site of polarized growth. Buffer containing 50 µM biotin-Hst-5 was added to the cells and uptake of PI was followed by time-lapse fluorescence microscopy at 37°C with frames recorded every twenty seconds. Frames recorded at 0, 20, 40, and 120 seconds are shown. The yellow arrows indicate the formation of a single breach site on the yeast.
The general mode of action of most α-helical cationic antimicrobial peptides is initiated by their net positive charge that attracts them to an anionic microbial surface. Following this electrostatic binding, the α-helical peptide is inserted into the plasma membrane, resulting in the release of cellular contents and/or lysis of the cell. Previous Hst-5 research has indicated that the mode of Hst-5 killing does not behave in this classical manner. Rather it has been suggested that Hst-5 binds the heat shock protein 70 (Ssa1/2) located on the cell wall and is subsequently transported across to the cytoplasm in a nonlytic manner [12],[20],[49]. Intracellular targeting by cytoplasmic Hst-5 to the mitochondria and the plasma membrane leads to cell death [6],[10],[49],[50]. Here we show for the first time that the uptake of Hst-5 is actually a dichotomous event. Under low physiological concentrations the peptide is internalized to the vacuole via receptor-mediated endocytosis. Whereas under moderate to high physiological conditions, uptake of Hst-5 to the cytoplasm is initiated by a direct translocation through a spatially restricted site on the plasma membrane, causing the initial fungicidal activity to the yeast by damage to the cell membrane. Uptake of Hst-5 is both a dynamic and stochastic process within the Candida population at low to moderate concentrations. The rapid uptake of Hst-5 into the cytoplasm and its subsequent killing of C. albicans most likely prevents the slower process of receptor-mediated endocytosis from happening in the same cells. On the other hand, when Hst-5 is below the critical concentration necessary to induce cytoplasmic translocation, the endocytic removal of Hst-5 from the cell surface likely lowers the build-up of Hst-5 on the cell surface, and therefore impedes the candidacidal activity of the antimicrobial peptide. These two opposing internalization pathways give rise to two cell populations with distinct Hst-5 localizations and outcomes.
Net cationic charge of Hst-5 plays a greater role than just the initial binding affinity to the surface of C. albicans. A single lysine substitution for a histidine or amidation of the C-terminus of Hst-5 analogs increases the candidacidal activity of the peptide by almost two-fold [35],[36]. Whereas, reduction of the net positive charge of Hst-5 by either amino acid substitution or an increase in pH markedly reduced the cytoplasmic translocation of the antimicrobial peptide without affecting binding to the cell surface. In support of this idea Dathe et al. showed that increasing the charge of a magainin 2 analog from +3 to +5 increased its antibacterial activity [51]. It has been postulated that mechanism of translocation of cationic peptides is based on the strong trans-negative membrane potential common to many pathogenic organisms. It is thought that the electrochemical gradient helps orient the cationic peptide to the membrane so that it may gain entrance to the polar membrane core and/or translocate across the exoplasmic to the cytoplasmic membrane leaflet [34]. The permeabilization of cellular membranes by magainin and platelet microbicidal proteins (PMPs) has been shown to require acidic phospholipids and a large membrane potential [52],[53]. Furthermore, while not affecting the initial binding event of Hst-5, C. albicans cells treated with uncouplers of the electrochemical gradient, such as carbonylcyanide-m-chlorophenylhydrazone, dinitrophenol, and sodium azide, as well as petite respiration-deficient mutants have increased resistance to Hst-5 [12],[18]. The membrane potential of S. cerevisiae has been measured at −71 mV, a value closer to that of mammalian cells (−90 to −110 mV), whereas C. albicans membrane potential is −120 mV, a value more in line with bacterial pathogens (−130 to −150 mV) [34],[54],[55]. The trans-negative electrochemical gradient may potentially act as an underlying mechanism providing Hst-5 resistance to S. cerevisiae and Hst-5 susceptibility to C. albicans. This mechanism could provide species-specific targeting of the peptide while preserving nonpathogenic microbial communities and host tissue.
One of the most intriguing aspects of this study was the demonstration that the primary event in the inevitable killing of C. albicans was due to the concentration dependent formation of a single disruption site on the plasma membrane, visualized by the rapid influx of the fluorochromes PI and RB and Hst-5 through the disruption on the cell surface of C. albicans. The existence of a breach on the plasma membrane is also evident from the rapid expansion of the vacuole and the loss of cell volume. In S. cerevisiae, the vacuole expands when cells are shifted to a hyposmotic condition [56]. The rapid expansion of the vacuole in Hst-5 treated cells could be a response to the loss of ions and other small solutes from the cytoplasm. The disruption site is likely responsible for the rapid efflux of ATP and K+ that are linked to the killing of C. albicans. The size of the holes should be small, as only proteins smaller than 4 kDa were found to be released into the culture [28]. To our knowledge only one other antimicrobial peptide, the amphibian skin peptide dermaseptin s3, has been observed to induce a single breach site on the surface of a microorganism [57]. The dependence of Hst-5 internalization on the membrane potential may provide an explanation for a single rupture per cell, as once there is one site of perturbation, the membrane potential is lost, and therefore, prevents a second rupture. The dependence on the existence of the membrane potential may explain why Hst-5 had little lytic effect on vesicles in vitro [9]. Moreover, Hst-5 is capable of targeting and disrupting the electrochemical gradient of isolated mammalian mitochondria, as well as the mitochondria of C. albicans and Leishmania species [6],[58],[59]. The site of the rupture seems to be random, as it does not correlate to regions where daughter cells bud off or to the region of polarized growth. The randomness of the solitary disruption site further indicates that Hst-5 may act on the plasma membrane directly, but not through a particular cellular landmark. However, at this time we cannot definitively exclude the possibility that the formation of the Hst-5 spatially restricted site is due to its greater affinity to an unknown cellular marker on the surface of C. albicans rather than the strong electrochemical gradient of the plasma membrane.
Nonetheless, two additional lines of evidence suggest that the Hst-5 interaction with the plasma membrane initiates a mode of action that weakens the stability of membrane bilayer. First, our research indicates that the deletion of the class I myosin and the depolymerization of F-actin by CytoA and LatA markedly reduces the threshold necessary for the cytoplasmic translocation of Hst-5. The cortical actin cytoskeleton is important for the stability of the plasma membrane under hyperosmotic conditions, as S. cerevisiae act1 mutants and C. albicans myo5 mutants are extremely sensitive to salt stress [33],[60]. Furthermore, the high-osmolarity glycerol pathway (Hog1p), involved in adaptation to osmotic and oxidative stress [61]–[63], is required to prevent severe growth defects in the C. albicans myo5 mutants [60]. Second, Hst-5 is shown to activate the Hog1 pathway in C. albicans [15]. In S. cerevisiae, the Hog1 pathway is activated by a reduction of turgor pressure in response to hyperosmotic stress that induces water efflux or by treatment with nystatin, a membrane-permeabilizing antifungal drug that causes leakage of low molecular weight cytosolic components [64]. Moreover, Hst-5 shows strong synergistic killing of C. albicans when combined with amphotericin B [65],[66]. The synthetic interaction between Hst-5 and amphotericin B and the fact that both activate the Hog1 pathway suggests that Hst-5 and polyene macrolides (eg. amphotericin B and nystatin) have overlapping functions.
In summary, Hst-5 is not a classical channel forming cationic peptide. However, we do show in this report that Hst-5 induces perturbation at a spatially restricted site on the plasma membrane. Unlike classical channel forming antimicrobial peptides, this action requires an energized membrane and causes disruption at one region of the plasma membrane. This mechanism of cell membrane disruption may provide species-specific killing with minimal damage to microflora and the host, and may be used by many other weakly amphipathic antimicrobial peptides.
Materials and Methods
Yeast strains and growth conditions
The following fungal strains were used in this study: C. albicans SC5314 (wild-type clinical isolate); C. albicans vps36 (BWP17 Cavps36Δ::UAU1/Cavps36Δ::URA3) and C. albicans snf7 (BWP17 Casnf7Δ::UAU1/Casnf7Δ::URA3) (gifts from A. Mitchell, [67]); C. albicans COU46 (CAI4 Camyo5::hisG/Camyo5::hisG) (gift from M. Whiteway, [68]); C. albicans SPA2-GFP (BWP17 SPA2/SPA2-GFP-URA3); C. glabrata BG2 (gift from B. Cormack, wild-type clinical isolate, [69]); and S. cerevisiae BY4741 (MATa, leu2Δ0, met15Δ0, and ura3Δ0). All of the yeast strains were maintained on YPD plates [1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose]. Prior to Hst-5 localization assays, the cells were grown overnight at 30°C in 5 ml YPD broth. A 1/50 dilution of the overnight culture was suspended in fresh 5 ml YPD and grown for an additional 4 hours at 30°C to obtain a mid-log phase culture at which time the optical density was determined (OD595 of 1.0 = 3×107 cells/ml) using a Beckman Coulter DU 800 spectrophotometer to obtain a cell population of 106 cells/ml.
Unconjugated Hst-5 and FITC- and biotin-labeled Hst-5 (DSHAKRHHGYKRKFHEKHHSHRGY) and FITC-labeled Hst-5 m68 (DSHAKRHHGYKREFHEKHHSHGGY) were synthesized and purified by Genemed Synthesis, Inc. (San Francisco, CA). The identity and purity of the peptides were confirmed by mass spectrometry. Both FITC-Hst-5 and biotin-Hst-5 have been shown to have similar levels of candicidal activity when compared against unlabeled Hst-5 [10],[13],[49].
Hst-5 localization studies
The intracellular localization of FITC-Hst-5 (5, 10, 20, and 50 µM) and FITC-m68 (5, 10, 20, 50, and 200 µM) was investigated either alone or in a double-labeling experiment using FM4-64 (Molecular Probes, Inc. Eugene, OR). Yeast cells in 50 µl (∼106 cells/ml) were incubated for 30 minutes at 30°C with 10 µM FITC-Hst-5 and 10 µM FM4-64; the cells were then washed twice with 10 mM NaN3 and 10 mM NaF in 20 mM PBS buffer, and analyzed immediately by wide-field fluorescence microscopy.
To depolarize F-actin, cells were treated with either 5 µM cytochalasin A or 50 µM latrunculin A (Sigma, St. Louis, MO) for 1 hour at room temperature. The control cells (wild-type and myo5) were treated with the equivalent volume of the DMSO solvent (0.5%). The cells were then exposed to 5 µM of FITC-Hst-5 for 30 minutes at 30°C. The cells were then washed twice with buffer containing NaN3 and NaF and analyzed by wide-field fluorescence microscopy and flow cytometry.
For live cell imaging, 300 µl of a 100 µg/ml solution of concanavalin A (MP Biomedicals, LLC. Solon, Ohio) was coated onto a sterile 0.17 mm glass bottom dish (WillCo Wells BV, Amsterdam, Denmark). The wells were incubated for 1 hour at room temperature and then washed three times with water. A 300 µl buffer suspension of ∼2×106 C. albicans cells were aliquoted onto the well and incubated at room temperature. After settling and binding for 15 minutes unbound cells were washed away [70]. Buffer containing 50 µM of either unconjugated Hst-5 or FITC-Hst-5 and 5 µg/ml PI or RB was added to the cells and uptake of fluorescence was followed by time-lapse confocal microscopy. To determine the position of site specific breach by Hst-5 in relation to known cellular markers, cells were either observed using 2 µg/ml calcofluor white (Sigma, St. Louis, MO) and 5 µg/ml PI in PBS buffer or the C. albicans Spa2-GFP strain was grown in the presence of PI and 2% fetal calf serum in water. Buffer containing 50 µM biotin-Hst-5 was added to the cells and uptake of PI was followed by time-lapse wide-field fluorescence microscopy at room temperature and 37°C, respectively.
Fluorescence microscopy
Wide-field fluorescence images were obtained on either Zeiss Axioplan 2 or the inverted Zeiss Axio Observer.Z1 Microscope (Carl Zeiss MicroImaging, Inc. Thornwood, NY) fluorescent system, equipped with the AttoArc HBO 100 and the X-Cite series 120 mercury lamps, respectively. Images were taken using a 100× NA 1.4 objective. Both fluorescence microscopes were equipped with GFP, RFP, and DAPI filter sets.
Data sets were obtained as 10–20 optical sections per wavelength spaced 0.2 µm apart along the Z-axis. Out of focus information was removed using a constrained iterative deconvolution algorithm. During the experiment cells were kept at either a constant 30° or 37°C using the TempModule S system on the microscope. Processing was done on a PC using the software packages Axiovision 3.1 and 4.6.3, as well as Photoshop (Adobe Systems Inc., Mountain View, CA).
Confocal laser scanning microscopy was performed on an inverted LSM510 laser scanning microscope (Carl Zeiss, Göttingen, Germany) using a Plan-Apo 100×/1.4 NA lens. For the simultaneous detection of fluorescein-labeled peptides and the fluorochromes PI or RB, the 488-nm line of the argon ion laser and the light of a 543-nm helium neon laser were directed over an HFT UV/488/543/633 beam splitter, and the fluorescence was detected using an NFT 545 beam splitter in combination with a BP 500–550 band pass filter for fluorescein detection and an BP 565–615 band pass filter for PI and RB detection.
FACS analysis
The distribution of FITC-labeled Hst-5 over the cell population was investigated by using a dual laser fluorescence-activated cell sorter (BD FACSCalibur System, Becton Dickinson, San Jose, CA). The results were analyzed with the software package CellQuest Pro (version 5.1.1) provided by Beckton Dickinson.
Flow cytometric cell sorting and candidacidal activity of histatin-5
C. albicans cells were incubated for 30 minutes with 10 µM of FITC-Hst-5 at 30°C. The cells were then washed twice with 20 mM PBS buffer and under went flow cytometric cell sorting using the DAKO Cytomation MoFlo Flow Cytometer (DAKO, Glostrup, Denmark). The results were analyzed on a PC using the software package Summit (version 4.0) provided by DAKO. The cells were sorted by gating the two peaks of the histogram representing vacuolar and cytoplasmic localization of Hst-5. Cellular localization of Hst-5 was confirmed with fluorescence microscopy. The sorted cells were then plated onto YPD plates and incubated overnight at 30°C (data as a mean±1SD of triplicate cultures).
Determination of charge for Hst-5 and m68
Determination of charge for Hst-5 and m68 were done using PROTEIN CALCULATOR v3.3 (www.scripps.edu/∼cdputnam/protcalc2.html). The pKa values for the individual amino acids are from Stryer Biochemistry, 3rd edition. The software was designed by Chris Putnam at the Scripps Research Institute email@example.com.
Supporting Information
Figure S1.
Unlabeled Hst-5 induced internalization of the fluorchrome propidium iodide from spatially restricted sites on the cell surface. 50 µM Hst-5 (unconjugated) was added to the buffer containing PI, and uptake of fluorescence was followed by time-lapse confocal microscopy at room temperature, with frames recorded every 9 seconds for 7 minutes and 30 seconds. Six frames recorded at 9, 27, 45, 90, and 297 seconds are shown.
Video S1.
Cytoplasmic translocation of FITC-Hst-5 into the yeast C. albicans. The uptake of fluorescence after the addition of 50 µM FITC-Hst-5 to C. albicans cells. The images were recorded by time-lapse confocal microscopy at room temperature with frames recorded every 9 seconds for a total of 7 minutes and 30 seconds.
Video S2.
Rapid vacuole expansion and deformation of C. albicans after the addition of Hst-5. The morphological change to the yeast C. albicans after the addition of 50 µM Hst-5. The images were recorded by time-lapse microscopy at room temperature with frames recorded every 15 seconds for a total of 15 minutes.
Video S3.
Cytoplasmic translocation of propidium iodide into the yeast C. albicans. The uptake of PI after the addition of 50 µM FITC-Hst-5 to C. albicans cells. The images were recorded by time-lapse confocal microscopy at room temperature with frames recorded every 9 seconds for a total of 7 minutes and 30 seconds.
Video S4.
Localization of FITC-Hst-5 and propidium iodide using three-dimensional imaging. Buffer containing 50 µM FITC-Hst-5 and 5 µg/ml PI was added to the cells, and uptake of fluorescence was followed by wide-field time-lapse microscopy. The three-dimensional image was acquired from deconvolution using fifteen 0.2 micron optical sections.
We thank Drs. Aaron Mitchell (Columbia University, New York, United States of America), Malcolm Whiteway (Biotechnology Research Institute, Canada), and Brendan Cormack (Johns Hopkins University, Maryland, United States of America) for generously providing yeast strains.
Author Contributions
Conceived and designed the experiments: ABM HL. Performed the experiments: ABM. Analyzed the data: ABM HL. Contributed reagents/materials/analysis tools: ABM. Wrote the paper: ABM HL.
- 1.
Kwon-Chung KJ, Bennett JE (1992) Medical mycology. Philadelphia: Lea & Febiger.
- 2. vanderSpek JC, Wyandt HE, Skare JC, Milunsky A, Oppenheim FG, et al. (1989) Localization of the genes for histatins to human chromosome 4q13 and tissue distribution of the mRNAs. Am J Hum Genet 45: 381–387.
- 3. Raj PA, Edgerton M, Levine MJ (1990) Salivary histatin 5: dependence of sequence, chain length, and helical conformation for candidacidal activity. J Biol Chem 265: 3898–3905.
- 4. Xu T, Levitz SM, Diamond RD, Oppenheim FG (1991) Anticandidal activity of major human salivary histatins. Infect Immun 59: 2549–2554.
- 5. Raj PA, Marcus E, Sukumaran DK (1998) Structure of human salivary histatin 5 in aqueous and nonaqueous solutions. Biopolymers 45: 51–67.
- 6. Helmerhorst EJ, van't Hoff W, Breeuwer P, Veerman EC, Abee T, et al. (2001) Characterization of histatin 5 with respect to amphipathicity, hydrophobicity, and effects on cell and mitochondrial membrane integrity excludes a candidacidal mechanism of pore formation. J Biol Chem 276: 5643–5649.
- 7. Oren Z, Shai Y (1998) Mode of action of linear amphipathic alpha-helical antimicrobial peptides. Biopolymers 47: 451–463.
- 8. Lear JD, Wasserman ZR, DeGrado WF (1988) Synthetic amphiphilic peptide models for protein ion channels. Science 240: 1177–1181.
- 9. Den Hertog AL, Wong Fong Sang HW, Kraayenhof R, Bolscher JG, Van't Hof W, et al. (2004) Interactions of histatin 5 and histatin 5-derived peptides with liposome membranes: surface effects, translocation and permeabilization. Biochem J 379: 665–672.
- 10. Helmerhorst EJ, Breeuwer P, van't Hoff W, Walgreen-Weterings E, Oomen LC, et al. (1999) The cellular target of histatin 5 on Candida albicans is the energized mitochondrion. J Biol Chem 274: 7286–7291.
- 11. Helmerhorst EJ, Troxler RF, Oppenheim FG (2001) The human salivary peptide histatin 5 exerts its antifungal activity through the formation of reactive oxygen species. Proc Natl Acad Sci U S A 98: 14637–14642.
- 12. Koshlukova SE, Lloyd TL, Araujo MW, Edgerton M (1999) Salivary histatin 5 induces non-lytic release of ATP from Candida albicans leading to cell death. J Biol Chem 274: 18872–18879.
- 13. Xu Y, Ambudkar I, Yamagishi H, Swaim W, Walsh TJ, et al. (1999) Histatin 3-mediated killing of Candida albicans: effect of extracellular salt concentration on binding and internalization. Antimicrob Agents Chemother 43: 2256–2262.
- 14. Baev D, Li XS, Dong J, Keng P, Edgerton M (2002) Human salivary histatin 5 causes disordered volume regulation and cell cycle arrest in Candida albicans. Infect Immun 70: 4777–4784.
- 15. Vylkova S, Jang WS, Li W, Nayyar N, Edgerton M (2007) Histatin 5 initiates osmotic stress response in Candida albicans via activation of the Hog1 mitogen-activated protein kinase pathway. Eukaryot Cell 6: 1876–1888.
- 16. Ruissen AL, Groenink J, Helmerhorst EJ, Walgreen-Weterings E, Van't Hof W, et al. (2001) Effects of histatin 5 and derived peptides on Candida albicans. Biochem J 356: 361–368.
- 17. Veerman EC, Valentijn-Benz M, Nazmi K, Ruissen AL, Walgreen-Weterings E, et al. (2007) Energy depletion protects Candida albicans against antimicrobial peptides by rigidifying its cell membrane. J Biol Chem 282: 18831–18841.
- 18. Gyurko C, Lendenmann U, Troxler RF, Oppenheim FG (2000) Candida albicans mutants deficient in respiration are resistant to the small cationic salivary antimicrobial peptide histatin 5. Antimicrob Agents Chemother 44: 348–354.
- 19. Raj PA, Soni SD, Levine MJ (1994) Membrane-induced helical conformation of an active candidacidal fragment of salivary histatins. J Biol Chem 269: 9610–9619.
- 20. Li XS, Reddy MS, Baev D, Edgerton M (2003) Candida albicans Ssa1/2p is the cell envelope binding protein for human salivary histatin 5. J Biol Chem 278: 28553–28561.
- 21. Riezman H, Munn A, Geli MI, Hicke L (1996) Actin-, myosin- and ubiquitin-dependent endocytosis. Experientia 52: 1033–1041.
- 22. Geli MI, Riezman H (1998) Endocytic internalization in yeast and animal cells: similar and different. J Cell Sci 111(Pt 8): 1031–1037.
- 23. Mukhopadhyay D, Riezman H (2007) Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315: 201–205.
- 24. Duchardt F, Fotin-Mleczek M, Schwarz H, Fischer R, Brock R (2007) A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic 8: 848–866.
- 25. Rajarao GK, Nekhotiaeva N, Good L (2002) Peptide-mediated delivery of green fluorescent protein into yeasts and bacteria. FEMS Microbiol Lett 215: 267–272.
- 26. Vylkova S, Sun JN, Edgerton M (2007) The role of released ATP in killing Candida albicans and other extracellular microbial pathogens by cationic peptides. Purinergic Signal 3: 91–97.
- 27. Gyurko C, Lendenmann U, Helmerhorst EJ, Troxler RF, Oppenheim FG (2001) Killing of Candida albicans by histatin 5: cellular uptake and energy requirement. Antonie Van Leeuwenhoek 79: 297–309.
- 28. den Hertog AL, van Marle J, van Veen HA, Van't Hof W, Bolscher JG, et al. (2005) Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane. Biochem J 388: 689–695.
- 29. Staub O, Rotin D (2006) Role of ubiquitylation in cellular membrane transport. Physiol Rev 86: 669–707.
- 30. Gill DJ, Teo H, Sun J, Perisic O, Veprintsev DB, et al. (2007) Structural insight into the ESCRT-I/-II link and its role in MVB trafficking. Embo J 26: 600–612.
- 31. Odorizzi G, Katzmann DJ, Babst M, Audhya A, Emr SD (2003) Bro1 is an endosome-associated protein that functions in the MVB pathway in Saccharomyces cerevisiae. J Cell Sci 116: 1893–1903.
- 32. Geli MI, Riezman H (1996) Role of type I myosins in receptor-mediated endocytosis in yeast. Science 272: 533–535.
- 33. Oberholzer U, Nantel A, Berman J, Whiteway M (2006) Transcript profiles of Candida albicans cortical actin patch mutants reflect their cellular defects: contribution of the Hog1p and Mkc1p signaling pathways. Eukaryot Cell 5: 1252–1265.
- 34. Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55: 27–55.
- 35. Rothstein DM, Spacciapoli P, Tran LT, Xu T, Roberts FD, et al. (2001) Anticandida activity is retained in P-113, a 12-amino-acid fragment of histatin 5. Antimicrob Agents Chemother 45: 1367–1373.
- 36. Helmerhorst EJ, Van't Hof W, Veerman EC, Simoons-Smit I, Nieuw Amerongen AV (1997) Synthetic histatin analogues with broad-spectrum antimicrobial activity. Biochem J 326(Pt 1): 39–45.
- 37. Tsai H, Raj PA, Bobek LA (1996) Candidacidal activity of recombinant human salivary histatin-5 and variants. Infect Immun 64: 5000–5007.
- 38. Galgut PN (2001) The relevance of pH to gingivitis and periodontitis. J Int Acad Periodontol 3: 61–67.
- 39. Smith RM, Baibakov B, Lambert NA, Vogel SS (2002) Low pH inhibits compensatory endocytosis at a step between depolarization and calcium influx. Traffic 3: 397–406.
- 40. Nikawa H, Jin C, Fukushima H, Makihira S, Hamada T (2001) Antifungal activity of histatin-5 against non-albicans Candida species. Oral Microbiol Immunol 16: 250–252.
- 41. Helmerhorst EJ, Venuleo C, Beri A, Oppenheim FG (2005) Candida glabrata is unusual with respect to its resistance to cationic antifungal proteins. Yeast 22: 705–714.
- 42. De Smet K, Reekmans R, Contreras R (2004) Role of oxidative phosphorylation in histatin 5-induced cell death in Saccharomyces cerevisiae. Biotechnol Lett 26: 1781–1785.
- 43. Isola R, Isola M, Conti G, Lantini MS, Riva A (2007) Histatin-induced alterations in Candida albicans: a microscopic and submicroscopic comparison. Microsc Res Tech 70: 607–616.
- 44. Auger P, Marquis G, Dallaire L (1979) Viability assessment by dye exclusion. A fluorescent method for fungal cells. Arch Dermatol 115: 1195–1196.
- 45. Darzynkiewicz Z, Traganos F, Staiano-Coico L, Kapuscinski J, Melamed MR (1982) Interactions of Rhodamine 123 with Living Cells Studied by Flow Cytometry. Cancer Res 42: 799–806.
- 46. Driscoll J, Duan C, Zuo Y, Xu T, Troxler R, et al. (1996) Candidacidal activity of human salivary histatin recombinant variants produced by site-directed mutagenesis. Gene 177: 29–34.
- 47. Alvarez FJ, Douglas LM, Konopka JB (2007) Sterol-rich plasma membrane domains in fungi. Eukaryot Cell 6: 755–763.
- 48. Zheng XD, Wang YM, Wang Y (2003) CaSPA2 is important for polarity establishment and maintenance in Candida albicans. Mol Microbiol 49: 1391–1405.
- 49. Koshlukova SE, Araujo MW, Baev D, Edgerton M (2000) Released ATP is an extracellular cytotoxic mediator in salivary histatin 5-induced killing of Candida albicans. Infect Immun 68: 6848–6856.
- 50. Baev D, Li X, Edgerton M (2001) Genetically engineered human salivary histatin genes are functional in Candida albicans: development of a new system for studying histatin candidacidal activity. Microbiology 147: 3323–3334.
- 51. Dathe M, Nikolenko H, Meyer J, Beyermann M, Bienert M (2001) Optimization of the antimicrobial activity of magainin peptides by modification of charge. FEBS Lett 501: 146–150.
- 52. Matsuzaki K, Sugishita K, Fujii N, Miyajima K (1995) Molecular basis for membrane selectivity of an antimicrobial peptide, magainin 2. Biochemistry 34: 3423–3429.
- 53. Yeaman MR, Bayer AS, Koo SP, Foss W, Sullam PM (1998) Platelet microbicidal proteins and neutrophil defensin disrupt the Staphylococcus aureus cytoplasmic membrane by distinct mechanisms of action. J Clin Invest 101: 178–187.
- 54. Vacata V, Kotyk A, Sigler K (1981) Membrane potentials in yeast cells measured by direct and indirect methods. Biochim Biophys Acta 643: 265–268.
- 55. Prasad R, Hofer M (1986) Tetraphenylphosphonium is an indicator of negative membrane potential in Candida albicans. Biochim Biophys Acta 861: 377–380.
- 56. Hohmann S (2002) Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 66: 300–372.
- 57. Coote PJ, Holyoak CD, Bracey D, Ferdinando DP, Pearce JA (1998) Inhibitory action of a truncated derivative of the amphibian skin peptide dermaseptin s3 on Saccharomyces cerevisiae. Antimicrob Agents Chemother 42: 2160–2170.
- 58. Petruzzelli R, Clementi ME, Marini S, Coletta M, Di Stasio E, et al. (2003) Respiratory inhibition of isolated mammalian mitochondria by salivary antifungal peptide histatin-5. Biochem Biophys Res Commun 311: 1034–1040.
- 59. Luque-Ortega JR, Van't Hof W, Veerman EC, Saugar JM, Rivas L (2008) Human antimicrobial peptide histatin 5 is a cell-penetrating peptide targeting mitochondrial ATP synthesis in Leishmania. Faseb J.
- 60. Balguerie A, Bagnat M, Bonneu M, Aigle M, Breton AM (2002) Rvs161p and sphingolipids are required for actin repolarization following salt stress. Eukaryot Cell 1: 1021–1031.
- 61. Alonso-Monge R, Navarro-Garcia F, Molero G, Diez-Orejas R, Gustin M, et al. (1999) Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans. J Bacteriol 181: 3058–3068.
- 62. Alonso-Monge R, Navarro-Garcia F, Roman E, Negredo AI, Eisman B, et al. (2003) The Hog1 mitogen-activated protein kinase is essential in the oxidative stress response and chlamydospore formation in Candida albicans. Eukaryot Cell 2: 351–361.
- 63. San Jose C, Alonso R, Perez-Diaz RM, Pla J, Nombela C (1996) The mitogen-activated protein kinase homolog HOG1 gene controls glycerol accumulation in the pathogenic fungus Candida albicans. Journal of Bacteriology 178: 5850–5852.
- 64. Reiser V, Raitt DC, Saito H (2003) Yeast osmosensor Sln1 and plant cytokinin receptor Cre1 respond to changes in turgor pressure. J Cell Biol 161: 1035–1040.
- 65. van't Hoff W, Reijnders I, Helmerhorst E, Walgreen-Weterings E, Simoons-Smit I, et al. (2000) Synergistic effects of low doses of histatin 5 and its analogues on amphotericin B anti-mycotic activity. Antonie Van Leeuwenhoek 78: 163–169.
- 66. Tanida T, Okamoto T, Ueta E, Yamamoto T, Osaki T (2006) Antimicrobial peptides enhance the candidacidal activity of antifungal drugs by promoting the efflux of ATP from Candida cells. J Antimicrob Chemother 57: 94–103.
- 67. Xu W, Smith FJ Jr, Subaran R, Mitchell AP (2004) Multivesicular body-ESCRT components function in pH response regulation in Saccharomyces cerevisiae and Candida albicans. Mol Biol Cell 15: 5528–5537.
- 68. Oberholzer U, Marcil A, Leberer E, Thomas DY, Whiteway M (2002) Myosin I is required for hypha formation in Candida albicans. Eukaryot Cell 1: 213–228.
- 69. Fidel PL Jr, Cutright JL, Tait L, Sobel JD (1996) A murine model of Candida glabrata vaginitis. J Infect Dis 173: 425–431.
- 70. Colman-Lerner A, Gordon A, Serra E, Chin T, Resnekov O, et al. (2005) Regulated cell-to-cell variation in a cell-fate decision system. Nature 437: 699–706.


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Derby Mercury - 7 March 1805
TO BE SOLD BY AUCTION,At the house of Mr. Robert Goodall, the sign of the Saracen's Head, Shirley, in the County of Derby, on Thursday the 14th day of March, 1805, betwixt the hours of one and three o'clock in the afternoon;
By Mr. HARLOW,
By Mr. HARLOW,
LOT 1.TWO pieces of rich Meadow land situate at Shirley, within one mile of the Turnpike Road leading from Ashborne to Derby, containing by estimation six acres or thereabouts, be the same more or less, in the respective possessions of Mr. Humphrey Thompson, and Mr. William Jackson; The Land Tax is redeemed, and immediate possession may be had as soon as purchased if required.
LOT 2.A stack of excellent HAY, standing on the aforementioned land, containing by estimation, nine tons, or thereabouts, be the same more or less.
Further particulars may be had by applying to Mr. HUMPHREY THOMPSON, at Spend Lane, near Ashborne.
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Now we all have our New Year resolutions. Some people want
to lose weight, others hope to get better organized and many desire to spend
more time with their families and friends. Everyone has areas of their lives
that could use some restoring and most have the instinctual need to simply
become more balanced.
For the past two years, I have started off the beginning of
January with a New Year post designed to not only motivate, but to also support
those of you who have made personal and/or work related resolutions to achieve
a more purposeful and productive existence. Here are two previous posts from
2010 and 2011 that you may find still find inspiring in 2012:
So, after two very relaxing and enjoyable weeks off from
school, I am now preparing to return to preschool and the demands of work life.
I already have my "to do" list written, with evaluation reports and IEPs to
write, quarterly progress notes due next week and meetings scheduled over the
next several days.
Although there are the immediate responsibilities that wait,
I do not want to lose sight of the areas of my work life that often are rushed
or pushed aside. So here is my high-five list for the workplace in 2012. Each
addresses an area that empowers me to be a better therapist and coworker!
Happy New Year everyone!
May 2012 be a year of growth and opportunity, peace and joy for you!


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The CAMERICH Dining Table collection has something for everyone, from simple, more traditional styles to striking modern silhouettes; all beautifully hand-crafted to bring a practical yet stylish focal point to your dining space. This exclusive range of tables is available in a variety of different sizes and exquisite wood finishes.
Perpetually modern, Joint Dining Table exhibits a striking stance that is sculpturally stunning from every angle. Table top is available Grey Oak Veneer, Carrara Marble, or Mystique Brown Marble. Marble options offer beautiful veining, unique to each piece. Legs are well constructed in solid ash wood, and available in Grey Oak and Brown Walnut finishes. Both top and legs are further reinforced with internal steel frame.
· Top and solid ash wood legs reinforced with internal steel frame
· Available in Grey Oak finish
· The table comes in four sizes 160x85x74cm, 180x100x74cm, 210x100x74cm and 240x100x74cm


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How To Make Money From The Internet
So, today I am going to be talking about how to make money from the internet, which is in fact, not as hard as you might think, it does involve a lot of work, but ultimately, it is very worthwhile! The time you spend creating wealth is not wasted, because it will continue for months and years into the future, creating a "passive" income.
The first thing you will need to decide is what will be your "niche". I am not going to discuss this here, you can read more about it here. Once you have decided which "niche" you are going to enter, you then need to create a website, and host it, you can do that here,
Obviously, you will need a web site, with a few plugins, which are free, for the most part anyway, I have a paid plugin on this site, which I use for e-mail capture, but when you are first starting out, there are a few ways you can get round this, by using free programs, such as MadMimi, unfortunately, MadMimi don`t like the make money online niche, they consider it to be a "spammy" industry, which is fine.
6 Ways To Make Money From Your Site
Affiliate Programs – This is far and away the best way to make money from your site.
The principle is so simple, I am honestly surprised more people don`t do this, in essence all you do is find an appropriate affiliate program, join it, and then promote it on your site!
The secret is to get people who are just about to buy an item, and are doing additional research on it. This could be a camera, a car, microwave oven!
If you join one of the better known affiliate programs, such as Amazon, if they visit Amazon using your link, then anything they buy, you get commission for! The beauty is in the fact, that you have written a bit about the item, which will continue to sell for you, far into the future.
Create your own product – This can sound very daunting, but in actual fact it is remarkably simple to do, especially in the digital age, if you were to create a pdf file, and sell it as an e-book, you would make money from it, especially if there was very little competition for the information contained in it.
Due to geographical differences, some products aren`t available everywhere, so, if you were talking about the best way to repair the gel coat on a boat, for example, the fact that a piece of linoleum will leave the finished repair as smooth as glass, would be worth something to a diy er! You can learn how to write an e-book here
Adsense advertising – This a way that google will place adverts on your site, if you have enough traffic, some of it will click through to the advert, which will generate revenue for you. I t should be noted that here, in the UK, google will pay out when you have earned £60. If you choose to have google adsense on your site, it is very important that you do not click the ads yourself! You will quickly lose this way of generating money if you do! It is a free program to join and approval is usually very quick.
Build A List – This is one of the easiest things to do, so long as your site gets reasonable traffic, no doubt you have the heard "the money is in the list" and this is very true, but the fortune is in the follow up, and it is vital that the messages your autoresponder sends out are relevant, and address the problems that your readers have in their chosen niche, pointless sending an e-mail that promotes gardening gloves to a single mum! You need to create a profile of who your visitor is, and what problems they want to solve.
Relevant advertisers – These are companies, within your niche, who produce and sell products, that your visitors could conceivably want to buy, they are not in competition with adsense, as they are niche related, and not retargetting, as google does, where the same advert follows you around on the internet!
Amazon Products – Everybody is used to buying on Amazon these days, so it is no wonder that most people actively promote them, the trick is to address the problem that the reader has! Someone has typed, for example, "best way to get fit at home" then, you would write an article that addresses that particular problem, tell them what you did, and how this wonderful multi gym will help them, that you found on Amazon, concentrate on the F.A.B principles, features, advantages, benefits! tell them they can read reviews here, and link it to amazon, once the cookie is set, it lasts for 24 hours, so if they then go on to buy a refrigerator, you earn a commission.
Ok, so now you know six ways to make money from your site, now you will need to get traffic to it, and by far the best way, is to do proper seo on the articles you have written. you can read more about that subject here
You will obviously need to learn other aspects of internet marketing, such as marketing! you can get expert help and advice from the Wealthy Affiliate Program, a program which I wholeheartedly endorse! I have a review of the program on this site, but if you would much rather just create a free account and get cracking, I would Love to welcome you, you can get access here
I believe in you!
Till next time,


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With one of the best patios in Park City, The Bridge Café & Grill, on Main Street, is the place to soak in the spring sun. Enjoy stunning mountain views as you indulge in their all day breakfast menu or one of their Brazilian-inspired twists on classic dishes. This includes the national drink of Brazil: the caipirinha, which is impossibly refreshing and tastes as if lemonade and a mojito had a love child. As we welcome summer salad season, we love the creativity in their passion fruit vinaigrette. When you enjoy this salad on the patio, hopefully you get a side of sunshine.
- 1/4 cup passion fruit puree
- 1/2 cup olive oil
- 1 tablespoon lime juice
- 1 tablespoon shallots, chopped
- 1 clove garlic, chopped
- 2 tablespoons honey
- 1/4 teaspoon salt, black pepper and paprika
- Add all ingredients to a blender or food processor and blend until the mixture is smooth.
- Use the dressing to top the salad of your choice.
CAMARAO A BRAZUCA: sautéed shrimp with garlic, fresh tomatoes, herbs, onions, dende oil and chili flakes, served with toasted garlic bread
BRAZILIAN PASSION SALAD: fresh garden greens tossed with homemade passion fruit vinaigrette, red onions, grape tomatoes and potato sticks, served with grilled garlic bread
BRAZUCA OMELET: made-to-order omelet with up to five items, served with home fries and your choice of toast
BRAZILIAN MOCKTAIL: lemonade and soda water flavored with passion fruit, guava or strawberry
American cuisine with Brazilian flair.
Spring hours: Mon.-Thurs. 8 a.m.-2 p.m. and Fri.-Sun. 8 a.m.-5 p.m. | 825 Main St. #201
(435) 658-5451 | thebridgecafeandgrill.com


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Review for Festival Of The Spoken Nerd: You Can't Polish A Nerd
I was jumping for joy at another unsolicited review disc. That's not something I do for the average unexpected thump on the doormat, and most such discs, either end up at the bottom of a mile-high pile of anime check discs, or more likely in the circular file. But this was a review copy of the new Festival of the Spoken Nerd release; this went to the top of the anime pile. It also turned out to be an object lesson in not extrapolating from two points of data. After reviewing Full Frontal Nerdity, and then after 2½ years, Just for Graphs, I facetiously remarked that I would expect the review disc for You Can't Polish a Nerd in May 2020. But here I am, just ten months later, about to lose myself in Festival of the Spoken Nerd's most recent show, filmed for this release at the Arts Depot in London on the nights of the 27th and 28th June 2018.
For the uninitiated, Festival of the Spoken Nerd combine cutting edge science with comedy; entertaining and enthralling with theories, experiments and songs, and the odd pop-culture reference as well. They juggle too. As well as this DVD release, you can buy the show on HD download, and on 3½" floppy disc. Yes, the entire 109 minute show has been digitised and divided onto 227 floppy discs, and you can buy one (plus download) for £12.57, or buy the lot for the budget price of £1440 (Just for Graphs came on limited edition VHS, I expect their next show will be on VHD, video on vinyl).
So join Geek Songstress Helen Arney, Experiments Maestro Steve Mould, and Stand-up Mathemetician Matt Parker as they talk about the melancholy of soap in a microwave, sing the periodic table, demonstrate gravitational waves with Lycra, turn the world Escher and much, much more.
The image is presented in 1.78:1 anamorphic format on the Region 0 PAL disc, and it's what you would expect from a live performance. The important thing is that everything that you need to see is clearly visible, and there are no technical issues with the disc.
What is it with the love for obscure formats? Not PCM, not Dolby Digital, and not DTS, we get MPEG 2 audio in 2.0 format; English of course, with optional subtitles. The Safe For Schools soundtrack accessible from the Alternative Soundtracks menu inserts comedy noises over the minor swears to completely sanitise the show for our delicate youth. The all important dialogue is clear throughout.
You get one disc in an Amaray case and a 16 page booklet with more experiments to try at home, and various other goodies.
On the disc you'll find a DVD Unboxing Video with a difference. Quite honestly, I'm surprised that it fits on just one disc. ∞ mins.
You also get three commentaries, Helen comments on Steve's commentary, Steve comments on Matt's commentary, and Matt comments on Helen's commentary. All of which raises the question, which commentary was recorded first?
I absolutely adored Full Frontal Nerdity when I first reviewed it, an extravaganza of science comedy that both entertained and informed the way the BBC can only dream of. It presented me with science that astounded and intrigued, tantalised my nerdy taste buds that had lain dormant for many a year. So you can imagine how delighted I was when Just For Graphs turned up, and while it was more science and more of the funny, I have to admit that there was a slight deflating feeling with it. Just for Graphs presented me with science that I was aware of, things that I knew about, and I missed the sense of wonder and discovery that I had with Full Frontal Nerdity, even if I enjoyed Just for Graphs just as much. And now that You Can't Polish a Nerd is here, I have to say... Mind Blown!
I have to admit that it did take me a little while to warm to You Can't Polish a Nerd. The webcam in a microwave was typical Steve Mould anarchy, and the Space Traveller song covered the same philosophical ground as Eric Idle did at the end of The Meaning of Life, but 30 years of cosmological research and discovery surely deserves a more up to date lyric. But once we got to the Freezer/Plane conundrum which illustrates the necessity for clear communication in science, I was hooked into the show.
I could go on listing all the good bits, but that would entail detailing the whole show, but things that did indeed blow my mind included the measurement of ℼ with a pie, the demonstration of gravitational waves, the real-time demonstration of recursion, and the call-back to my favourite mp3 player, Winamp (note that this show was recorded before the release of Winamp 5.8).
Speaking of recursion, I'm basically going to say the same thing I said in my previous two reviews. The beauty of Festival of the Spoken Nerd is that they make important and occasionally complex scientific ideas entertaining and easy to understand. It's because the three stars of the show are so personable, and have naturally funny bones, skilled communicators that you stay hooked to the show. And they provide their own heckles. With You Can't Polish a Nerd, they are on top form, and this is a DVD that any self-respecting nerd will want in their Christmas stocking, or indeed Bonfire Night stocking or Diwali stocking.
If you want to see Festival of the Spoken Nerd live (with guests), then you can get tickets for An Evening of Unnecessary Detail at the Backyard Comedy Club on the Festival of a Spoken Nerd website.


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We are Rothesay's original brewing company, located at 47 Clark Road in the heart of the Kennebecasis Valley. We believe in brewing incredible craft beer, working with locally-focused partners, and making our community more foggin' awesome each day.
Foghorn Brewery is the dream of Steve & Esty, two New Brunswick dudes from very different backgrounds. Steve comes off a twelve-year post-secondary academic career resulting in two undergrad degrees, one masters degree & most of a PhD. You'd think all of this education would be enough to deter him away from his dream of owning a brewery. Esty's reputation as a New Brunswick brewer is unrivaled only by his passion for craft beer. After putting his name on some of New Brunswick's best known craft beers, Esty figured that the Valley was ripe for a new brewery. Esty & Steve connected, and Foghorn Brewing Company was born.
Our Beers
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text: Cabi Clothing Share: Retail Cabi Clothing Visit Website Request Info 2808 Addison Circle South Rochester Hills, MI 48306 248-990-0515 darnellterese.cabionline.com About Map About I am a personal stylist for Cabi clothing. A women's clothing line that was established in California. I bring the collection to you and your friends and help you with styling tips and trends. What's Nearby? Distance: 5 10 15 20 25 50 mi.


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As an MCI student, you have access to the following reading resources:
1. Our London MCI Library, which contains educational and Montessori related books, magazines, and journals. EYE students can borrow 2 books at a time for 1 week and FdA students can borrow up to 3 books at a time for 1 week; book loans can be renewed once. For full terms and conditions of using the Mallinson Library please see the relevant section in your course handbook. Click here for a full catalogue of our books.
2. The section called Reading Files from Workbooks, on your course on the VLE. You'll find this section by going into your course, then clicking on Resources on the menu on the left. Look for the link called Reading Files from Workbooks and click on it. On that page you'll find all digital reading resources of your course, organised in units/modules.
3. The online EBSCO e-Library, which contains a rich variety of educational journals, magazines, and e-books. Simply right-click on the e-Library button at the navbar of your course page, and select Open in a new tab. To find out more on how to access and use the EBSCO e-Library download this printable guide. You can also watch our video guidelines:
4. The Resources section on our VLE Meeting Point (underneath Noticeboard), which contains detailed Montessori reading lists and topics, as well as archived issues of the Montessori International magazine. Click here for a direct link to Meeting Point.
5. In addition, FdA students have access to the LMU online library resources. To find out how to access and use the LMU online resources download this printable guide.
The full Harvard Referencing Guide.
All digital resources provided by MCI are covered by UK copyright law, our CLA licence, and our partner agreements (LMU, EBSCO). According to these you are only allowed to:
- Download those digital resources for personal use
- Print a single copy
You are not allowed to share or disseminate those digital resources, or print multiple copies.


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The security challenges facing organizations today can't be solved with traditional approaches. Digital transformation is creating a borderless digital attack surface, point solutions are creating unprecedented complexity, and threats are constantly evolving.
Join us to learn how FortiOS 6.0, the newest version of the most deployed network security operating system in the world, solves today's security challenges with broad coverage, massive integration, and time-saving automation. Specifically, learn the details of more than 200 new features and how they enable the Fortinet Security Fabric.
After the presentation you are invited to stay for an executive lunch briefing: "Intent Based Network Security" with John Maddison, Fortinet's SVP of Products and Solutions. The digital attack surface is larger and more dynamic than ever. As IoT/OT devices attach and detach to the network and as apps and workloads are spun up and down, it's imperative to have a cybersecurity framework in place to mitigate risk. Orchestrated segmentation is one of the most fundamental ways to gain visibility into any network and to protect it. John will explore how to provide automated security policies that are in line with infrastructure changes in today's dynamic environments.
Thursday, June 14, 2018
Hudson Event Space, Loft 3
200 8 Avenue SW, 6th Floor
Calgary, AB T2P 1B5


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274 Ringinglow Road, Sheffield, S11 7PX
T: 0114 321 0440 E: email@example.com
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The Concept Centre is the heart of the Concept Cinema System. This new loudspeaker boasts a precisely optimised implementation of the revolutionary 'Gelcore' cabinet technology developed for the ground breaking Concept 20 and Concept 40 models. The result is a centre channel speaker that delivers astonishingly clear and dynamic dialogue.
Both 5.1 and 7.1 systems can be configured from the other speakers in the concept range combined with our 2070i sub. In addition, if an installation requires overhead speakers (Dolby Atmos or DTS:X), we recommend the use of the Q Acoustics Install Performance In-ceiling range.
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On most social media sites, gaming platforms, communication apps and internet forums, users can opt to have any user name, or "handle", that they choose. For example, on Facebook, most people use their real name (and it has to be "a name", as in first name, last name), but on Twitter, Instagram, Tumblr and most forums and online gaming networks, a user named Joe Smith is as likely to be named "AngryPirate" as he is some version of "Joe Smith". If your teen is using a pseudonym on any given social network or app, you should ask her why.
We're not saying that using a pseudonym is wrong; we're saying that getting to the bottom of why your teen chooses a name other than her own when online is a great way to kick start a conversation about what your kid is doing online.
There are a number of reasons why pseudonyms are used. Some of them can be suspect, and some are pretty straightforward and not at all unsavory:
Cyberbullying – If your teen has been accused of cyberbullying, or is inclined to do it and wants to stay out of trouble, she might use a pseudonym. She probably won't admit it initially, but a little prodding might produce some answers.
Other inappropriate behavior – The reason might be that she tends to use racy or inappropriate language, post outlandish opinions in order to get a response (trolling), or associate with an online group whose interests are suspect or unpopular.
Avoiding bullying – Most kids witness some form of cyberbullying on a weekly basis. If your teen uses a social network or app anonymously, either because she has been bullied in the past, or wants to avoid it in the first place, that's totally fine. If she was bullied in the past and this is the first time you've heard about it, now is a great time for a deeper discussion.
Stalking – Your teen might be using an account to stalk or spy on another teen. This could be a precursor to something worse so feel free to discuss it.
Humor – It could just be an inside joke among friends, or something that your teen finds funny or ironic.
Everybody does it – On most gaming networks and websites like Reddit, almost nobody uses their real name. Since that is the standard, it's fine for your teen too, as long as behavior is appropriate.
Innocent desire to be anonymous – This is pretty broad, but for a number of reasons, there are people who wish to engage online, harmlessly, and not be tied to their real identity. These people tend to do more listening than talking. This is also fine as long as no lines are crossed.
In our opinion, any reason to talk to your teen about what she is doing online is a good one. Get involved, guide good behavior and everyone should sleep better at night.
Contact ThirdParent any time for help and resources for monitoring teen internet activity.
Follow us on Twitter or Facebook for more news and information on keeping your teens safe online. You can sign up for our weekly newsletter on the right.


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Sunscreen is one of those products that should never be put in a drawer and forgotten. It is vital to ensuring your skin's overall health as well as helping to protect its healthy appearance. Unfortunately, a variety of factors contributes to people... read more
Summer is one of those times of the year where we're continually told to enjoy ourselves. It makes sense given it is the definite break from the cold or chilly weather for a while for many people. The warmer temperatures certainly do wonder to help ... read more
Although there are constantly new skin care products being released onto the market, each highlighting a specific ingredient with an abundance of health and beauty claims, research shows that the majority of these hypes aren't actually backed by sci... read more
Although skin cancer may be the most prevalent form of cancer in the United State, it is also the most preventable, meaning that there are certain steps that you can take when it comes to your skin care to keep skin cancer at bay. In honor of skin cancer ... read more


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By jspuhler
High retention and satisfaction rates are a result of good hiring choices. You can improve those rates by improving processes between hiring managers and recruiters.
In smaller companies, HR may consist of one or two people who recruit, hire, onboard, manage benefits, and offboard. The high touch atmosphere of a smaller company lends itself to strong, informed relationships and hiring decisions.
As a company grows, or with larger organizations, roles necessarily differentiate, leaving hiring managers and recruiting groups in different units, or even outsourced. The gap between hiring needs and candidate pools becomes greater and leads to disconnect if the relationship between the two is not recognized or valued.
A cost-effective, efficient recruiting cycle with a reputation for good quality talent acquisition does not happen without work. How can you build end-to-end success when you have an open position?
Communication is key
A culture of frequent and open communication means hiring needs are communicated, and appropriate feedback is given about candidate offerings. Consider these common problems between recruiters and hiring staff, and how they might be resolved:
It is the responsibility of the recruiter to inform and align hiring managers to current realities of the job market. Within the last several years, the availability of talent has tightened, requiring recruiters to plumb new sources, consider new recruiting channels, and take more time to find appropriate candidates. Education of hiring managers about current talent pools is essential.
Whether you are a recruiter, or hiring staff, it is your job to understand open job positions. Highly successful recruiting teams work to understand the skills, experience, and education needed to fill a position as well as align with company culture. Hiring managers need patience, flexibility, and time to educate recruiters responsible for developing a suitable candidate pool. Best practices involve the hiring manager and recruiter working together to develop a job description.
Live or one-way videos offer employers and outside agencies the opportunity to make some of the same assessments they would if they met the candidate in person. The cost of hiring is significantly reduced when video screening is in place. Video allows hiring managers and recruiting staff to view candidate material together, making notes, refining a job description, and creating understanding about organizational talent needs.
Yet, that kind of advance notice does not often happen. Recruiters can buffer for hiring managers by engaging in strong source and social media networking. By identifying existing talent pools, using personal outreach and referrals, and the deft management of social media, recruiters can create candidate audiences to tap when potential positions arise.
Effective communication is the better part of any relationship. With talent acquisition, technology is part of that conversation, serving to eliminate redundant tasks, manage details, source talent, and speed the recruiting cycle toward onboarding.
When you have questions about staffing, and other HR software capabilities, we can help. Brightmove offers sophisticated, scalable solutions to help you hire and retain the talent your company needs to stay competitive.
Source: Brightmove
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Kurnool, a city and district headquarter of Andhra Pradesh. is known as the "Gateway to Rayalaseema". This city got its name from Kandenavolu, and was also known as the city of Kandena, which means grease in Telugu, as this was the area where the cartwheels used to get greased. The Ketavaram rock paintings, dating back to the Paleolithic Era are a major tourist attraction in this district. Tungabhadra River and Handri River flow through this area.
Places to Stay
Kurnool offer budget hotels to travellers visiting its city. Some of these are Hotel Raga Sudha (Tariff – Rs.497), Woodlands Lodge (Tariff – Rs.664), Hotel Durga (Tariff – Rs.218) and Lodge Prasuna (Tariff – Rs.365).
The official language spoken in Kurnool is Telugu. The other language spoken here is Urdu.
The important festivals celebrated in Kurnool are Makar Sankranti in January, Maha Shivratri in February, Ugadi in March, Vinayaka Chaviti in August, Dussehra in September-October, Navratri in September-October and Diwali in October-November.
Places of Interest
Kurnool is famous for its tourist attractions in and around the area. They include Jogulamba Temple (26 km), Sitaramapuram Reservoir (17 km), Chinna Tekuru (15 km) and Kodumur 33 km away from Kurnool.
How to Get There
Rajiv Gandhi International Airport in Hyderabad, which is 200 km away from Kurnool, is the nearest airport.
Kurnool Railway Station is located just 2 km away from the city centre.
Kurnool is well connected through state-run and private buses from various towns and states. Private bus operators such as Meghana Travels, Reddy Express Tours & Travels, Kallada Travels and Kesineni Tours & Travels run daily Volvo buses from Hyderabad to Kurnool. Avail of bus ticket booking coupon when you book your tickets online.
Known to be the commercial city of Andhra Pradesh, Vijayawada is surrounded by two rivers t Krishna river on the east and west and the Budameru River on the north. The city which means victory is known to have the largest junction of South India. The place is important state since it is situated on stretch of the Chennai-Howrah and Chennai-Delhi rail routes. It is also has a highway 5 and 9 connecting the city. One of the most popular temples in Vijayawada is the Kanaka Durga temple. It is located on the Indrakeeladri hill that overlooks the river Krishna.
Budget Hotels in Vijayawada
There are good budget hotels in Vijayawada,Some of them are Hotel Sai Siddartha (Tariff – Rs.550), Quality Hotel D V Manor (Tariff – Rs.990) and Lotus Green Park (Tariff – Rs.1000).
The official language of Vijayawada is Telugu. The other languages spoken include English
Events in Vijayawada
Deccan festival is one of the famous festivals in Vijayawada. It is celebrated for five days in the month of February.
Places to See in Vijayawada
There are several places of tourist attraction in Vijayawada. Some of the places include Kondapalli Fort, Vijayawada, Vijayawada (8kms from Vijayawada), Mogalarajapuram Caves (5 kms from Vijayawada), Undavalli Caves (7kms from Vijayawada) and Victoria Museum (20 kms from airport).
How to Get There
Vijayawada Airport is located about 20 kms away from the heart of city.
Vijaywada railway junction is the most important railway station of Southern railway network.
Vijayawada bus terminus is the biggest in Asia. It stands next Chennai . Some of the best bus operators that offer good experience for passengers include Sidhi Vinayak Tours N Travels, Barathi Travels amd Abi Tours & Travel. Avoid standing in queues in bus counters and go for online bus ticket booking facility instead.


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Age of Empires is a civilization-building strategy game with few competitors. Designed at Microsoft by the same people who crafted the award-winning Civilization, Age of Empires features advanced gameplay that's comprehensive enough for hard-core fans of strategic gaming, yet simple enough for the newest gamer to pick up. It also has breathtaking 3D graphics, unusual in this area of the gaming world. Start the game with only your powers of persuasion, and then get your tribe learning essential skills and adapting and developing important technologies to advance. In time, you'll reach out to other civilizations through peaceful trade or through combat and annexation. Age of Empires is anything but predictable--single out a scenario to play and repeat, and you'll be greeted with a different situation every time. This demo version allows you to play one campaign.
|Free to try
|File Size
|Operating System
|Windows 95 Windows NT Windows
|System Requirements
|Pentium 90, Windows 95/NT, 16MB RAM, 80MB disk space (game), 50MB disk space (swap file), local SVGA bus video display (with 1MB VRAM), DirectX Drivers 5.0


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Craft is a consumer web / mobile application building the "Source of truth" on companies. Today our technology structures and presents over 50 million data-points on 264,000 companies in 230 sectors, including 400,000 job openings updated daily. Our in-depth profiles cover both private and public companies, and are used by sophisticated job-seekers, investors, service providers and marketers to map and track the business landscape.
We're working in an Agile environment, with investment from leading technology investors in the UK and Silicon Valley.


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Do you find your forklift downed by consistent maintenance issues and malfunctions? Are you looking for ways to reduce costs and enhance daily operations in your warehouse? Proper forklift maintenance is a foundation of streamlined business processes and warehouse operations. Keeping on top of your forklift will help initiatives flow more smoothly. There are steps you can take right now to reduce the likelihood of negative impacts. Focusing your attention on these four items will assuredly make immediate positive impacts on your warehouse:
1) Malfunctions of Brakes, Wheels and Tires – Many experts agree that checking tire pressure is instrumental in avoiding accidents. Underinflated tires can result in poor traction, which can ultimately be the cause of an incident in the workplace. Furthermore, overinflated tires can blow out and thus ramp up maintenance and repair costs. Keeping an appropriate tire gauge handy and checking the pressure before each and every use can circumvent and prevent these hazards. In addition to this, brake malfunction can provoke calamity. It is imperative to deal with any brake issues as they emerge and do so as soon as possible. If a forklift operator notices the brakes getting softer and are forced to push harder to engage the system, this is a warning sign that should not be ignored. Lastly, checking the play in the wheel is essential as well. Doing so prevents steering-related incidents and assures the steering column is sound and ready for work.
2) Emissions – In many cases, automobile owners find themselves beleaguered by emissions costs when they go for an inspection. The same holds true here. Failing to adhere to emissions imperatives and directives can result in fines from various organizations. The integration of green technology has been delivered as a solution to tackle these issues. More often than not, emissions-related problems are due to a poor catalytic converter, which releases too much hydrocarbons into the atmosphere.
3) Leakages – Whether it is the battery, coolant or hydraulic fluids, it is essential to check fluid levels before operation. Failing to do so can result in a loss of power, failed forks, or overheating. All of these can be disastrous in the critical phases of a heavy operation. According to TotalTrax, reports estimate that multi-shift class I and II forklifts cost about $20,000 to maintain and operate on a yearly basis. Class III forklifts come in just under $13,000 per year in annual costs. One sure-fire method to reduce these earmarks is to check for leakages, which can identify and fix what could become a huge problem.
4) Outdated Safety Legacies – As has been championed in many articles, it is essential to keep up with the times in terms of safety practices. Every year, new ideologies and methodologies emerge to the forefront, and as a result many standardized operating procedures can become obsolete very quickly. Keeping an ear to the street can optimize maintenance regimes and directly influence any warehouse operation in the most positive of ways.