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Cryptococcus neoformans is a heterothallic fungal pathogen of humans and animals. Although the fungus grows primarily as a yeast, hyphae are produced during the sexual phase and during a process called monokaryotic fruiting, which is also believed to involve sexual reproduction, but between cells of the same mating type. Here we report a novel monokaryotic fruiting mechanism that is dependent on the cell cycle and occurs in haploid cells in the absence of sexual reproduction. Cells grown at 37°C were found to rapidly produce hyphae (∼4 hrs) and at high frequency (∼40% of the population) after inoculation onto hyphae-inducing agar. Microscopic examination of the 37°C seed culture revealed a mixture of normal-sized and enlarged cells. Micromanipulation of single cells demonstrated that only enlarged cells were able to produce hyphae and genetic analysis confirmed that hyphae did not arise from α-α mating or endoduplication. Cell cycle analysis revealed that cells grown at 37°C had an increased population of cells in G2 arrest, with the proportion correlated with the frequency of monokaryotic fruiting. Cell sorting experiments demonstrated that enlarged cells were only found in the G2-arrested population and only this population contained cells able to produce hyphae. Treatment of cells at low temperature with the G2 cell cycle arrest agent, nocodazole, induced hyphal growth, confirming the role of the cell cycle in this process. Taken together, these results reveal a mating-independent mechanism for monokaryotic fruiting, which is dependent on the cell cycle for induction of hyphal competency.
Author Summary
Fungi typically grow vegetatively as either yeast or hyphae. Many of the major human fungal pathogens can generate both morphologies and are referred to as the dimorphic fungi. Cryptococcus neoformans is a yeast-like fungus that has not been traditionally thought to be dimorphic since hyphae production typically occurs during the mating process between cells of opposite mating types. However, C. neoformans also can generate the hyphal state from haploid cells (called monokaryotic or haploid fruiting) in the absence of the opposite mating type. Recent studies have shown that the mechanism behind this process also involves mating, however, the mating reaction occurs between cells of the same mating type. Here we describe a unique mechanism responsible for monokaryotic fruiting that is independent of mating and does not proceed through a diploid intermediate. Instead, the key requirement for hyphal induction appears to be cell cycle arrest. Importantly, arrested cells display an enlarged cell phenotype, which has been observed in vivo in recent reports and has been hypothesized to be a novel protection strategy against host defenses. C. neoformans appears to have an extensive morphological repertoire, which likely contributes to its success as both a pathogen and a saprophyte.
Citation: Fu J, Morris IR, Wickes BL (2013) The Production of Monokaryotic Hyphae by Cryptococcus neoformans Can Be Induced by High Temperature Arrest of the Cell Cycle and Is Independent of Same-Sex Mating. PLoS Pathog 9(5): e1003335. https://doi.org/10.1371/journal.ppat.1003335
Editor: Tamara L. Doering, Washington University School of Medicine, United States of America
Received: August 28, 2012; Accepted: March 18, 2013; Published: May 2, 2013
Copyright: © 2013 Fu et al. 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 material is based upon the work supported by the Army Research Office of the Department of Defense under Contract No. W911NF-11-1-0136. FACS and cell sorting were performed in the UTHSCSA Flow Cytometry Shared Resource Facility, which is supported by UTHSCSA, NIH-NCI P30 CA54174, and UL1RR025767. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Cryptococcus neoformans is a basidiomycetous fungal pathogen of humans and animals that typically causes opportunistic infections in patients with cellular immune defects [1]. Infection initiates in the lungs and frequently disseminates to the brain where it manifests as a fatal meningoencephalitis if untreated. AIDS patients are at increased risk for infection, though infection rates have decreased significantly with better AIDS management [2]. However, in spite of the reduction in AIDS-related cases, cryptococcosis remains a frequent life-threatening opportunistic mycosis for these patients in underdeveloped countries, and is a recently emergent disease in the United States Pacific Northwest [3] for as yet, unexplained reasons.
Naturally occurring strains of C. neoformans are heterothallic with two mating types, MATa and MATα, with both mating types being pathogenic, although most clinical isolates are MATα [4]. Although the taxonomy has been changing, four serotypes have been described (A, B, C, D) with serotypes A and D often referred to as C. neoformans variety grubii and variety neoformans respectively, and serotypes B and C being collectively referred to as C. neoformans variety gattii, or more recently, C. gattii [5]. For all serotypes, throughout the course of infection and under normal culture conditions, the fungus grows as an encapsulated yeast. Under appropriate in vitro conditions, however, the fungus can produce two kinds of hyphae; dikaryotic hyphae during MATa×MATα sexual reproduction and monokaryotic hyphae (from individual MATa or MATα strains) during monokaryotic fruiting [6]. Basidiospores can be produced from both hyphal types. Environmental factors required for sexual reproduction and monokaryotic fruiting are similar and include culture under low temperature (25°C), low moisture, and nutrient limitation [6]. Many genes, including homologs of the Saccharomyces cerevisiae pheromone response pathway, are required to produce both types of hyphae [7], [8], [9], [10], [11]. There are, however, distinct differences between the two hyphal types. Structurally, dikaryotic hyphae have fused clamp connections and a pair of nuclei (one MATa and one MATα) per hyphal compartment while monokaryotic hyphae have unfused clamp connections and a single nucleus per hyphal compartment. Sexual reproduction in C. neoformans has been characterized in detail and largely follows the pheromone response paradigm that has been developed from decades of S. cerevisiae research [12]. Less clear is the mechanism by which monokaryotic fruiting occurs.
Recent studies have concluded that monokaryotic fruiting in C. neoformans variety neoformans can result from α-α mating [13] and may be an important part of the natural life cycle of this fungus [14], with possible implications for human disease [15]. We recently reported that high temperature seed culture conditions could induce very robust monokaryotic fruiting in C. neoformans variety neoformans [11] and assumed these growth conditions enhanced α-α cell fusion. However, this assumption proved to be false. Instead, we found that cells arrested in the G2 stage of the cell cycle were competent to undergo monokaryotic fruiting at high frequency, in the absence of α-α cell fusion. Importantly, this mechanism proceeds through enlarged cells, a morphological phenotype that has been increasingly observed in vivo and is hypothesized to serve as a strategy for avoiding host defenses [16], [17], [18]. These results demonstrate that C. neoformans has evolved a number of different mechanisms for modifying cellular morphology to suit its specific environment, with some of these mechanisms contributing to the success of this fungus as a pathogen.
Seed culture conditions determine competence for monokaryotic fruiting
Previous studies of monokaryotic fruiting typically were performed by patching cells from a seed culture onto filament agar and then screening for a hyphal fringe around the periphery [6], [11], [19]. Our recent observation of the role of temperature in this process [11] led us to test whether or not high temperature increases the intensity of hyphae production after inoculation onto filament agar, or increases the number of cells that produce hyphae. A suspension of cells from a 24 h, 37°C seed culture was spread onto filament agar to observe monokaryotic fruiting in individual cells, which would reveal whether or not all cells from the seed culture were capable of undergoing monokaryotic fruiting. Figure 1A demonstrates that only part of the population grown at 37°C was able to undergo monokaryotic fruiting and that hyphae began to appear as early as 4 hours after plating onto filament agar (Fig. 1B). This phenomenon was not a strain artifact nor was it restricted to a single serotype as all four serotypes were found to be able to produce hyphae under the above inducing conditions (Fig. 1C–F). These results suggest that only a specific type of cell is capable of undergoing monokaryotic fruiting, and that this capability requires seed culture conditions that enable these cells to become competent for hyphal production.
(A) Individual colonies visualized at 2× on a spread plate after 4 days growth at 25°C from a seed culture (JEC-21) inoculum grown at 37°C for 24 h on YPD. Bar = 1 mm. (B) Individual cells under the same seed culture and plating conditions as in (A), but visualized after 4 h at 200× magnification. Bar = 10 µm. (C–F) Monokaryotic fruiting in all serotypes of C. neoformans. (C) WSA-79 (serotype D, also known as C. neoformans variety neoformans), (D) WSA-522 (serotype A, also known as C. neoformans variety grubii), (E) WSA-533 (serotype B, also known as C. neoformans variety gattii, or C. gattii), (F) WSA-2507 (serotype C, also known as C. neoformans variety gattii, or C. gattii). Colonies visualized at 200×. Bar = 10 µm.
Temperature-induced hyphal competency does not result from α-α cell fusion
Lin et al., have demonstrated that same-sex mating is one way in which monokaryotic fruiting can occur [13]. However, because the previous experiment showed that individual cells were still capable of monokaryotic fruiting in spite of being well separated from potential mating partners on spread plates, we hypothesized that either same-sex mating occurs during the seed culture growth period, or that another mechanism, which does not involve same-sex mating, could also lead to monokaryotic fruiting.
To investigate these two possibilities, we first screened for evidence of α-α mating during monokaryotic fruiting using the α-α cell fusion assay to test different combinations of complementing MATα auxotrophs that would be predicted to yield prototrophic colonies on unsupplemented MIN agar. No fusants were observed from MATα×MATα crosses plated onto MIN agar after growth as a seed culture on YPD at 37°C for 24 h. However, assisted α-α matings showed that each of these strains was capable of undergoing α-α fusion, ruling out an α-α cell fusion defect (Fig. 2A). The most likely explanation for these results was that because α-α fusion is a rare event [13], our conditions, although not detecting an α-α fusion, were still not excluding this possible mechanism. To exclude a cryptic fusion event as an explanation for our observations, we utilized a cpk1Δ mutant to further test whether or not α-α fusion was required for temperature-induced monokaryotic fruiting. Cpk1p, the MAP kinase in the C. neoformans pheromone response pathway, is required for α-a fusion during sexual reproduction as well as α-α fusion during monokaryotic fruiting [9], [13]. Therefore, if α-α fusion was the mechanism of monokaryotic fruiting in our system, the cpk1Δ mutant should not produce hyphae after plating onto filament agar because it could not fuse with the complementing strain. Our results showed that neither assisted nor unassisted α-α mating reactions (WSA-2126×WSA-591×WSA-65 or WSA-2126×WSA-591) with the cpk1Δ mutant (WSA-2126) showed evidence of α-α cell fusion (Fig. 2B). However, when WSA-2126 was tested for monokaryotic fruiting ability after high temperature seed culture, this strain fruited normally (Fig. 2B). These results confirmed that monokaryotic fruiting could occur independently of α-α fusion.
(A) Crosses between MATα strains with complementary auxotrophic markers do not yield fusants (WSA-1226×WSA-70, or WSA-1226×WSA-1) unless the mating is assisted with a MATa (WSA-68) helper strain (WSA-1226×WSA-70×WSA-68, or WSA-1226×WSA-1×WSA-68). However, the three MATα strains are capable of monokaryotic fruiting (insets). (B) To rule out an undetectable fusion event as an explanation for the results seen in (A), a fusion defective strain (WSA-2126) was included in the assay. This strain, which is a cpk1 mutant, did not yield fusants with WSA-591 alone or with a MATa (WSA-65) helper. Complementation of WSA-2126 with the CPK1 gene (WSA-3145) did not yield fusants with WSA-591 unless WSA-65 was included as a helper. However, all three MATα strains were capable of monokaryotic fruiting, including the cpk1 mutant (inserts) ruling out α-α cell fusion as the mechanism for monokaryotic fruiting.
Monokaryotic fruiting initiates from large cells, which are induced by high temperature seed culture
The production of enlarged cells in C. neoformans has been reported to occur when cells are exposed to opposite mating type cells in standard mating type mixes [11], and in confrontation assays, which are performed by streaking cells of opposite mating type in close proximity to each other [20]. Clinical studies have also found this cell type in vivo [16], [18], [21], [22]. Because only certain cells produced hyphae during the quantitative monokaryotic fruiting assay, we decided to determine if there were developmental differences among cells after high temperature seed culture. Microscopic observation of wet mounts prepared from cells scraped off of filament agar after growing as a seed culture at 37°C showed that filaments always originated from enlarged cells (Fig. 3A). When cells from 30°C and 37°C seed cultures were screened for enlarged cells, we only observed enlarged cells from the 37°C seed culture, although this incubation temperature produced both enlarged and smaller, normal-sized cells (Fig. 3B and 3C). To confirm that the enlarged cells were responsible for the production of hyphae during monokaryotic fruiting, large and small cells from a 37°C seed culture were micromanipulated onto filament agar and then monitored for hyphae production. Analysis of hyphae production from each single cell revealed that small cells only grew into yeast cells while the large cells grew as both yeast and hyphae (Fig. 3D). These results demonstrated that only a subset of the population grown at high temperature, which can be distinguished by the larger cell size, becomes competent to produce hyphae.
(A) Wet mount of cells scraped from filament agar after 24 h growth showing hyphae growing from an enlarged cell. (B) Wet mount of cells from YPD agar after 24 h growth at 30°C showing a homogenous population of normal-sized (2–5 µm) yeast cells. (C) Wet mount of cells scraped from YPD agar after 24 h growth at 37°C showing a mix of enlarged (filled white arrowhead) and normal-sized cells (open arrowhead). All images were obtained with DIC microscopy at 400× magnification. Bar = 10 µm. (D) Results of micromanipulation of enlarged and normal-sized cells from a 24 h, 37°C YPD agar culture scored for morphology after germinating on filament agar.
Enlarged cells are haploid, arrested in G2, and produce haploid hyphae
The need for enlarged cells prior to the initiation of monokaryotic fruiting suggested that the mechanism for production of this phenotype possibly involved changes in cell ploidy since yeast ploidy has been noted to be associated with cell size [23]. In C. neoformans, monokaryotic fruiting has been shown to result in ploidy changes of yeast cells produced specifically from the hyphal filaments [13]. These observations suggested to us that monokaryotic fruiting may occur through ploidy changes, which are manifested as enlarged cells that arise from an endoduplication event, as has been previously suggested [24].
Cell cycle analysis of seed cultures grown at different temperatures (25°C, 30°C, 35°C, 37°C and 40°C) all revealed only 1n and 2n DNA content peaks, with no evidence of a 4n peak (Fig. 4A). This result showed that there was no ploidy change after high temperature (35°C, 37°C, 40°C) seed culture growth, demonstrating that the enlarged cells, which were responsible for monokaryotic fruiting, were haploid. Additionally, we found that as seed culture temperature increased from 25°C to 40°C, the percentage of cells in G1 decreased, the percentage of cells in G2 increased, and the percentage of cells in S phase was similar until dropping almost to 0 at 37°C (Fig. 4B). DAPI staining confirmed the relationship between cell size and G2 arrest as the staining patterns of large and small cells differed (Fig. 4C). The smaller cells displayed a compact nuclear staining pattern while the larger cells displayed a larger, diffuse staining pattern, which has been observed in other G2-arrested fungi [25], [26]. These results demonstrated that the effect of increasing temperature on monokaryotic fruiting involved the cell cycle, specifically G2 arrest.
(A) Cell cycle analysis of cells grown at various temperatures reveals that only peaks corresponding to a DNA content of 1N and 2N were observed, ruling out the formation of diploids. Peaks represent cell number. Scale represents propidium iodine fluorescent intensity. (B) Cell cycle analysis of JEC-21 grown at different temperatures on YPD agar for 24 hrs shows that with increasing temperature, the percentage of cells arrested at G2 increases. (C) DAPI stained (0.1 µg/ml) nuclei of control and G2-arrested cells. The top panel shows control cells grown at 30°C using DIC/Normarsky, DIC/Normarsky with DAPI staining, and the merged image. These cells were normal-sized with visibly stained nuclei. In contrast, the bottom panel of three images shows cells grown at 37°C, which yielded large and small cells. The size of the smaller cells was consistent with the control cells grown at 30°C. The staining pattern of these cells was also consistent with the control cell staining pattern in which the nuclei stained more compactly than the nuclei of the larger cells, which stained larger, but more diffuse. Cells were visualized at 400×. Bar = 5 µm.
In spite of the FACS results showing that hyphal-competent cells were haploid, we could not rule out a change in ploidy just before, or during growth in the hyphal phase. Unfortunately ploidy determination by FACS analysis of hyphae is physically restricted by the filamentous characteristics of the cells. However, hyphal ploidy can be determined using a blastospore assay [13]. The results of this assay revealed that 76 out of 78 blastospores (97%) were haploid, demonstrating that the hyphae produced during monokaryotic fruiting were haploid, which again excluded α-α mating or endoduplication as monokaryotic fruiting mechanisms in our system (Fig. 5A). As a further control, the two diploid blastospores were sub cultured repeatedly for an additional two weeks and then retested by FACS, which revealed that they remained diploid, ruling out the possibility that haploid blastospores could be segregation products of diploid blastospores. While a late endoduplication event could explain the two diploid spores, this possibility is unlikely since the 78 spores were picked from 78 independent hyphae. However based on the recovery of two diploid blastospores in our assay, and the rarity of basidiospore production during monokaryotic fruiting, we hypothesized that there could be two hyphal types produced during monokaryotic fruiting, which could be distinguished by ploidy. One type could be vegetative in nature and not undergo basidiosporogenesis (haploid) and a second type could be generated that ultimately produced basidiospores (diploid). To address these two possibilities, 37°C seed culture cells were spread onto filament agar and the resultant colonies screened for basidiospore chains. The blastospore assay was performed on blastospores recovered from hyphae with and without basidiospores. Cell cycle analysis again showed that all of the blastospores were haploid, regardless of whether or not the hypha produced basidiospores (Fig. 5B), which was consistent with our previous observations that excluded α-α mating or endoduplication as the mechanisms of monokaryotic fruiting. Together, these results demonstrate that endoduplication is not required for monokaryotic fruiting as these hyphae are produced from haploid cells and remain haploid, in spite of being able to occasionally generate diploid yeast cells.
(A) Blastospores recovered from hyphae produced during monokaryotic fruiting. JEC-21 is the haploid control strain used for the fruiting assays. WSA-3070 is one of two blastospores out of seventy-eight total blastospores recovered from the blastospore assay, which was found to be diploid, as evidenced by comparison to the JEC-21 FACS profile. WSA-3019 is one of the seventy-six blastospores recovered from the same assay, which was found to be haploid, as evidenced by comparison to the JEC-21 FACS profile. (B) FACS profiles of blastospores recovered from hyphae with and without basidiospore chains. Representative profile of a blastospore (WSA-3112) recovered from a hypha without basidiospores and a profile of a blastospore (WSA-3098) from a hypha with basidiospores. Comparison of these two profiles to the control haploid (JEC-21) and diploid (WSA-3002) profiles revealed them both to be consistent with a haploid cell. These results suggest that if diploidization occurs in hyphae during monokaryotic fruiting, it occurs late or even after hyphae have formed, and may be restricted to the basidium.
Monokaryotic fruiting proceeds only from G2-arrested cells
To determine whether only G2-arrested cells undergo monokaryotic fruiting, we sorted G1 and G2 phase, 37°C seed culture cells according to DNA content. Microscopic observation of sorted G1 and G2 cells revealed that G2 phase cells were much larger than G1 phase cells (Fig. 6). We next sorted live 37°C seed culture cells according to cell size and performed cell cycle analysis on the two populations. The results indicated that the smaller cells had a single DNA content peak at the 1n position while the enlarged cells had a single DNA content peak at the 2n position, which lead us to conclude that the small cells were G1 phase cells and the large cells were cells in G2 arrest (Fig. 7). The two populations were then assayed for monokaryotic fruiting ability, which revealed that monokaryotic fruiting was a property only of the G2 fraction (Fig. 7).
JEC-21 cells were grown on YPD agar at 37°C for 24 hrs, then were fixed and stained with propidium iodide. Cells were then sorted according to DNA content and then visualized by DIC microscopy at 400× in order to compare their sizes. A). Sorted G1 cell fraction. B). Sorted G2 cell fraction. Microscopy showed that the G2 fraction contained cells approximately 4× the size of cells in the G1 fraction. Bar = 5 µm.
(A) Cell cycle analysis of sorted JEC-21 cells revealed that the smaller cells were almost exclusively (90.57%) G1 phase cells and yeast-like in morphology when plated onto filament agar. (B) In contrast to the results observed for smaller cells, the population of enlarged cells recovered by sorting revealed that enlarged cells were primarily G2 phase (88.61%), and contained a sub population of cells (∼40–50%) able to undergo monokaryotic fruiting when these cells were plated onto filament agar. Approximately 20,000 cells were sorted for each cell size. Bar = 1.0 mm.
As a final confirmation that monokaryotic fruiting requires G2 arrest, cells were treated with nocodazole, a G2/M arrest agent that inhibits and disassembles microtubules [27]. Cells were grown as seed cultures at 30°C (the non-permissive seed culture temperature), then assayed for monokaryotic fruiting ability. The experiment showed that cells treated with nocodazole became competent to produce hyphae on filament agar in a dose-dependent manner even though they were grown as a seed culture under conditions that did not normally lead to monokaryotic fruiting, whereas untreated cells only grew as yeasts (Fig. 8). Taken together, these results demonstrate that G2-arrested cells can serve as a starting point for cells that undergo monokaryotic fruiting.
JEC-21 cells were cultured in YPD broth plus different concentrations of nocodazole at 30°C for 24 hrs, then dropped onto filament agar and incubated at 25°C for 5 days. (A) Qualitative agar assay for monokaryotic fruiting. Nocodazole concentrations consisted of a.) 0 µM, b.) 0.075 µM, c.) 0.15 µM, d.) 0.30 µM, e.) 0.60 µM, f.) 1.20 µM. Hyphal production begins at nocodazole concentrations ≥0.15 µM. Bar = 0.5 mm. (B) Quantitative agar assay for monokaryotic fruiting. Concentrations of nocodazole are indicated. The percent of colonies undergoing monokaryotic fruiting was calculated from the total colonies that grew out on the agar plates. Plates were assayed in triplicate.
In this study we have identified a novel mechanism in C. neoformans that leads to the production of hyphae, with or without basidiospores, by haploid cells (monokaryotic fruiting). This mechanism appears to be dependent on the cell cycle and initiates from cells in G2 arrest. It occurs in the absence of α-α mating and/or endoduplication, thereby demonstrating that monokaryotic fruiting can occur asexually. Previous studies have shown that sexual reproduction can occur between cells of the same mating type, resulting in monokaryotic fruiting [13], and that this phenomenon occurs in nature [24]. Under the specific conditions of this study, notably a 37°C seed culture temperature, we saw no evidence of an α-α cell fusion event, nor did we find evidence of endoduplication within the hyphae even though we screened hyphae that had produced basidiospores. During C. neoformans basidiosporogenesis, sexual reproduction results in meiosis in the basidium followed by successive mitotic divisions that yield the nuclei, which ultimately are inserted into spores as they form on the basidial surface [20]. Lin et al. observed that when fruiting was derived from an α-α fusant, sporogenesis was robust with spore chains that were long and phenotypically similar to α-a mating during sexual reproduction [13]. This process was found to be impaired in dmc1 mutants, which are meiotic mutants that still produce spores, but at a much lower frequency than sexually produced spores, and with truncated spore chains that sometimes occur as dyads (two rather than four chains) [13]. The phenotype of basidiospores produced in the dmc1 strains was strikingly similar to what we observed in this study and what was previously reported [6]. These observations may suggest that basidiosporogenesis can occur mitotically without meiosis, although we cannot exclude a duplication event in the basidium immediately followed by meiosis and sporogenesis. We did, however, test a dmc1 mutant and found that it was able to undergo monokaryotic fruiting under our conditions (data not shown).
Because our study was done in the same strain background as the study by Lin et al., we reviewed the conditions of both experiments and found some differences that may explain the contrasting differences in ploidy. Our study used a high temperature seed culture condition, which results in rapid hyphae production upon filament agar plating. The seed culture conditions in Lin's study were not clear, however, their plating medium was V8 agar, which is normally used for mating C. neoformans, and their incubation period was for a period of weeks, whereas we screened at 24 hrs and observed hyphae in as little as four hours, although cells also produced hyphae on V8 agar under our conditions. Both filament agar and V8 agar have high agar contents; however, V8 agar is an undefined medium with V8 juice as the basal ingredient. Filament agar, on the other hand, is a low-glucose, defined medium with Yeast Nitrogen Base without amino acids and without ammonium sulfate as the source of vitamins and cofactors. Although both media are starvation media, they are substantially different in composition, which may be one explanation for the differences in hyphal types that we observed. The seed culture conditions, or more precisely, the cell cycle stage may be another explanation.
Our initial investigation of the relationship between the cell cycle and monokaryotic fruiting focused on detecting what we presumed would be a transition to diploidy in yeast cells at some point during seed culture growth, which we believed would coincide with the appearance of enlarged cells in the seed culture and the association of this cell type with the hyphal progenitor. The high frequency of fruiting and enlarged cells in the seed culture suggested that detection of the diploidization event would be unambiguous. However, the data showed that instead of an α-α fusion or endoduplication event, which would result in a diploid cell, the actual mechanism that resulted in hyphae production was G2 arrest. The reason for the requirement of G2 arrest to induce hyphae is not clear, and while G2 arrest is required for monokaryotic fruiting, not all arrested cells produced hyphae. We suspect that the subpopulation of non-hyphal, G2-arrested cells consisted of cells that escaped G2, and then proceeded to bud rather than differentiate into a hypha. These two outcomes resemble the decision point that a pheromone-exposed, G2-arrested Ustilago maydis cell faces with regard to which of the two developmental paths it will follow (conjugation tube formation or budding) [26]. How the generation of occasional diploid blastospores occurs is also not clear. Given that C. neoformans hyphae produce typical basidiomycete-like clamp connections, the incomplete fusion of these structures in monokaryotic hyphae combined with an aberrant segregation event during the budding of blastospores off of the hyphal compartment may yield the diploid cells that we observed at low frequency.
Previous studies of the C. neoformans cell cycle have identified a number of stressors that cause G2 arrest, including oxygen depletion [28], stationary growth phase [29], and temperature [30]. Under our experimental conditions, hyphal competency occurs prior to transfer to hyphal inducing conditions (starvation on filament agar) and not during growth on filament agar. Therefore, stationary growth phase is not a factor nor is oxygen depletion since cultures were grown on the agar surface, and only for 24 hrs. Consequently, growth temperature seems to be responsible for inducing competency. Under our conditions, the 37°C incubation temperature differs from the original incubation temperature (30°C) for monokaryotic fruiting [6], which suggests that temperature is an important variable. In fact, while we did not see the temperature effect on all strains of C. neoformans, we were able to induce hyphae in all four serotypes. Interestingly, the reports of enlarged cells in vivo [16], [18], [21], [22] reflect growth at elevated temperature in the mammalian body. Other pathways have also been shown to influence C. neoformans cell size in vitro, including cAMP, RAS, and PKA [16], [31], [32], suggesting the possibility of conserved stimuli that may regulate these pathways. Presently, the STE12α signal transduction pathway seems to be the major or sole regulator of monokaryotic fruiting as this gene is required for monokaryotic fruiting regardless of inducing conditions. Fruiting still occurred normally in cpkΔ1 (pheromone response pathway), and cacΔ1 (cAMP pathway) mutants, ruling out these pathways as regulators of temperature-induced monokaryotic fruiting. Other pathways, such as the calcineurin signal transduction pathway cannot be ruled out, but are more complicated to test since some mutants in this pathway do not grow at high temperature [33].
A key characteristic of enlarged cells in vivo appears to be polyploidy, which has been hypothesized to arise when the M phase of the cell cycle is skipped [16], [18]. This enlarged cell phenotype appears to be a potential virulence factor as they are poorly phagocytized, if at all [18]. Perhaps increasing cell size evolved as a physical defense mechanism against predatory grazers, which in turn, protects cells from being phagocytized in vivo via the same mechanisms. It appears that the enlarged cell phenotype can be produced by multiple mechanisms: cell cycle arrest, a-α or α-α cell fusion, and endoduplication, each of which may have a different purpose. The developmental options available after cell cycle arrest may have been selected for in C. neoformans to enhance survival in its specific environmental niche while inadvertently creating an important human fungal pathogen. What remains to be determined is how high temperature generation of the large cell and monokaryotic fruiting phenotypes was incorporated into the evolution of C. neoformans. With the exception of C. neoformans, virtually all members of this genus grow poorly or not at all at mammalian ambient temperature. In contrast, all of the major human fungal pathogens grow at 37°C and virtually all of them have a hyphal phenotype. Perhaps the association of many basidiomycetes with rotting wood or decaying vegetation in general led to high temperature exposure and subsequent genetic selection during self-heating, compost-like conditions caused by microbial metabolism of organic matter. Once nutrients were consumed, hyphal extension towards additional nutrients and/or sporulation in the absence of nutrients could have completed the evolution of C. neoformans into a pathogen via development of a mechanism of infectious particle (basidiospores) dispersion combined with the ability to grow at elevated temperatures. Selection for the molecular linkage of pathways controlling cell cycle, nutrient sensing, and ultimately, differentiation, could have been the outcome of this lifestyle and allowed the fungus to coordinately regulate these pathways, thus enabling it to effectively exist as a saprophyte or pathogen.
Materials and Methods
Media and strains
YPD agar, MIN agar, V8 agar, and filament agar were prepared as described previously [6], [7], [11] with or without amino acids or nucleic acid supplements as required. JEC-21 is a wild type, MATα isolate that was used in the initial characterization of monokaryotic fruiting in C. neoformans [34]. WSA-79 is a serotype D clinical isolate from Maryland, WSA-522 is a serotype A clinical isolate from Thailand, WSA-533 is a serotype B environmental isolate from Australia, and WSA-2507 is a serotype C clinical isolate from Maryland. Additional strains, all of which were derived from the original JEC-21 - JEC-20 congenic pair [34], consisted of the following genotypes: WSA-1 (MATα lys2), WSA-70 (MATα ade2 lys2), WSA-591 (MATα ade2), WSA-1226 (MATα ura5), WSA-2126 (MATα ura5 cpk1Δ::ura5), WSA-3002 (MATα/MATα fusant from WSA-1226×WSA-70×WSA-68), WSA-3019 (MATα haploid blastospore), WSA-3070 (MATα/MATα diploid blastospore recovered from JEC-21 hypha without basidiospore chains), WSA-3098 (MATα haploid blastospore recovered from JEC-21 hypha with basidiospore chains), WSA-3112 (MATα haploid blastospore recovered from hypha without basidiospore chains), WSA-3145 (MATα ura5 cpk1Δ::ura5 CPK1::NEOr). MATa strains included WSA-65 (MATa ura5 lys1 ade2) and WSA-68 (MATa ura5 ade2 lys2).
Monokaryotic fruiting assay.
The standard assay for monokaryotic fruiting was performed as described [11] using YPD agar as the medium and a seed culture incubation temperature of 37°C for 24 h unless otherwise indicated. Isolates were harvested from plates and then streaked or spread (after suspension in distilled water) onto the appropriate agar plate, which was then incubated at 25°C until cells were photographed using an Olympus BX51, bright field, Spot Idea Camera, (Diagnostic Instruments, Inc., Sterling Heights, MI).
Quantitative monokaryotic fruiting assay.
Monokaryotic fruiting efficiency was determined by quantitative assay. This assay was performed by suspending ∼106 cells from a YPD seed culture grown at 37°C in 1 ml sterile water. This suspension was then serially diluted to yield approximately 2000 cells per ml. A 100 µl aliquot was then spread onto filament agar, which was incubated at 25°C for 24 h. The number of hyphal colonies was then compared to the total number of cells to determine the percentage of cells that were capable of undergoing monokaryotic fruiting.
Hyphal ploidy assays.
Hyphal ploidy was determined using the blastospore assay [13]. Single blastospores were recovered from hyphae produced by filament agar colonies using an Axiolab micromanipulator (Zeiss, Thornwood, New York). Each blastospore was isolated from independent hyphae-producing colonies, one blastospore per hypha. A second screen was used to compare blastospore ploidy from hyphae with and without basidiospores, which were part of the same colony. Colonies were identified that produced hyphae with basidiospores on a filament agar spread plate. Blastospores were then isolated from single hyphae that had produced four chains of basidiospores. Additional blastospores were also isolated from the same colony, but from single hyphae that had not produced basidiospores. All blastospores were immediately transferred to YPD agar and grown out for 72 hrs at 30°C. Each isolate was then screened for ploidy by FACS analysis as described below.
Nocodazole treatment
Nocodazole (Sigma-Aldrich, St. Louis, MO) stock was prepared in DMSO at 1.5 mM and then used to prepare different dilutions in YPD broth (YPD-0.075 µm nocodazole, YPD-0.15 µm nocodazole, YPD-0.30 µm nocodazole, YPD-0.60 µm nocodazole, YPD-1.2 µm nocodazole). JEC-21 cells were added to 1.5 ml nocodazole-YPD broth in 15 ml snap cap tubes (BD Biosciences, Franklin Lakes, NJ), at a final concentration of 1×106 cells/ml. Tubes were shaken at 200× RPM at 30°C for 24 hrs. Five µl of overnight culture were then dropped onto filament agar and incubated at 25°C for 5 days. The quantitative assay was performed by growing cells as above, and then plating cells onto filament agar as described in the quantitative monokaryotic fruiting assay. Pictures were taken with an Olympus SZX12 stereo microscope (Olympus, Center Valley, PA) at 2× magnification.
Cell fusions
To perform α-α cell fusions, 1×106 cells of complementary, auxotrophic, MATα strains were mixed, cultured on YPD agar at 37°C for 24 hrs, and then transferred onto filament agar plates. The plates were incubated at 25°C for 24 hrs. The mixture was scraped from the plate, suspended in 1.0 ml sterile H2O, and then 200 µl of cells from this suspension were spread onto MIN agar plates, which were then incubated at 30°C for 4 days to screen for fusants. Assisted mating reactions, in which two MATα strains were induced to fuse by including a MATa helper strain, were performed according to previously described methods [19]. Plates were photographed at 0.5× magnification.
Flow cytometry and cell sorting
Cells were harvested, fixed, and stained with propidium iodide (Sigma-Aldrich, St. Louis, MO), and then sorted as described by Sia et al. [35]. Cell cycle analysis was performed using a BD FACSCalibur flow cytometer (Becton Dickinson Biosciences, Sparks, MD). CellQuest Pro software was used for cell collection, and data analysis was performed using ModFit and FlowJo. G1, S, and G2 phases were identified using the Dean-Jett-Fox mathematical model. G2-arrested cells were identified as described [25], [26], [36], [37], [38]. This population of cells typically shows a large cell phenotype, an increased proportion of cells with 2C DNA content when compared to controls, and a DAPI staining pattern that shows larger nuclei vs. smaller condensed nuclei of G2 phase cells (see below). Cell sortings were performed on a BD FACSAria III (Beckton Dickinson Biosciences) cell sorter and analyzed using BD FACSDiva 6.1 software. Aliquots of sorted live cells were also used to perform the quantitative monokaryotic fruiting assay. All FACS analysis was performed at the Flow Cytometry Core Laboratory at The University of Texas Health Science Center at San Antonio.
Cells were stained with DAPI according to the method described by Fuchs et al. [39]. Briefly, 1×107 cells were harvested from 24 h YPD agar plates, which were grown at either 30°C or 37°C, washed twice with phosphate buffer (0.1 M KH2PO4, 1.25 mM EGTA, 1.25 mM MgCl2 at pH 6.9), then resuspended in 400 µl fixative (5% paraformaldehyde in wash buffer) followed by incubation at room temperature for 90 minutes. The cells were then washed three times in wash buffer and incubated with Lysing Enzymes (Trichoderma harzianum, Sigma-Aldrich, 1 mg/ml in sterile distilled water) for 20 min at 37°C. The cells were then washed once with sterile water, gently resuspended in 400 µl 0.3% Triton X-100 (Sigma-Aldrich), and permeabilized by incubation at room temperature for 15 minutes. The suspension was then pelleted and washed three times with PBS. Cells were stained in DAPI (Sigma-Aldrich) (0.1, 0.2, or 0.5 µg/ml) for 15 minutes, washed twice with PBS, and then resuspended in 200 µl prior to visualization.
Images were captured on a Zeiss AxioImager Z1 microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY) equipped with an AxioCam MR3_2 CCD camera, using the filters for DAPI (365 nm excitation, 395 nm beam splitter, 420–470 nm emission filter) and differential interference contrast (DIC, Nomarski contrast). Image analysis and adjustments were performed using Axiovision software (Zeiss, Version 4.8). Images were adjusted only for frame alignment (to overlay DAPI over DIC), brightness, and contrast (adjustment of +0.01 contrast units over default), and all images received the same treatment.
Author Contributions
Conceived and designed the experiments: BLW JF. Performed the experiments: JF. Analyzed the data: BLW JF. Contributed reagents/materials/analysis tools: JF IRM. Wrote the paper: BLW JF.
- 1.
Perfect JR (2006) Cryptococcus neoformans: a Sugar Coated Killer. In: Heitman J, Filler SG, Edwards JE, Mitchell AP, editors. Molecular Principles of Fungal Pathogenesis. Washington, DC: American Society for Microbiology. pp. 281–304.
- 2. Waters L, Nelson M (2005) Cryptococcal disease and HIV infection. Expert Opinion on Pharmacotherapy 6: 2633–2644.
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- 8. Wang P, Nichols CB, Lengeler KB, Cardenas ME, Cox GM, et al. (2002) Mating-type-specific and nonspecific PAK kinases play shared and divergent roles in Cryptococcus neoformans. Eukaryot Cell 1: 257–272.
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- 11. Fu J, Mares C, Lizcano A, Liu Y, Wickes BL (2011) Insertional mutagenesis combined with an inducible filamentation phenotype reveals a conserved STE50 homologue in Cryptococcus neoformans that is required for monokaryotic fruiting and sexual reproduction. Mol Microbiol 79: 990–1007.
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- 17. Garcia-Rodas R, Casadevall A, Rodriguez-Tudela JL, Cuenca-Estrella M, Zaragoza O (2011) Cryptococcus neoformans Capsular Enlargement and Cellular Gigantism during Galleria mellonella Infection. PLoS One 6: e24485.
- 18. Okagaki LH, Strain AK, Nielsen JN, Charlier C, Baltes NJ, et al. (2010) Cryptococcal cell morphology affects host cell interactions and pathogenicity. PLoS Pathog 6: e1000953.
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- 24. Lin X, Patel S, Litvintseva AP, Floyd A, Mitchell TG, et al. (2009) Diploids in the Cryptococcus neoformans serotype A population homozygous for the alpha mating type originate via unisexual mating. PLoS Pathog 5: e1000283.
- 25. Al-Feel W, DeMar JC, Wakil SJ (2003) A Saccharomyces cerevisiae mutant strain defective in acetyl-CoA carboxylase arrests at the G2/M phase of the cell cycle. Proc Natl Acad Sci U S A 100: 3095–3100.
- 26. Garcia-Muse T, Steinberg G, Perez-Martin J (2003) Pheromone-induced G2 arrest in the phytopathogenic fungus Ustilago maydis. Eukaryot Cell 2: 494–500.
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- 29. Takeo K, Tanaka R, Miyaji M, Nishimura K (1995) Unbudded G2 as well as G1 arrest in the stationary phase of the basidiomycetous yeast Cryptococcus neoformans. FEMS Microbiology Letters 129: 231–235.
- 30. Takeo K, Ohkusu M, Kawamoto S (2003) Effects of growth temperature upshift on cell cycle progression in Cryptococcus neoformans. Mycoscience 44: 465–471.
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- 32. Waugh MS, Nichols CB, DeCesare CM, Cox GM, Heitman J, et al. (2002) Ras1 and Ras2 contribute shared and unique roles in physiology and virulence of Cryptococcus neoformans. Microbiology 148: 191–201.
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- 34. Kwon-Chung KJ, Edman JC, Wickes BL (1992) Genetic association of mating types and virulence in Cryptococcus neoformans. Infection and Immunity 60: 602–605.
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We as a whole should spoil ourselves once now and again. Numerous ladies do stay aware of best spas and best facials at home. Once in a while, it is truly decent to have the facial accomplished for you. Facial medications do not need to be costly to make you feel decent. What are the various types of facials out there, you may inquire? All things considered, there is an amazing facial to dispatch called the Triple Oxygen Treatment. This facial is tried by research center and had accomplished incredible outcomes. Actually, the facial was lab tried, and had got great outcomes. It is evaluated very among the individuals who had encountered it.
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In the event that you will get one of these, then a steam shower is likewise decent for your wellbeing. The pipes system is so natural to utilize. On the off chance that you as of now have a shower, then you require a transom to carry out the employment. They are sufficiently modest to purchase and can work out more efficient than whatever else. Simply had, routine spa and facial sessions will be immensely valuable in keeping up your characteristic and young appearance.

subset: commoncrawl


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Turn Heads With This Green to Pink Lip Crayon
You may associate color-changing lipsticks with decades long gone. For example, in 1974 Avon debuted their popular line of Color Matcher lipsticks, and the '80s and '90s saw plenty more of the same from numerous brands. You may even recall that Claire's--yes, the accessories store everyone had in their local mall--sold the stuff in bulk alongside their roll-on glitter and infinite supply of earrings.
Even though the latter decades of the 20th century have come and gone, the "Surprise! I'm actually pink!" lip game is still going strong as we settle into the 21st century. You've got Smashbox's O-Gloss Intuitive Lip Gloss, Dior's Dior Addict Lip Glow Color Reviver Balm, Sephora's Color Reveal Glosses, and inexpensive NYX Mood Lip Glosses, considered dupes for the above. That's just a small sample list, of course.
Korean cosmetic brands, known for their whimsical spirit, have played along with this fun trend, too. Take Son & Park's Watermelon Lip Tint, for example. It's a bright, juicy green lip crayon that transforms into the prettiest shade of pink you've ever seen once it hits your lips. Seriously – it's the ideal shade for that naturally flushed glow thing we all strive for. Add to that fact that it smells divinely fruity and you've got a serious winner on your hands (and lips...).
The crayon does look funky in all its green glory, so plan for some surprised looks if you add it to your arsenal of lip goods. I'll tell you that it's definitely earned me some head turns when I pull it out of my purse. Talk about a conversation starter!
Now for a mini beauty science sidebar: The secret behind the enchanting color transformation in all of these color changing lippies is an ingredient called Red 27. Sounds simple enough, right? But how does it work? It's a colorless pigment while sitting in the waxy base of a gloss or lipstick, but once it hits the warmth and moisture of your lips, the dye brightens into a perfectly pleasing, flattering shade of pink. This is a chemical reaction in which the dye reacts to the pH level of your lips. Everyone pretty much has the same pH level, so don't expect a huge color variance from one person to another. However, the color will vary slightly depending on how light or dark your lips already are.
—Wendy Rose Gould
Wendy Rose Gould is a beauty writer and photographer who lives in the middle of the desert, where sunscreen and moisturizers mean more to her than perhaps to the average human. She vividly, and fondly, remembers the first time she used a B.B. cream: in Seoul during a one-year stint she did back in 2009. She currently serves as contributing editor at xoVain and is an editorial writer at Refinery29, ModCloth, Latest-Hairstyles.com and other outlets. For Wendy, a lazy makeup day usually involves at least six products and probably a cat eye. Follow Wendy at @Wendyrgould


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Andrew W.
Richmond, Canada, v7e3n5
Joined on May, 2011
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Education2011 BFA - Bachelor of Fine Arts in Theatre (Acting) at the University of Victoria
2011 BA - Bachelor of Arts in Writing at the University of Victoria, with co-op distinction.
ExperienceOn top of my degrees, I have worked as a Peer Helping Student Coordinator, leading drop-in offices designed to help students with learning and study skills, as well as to provide one-on-one counseling. I also spent a year with PEAK UVic, working as a study group leader in the residence buildings on campus.
I have a wide knowledge base. While I was a Fine Arts student, I also took courses all across campus (from GRS to COM to ANTH to PSYC to CHIN to ED-D and many more) and volunteered with Students for Literacy, Peer Helping, and other organizations. My co-op work terms lead me to science facilitation at the Telus World of Science, TRIUMF (a laboratory for particle and nuclear physics), and to the Herzberg Institute for Astrophysics. I am also an award-winning playwright (FIND, IGNITE 2009, 2012) and fiction writer (Martlet, 2010). I have many interests. :)
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|"Konstantin Boudnik (Updated) (JIRA)" <j...@apache.org>
|[jira] [Updated] (HDFS-1762) Allow TestHDFSCLI to be run against a cluster
|Wed, 05 Oct 2011 03:04:35 GMT
[ https://issues.apache.org/jira/browse/HDFS-1762?page=com.atlassian.jira.plugin.system.issuetabpanels:all-tabpanel ] Konstantin Boudnik updated HDFS-1762: ------------------------------------- Attachment: HDFS-1762.mapreduce.patch HDFS-1762.hdfs.patch HDFS-1762.common.patch These are patches for all three subprojects for 0.22 branch. I will create separate tickets in a bit. They should be mostly good for 0.23 except the ant build part which needs to be re-worked for maven. > Allow TestHDFSCLI to be run against a cluster > --------------------------------------------- > > Key: HDFS-1762 > URL: https://issues.apache.org/jira/browse/HDFS-1762 > Project: Hadoop HDFS > Issue Type: Test > Affects Versions: 0.22.0 > Reporter: Tom White > Assignee: Konstantin Boudnik > Attachments: HDFS-1762-20.patch, HDFS-1762.common.patch, HDFS-1762.hdfs.patch, HDFS-1762.mapreduce.patch > > > Currently TestHDFSCLI starts mini clusters to run tests against. It would be useful to be able to support running against arbitrary clusters for testing purposes. -- This message is automatically generated by JIRA. If you think it was sent incorrectly, please contact your JIRA administrators: https://issues.apache.org/jira/secure/ContactAdministrators!default.jspa For more information on JIRA, see: http://www.atlassian.com/software/jira
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Whoever said Love is the universal language never managed a multi-national gay porn studio. Frequently our poor Hungarian hunks are at a disadvantage due to the language barrier. So, in this edition we combined three of our Magyar Freshmen together to enable more lively interaction.
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Video: AVC at 7 818 Kbps,Aspect: 1440 x 810 (1.778) at 29.970 fps
Audio: AAC at 128 Kbps
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Founded in 1933 as the University of Kansas City (UKC) opened as a private college with 260 students. Today, UMKC serves over 14,000 students and has a living alumni base of more than 120,000 graduates.
UMKC has staked a name for itself as a top university with nationally and internationally ranked programs, a place where the arts and culture valued and cultivated, and where community service and scholarship meet alumni and civic pride to make the city and region a better place.
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What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
Calendar Report issue: It will jump back to November month and not to december when clicking Previoust buttonAsked by wisali on January 06, 2014 at 07:41 AM
Is there anyway to edit the css for the calendar report?
I am trying to display the iframe on my site and the problem is that it is not displaying properly
Also there is one more issue if you click to go back to previous month for some reason it jumps from Jan 2014 to Nov 2013 rather than Dec 2013, but if you click to go to next month from Nov 2013 it takes you to Dec 2013
Please see the example below
if i add this as an iframe to my site the arrows to go to next month/year or previous month/year are too close and as there is no text to explain what they are i would like to add my custom css or image to make sure it is clearer ?
Can you please advise if it is possible
This is a re-post of a comment on How to Create a Calendar Report?
Unfortunately it is not possible to change the layout of the report by injecting CSS codes.
Now, regarding to the issue of jumping to November when clicking on the back button I have already submitted a ticket to our dev team so that they can find a prompt solution to this issue. We will inform you via this thread if there is any update on this matter.
Thanks for you understanding
The bug has been fixed


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|Justin Sanders <jus...@justinjas.com>
|Re: Querying by date range when using TimeUUIDType ColumnFamily?
|Tue, 27 Apr 2010 19:50:37 GMT
You're going to have to build TimeUUIDs based on the date range you are scanning. Problem is most UUID libraries build version 1 UUIDs based on the current time. I was able to get this working in Python by changing the library to allow me to pass in a time. This isn't safe for creating unique UUIDs but works fine for doing slices. Justin On Tue, Apr 27, 2010 at 11:28 AM, Ed Anuff <firstname.lastname@example.com> wrote: > Assuming a ColumnFamily with a CompareWith of TimeUUIDType, is it possible > to call get_slice with an arbitrary date range? How would valid values for > the start and finish attributes of the slice range be constructed? > > Thanks > Ed >
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A Harker Heights man is in Bell County Jail in lieu of $1 million bond in connection with a January robbery in which the victim was hit in the head with a hammer.
Judge Garland Potvin arraigned Vance Blaine Luna, 24, on a charge of aggravated robbery Friday. Potvin said the high bail amount was set because Luna posed a flight risk.
Luna is accused of helping Carlos Derrell Collins Jr. escape from an apartment after Collins attacked another man with a hammer Jan. 14.
According to an arrest affidavit, the victim met Collins, whom he knew as "Steve" on Craigslist. The victim said he and Collins were lying naked on a bed when Collins struck him in the back of the head with an unknown object, the affidavit stated.
The victim said he blacked out "a little" and Collins ran off. Police recovered a hammer from the bedroom where the attack occurred. The victim later identified Collins as "Steve."
According to a second arrest affidavit, the hammer had the words "SPC Luna" carved on the handle. Luna's ex-girlfriend later told police Luna said he picked up Collins after the robbery, according to an arrest affidavit.
When questioned by police, Luna said they planned to rob the victim, and also said Collins only hit the victim once "because the hammer bounced out of his hand."
Collins was arrested and charged with aggravated robbery Jan. 19, and is in Bell County Jail on $1 million bond.
Earlier statements from the Killeen Police Department mentioned they wanted to "speak with" Collins regarding a homicide on Anderson Avenue. On Jan. 15, police found 32-year-old Sean William Schuman dead in his apartment. An autopsy determined Schuman died from blunt-force injuries to the head.
Speaking Friday, a KPD spokeswoman said she could not comment on whether Collins and Luna were suspects in Schuman's death, which is under investigation.
In an unrelated incident, Potvin arraigned Alfonza Loren Rudolph, 31, on a charge of fraudulent use of identifying information. Rudolph's bail was set at $20,000.
Area police reports indicated:
- A burglary of a habitation was reported at 12:45 a.m. Thursday in the 200 block of Bryce Avenue.
- A burglary of a habitation with forced entry was reported at 3:47 p.m. Thursday in the 2100 Block of Robert E. Lee Drive.
- A burglary of a habitation with intent to commit another offense was reported at 5:29 p.m. Thursday in the 2900 block of Illinois Avenue. The victim said her daughter's father broke into the residence and assaulted her.
- A burglary of a habitation was reported at 6:58 p.m. Thursday in the 4000 block of Littlerock Drive.
- A burglary of a habitation with forced entry was reported at 10:30 p.m. Thursday in the 3500 block of Ida Drive.
- Criminal mischief was reported at 1:20 a.m. Friday in the 2900 block of East Central Texas Expressway. An unknown person damaged a black 2003 Saturn Ion.
- A theft under $50 was reported at 1:03 a.m. Thursday in the 1400 block of Lowes Boulevard.
- Fraudulent destruction, removal or concealment was reported at 11:59 a.m. Thursday in the 1400 block of Lowes Boulevard.
- A theft, misdemeanor, was reported at 1:37 p.m. Thursday in the 600 block of North Gray Street.
- A forgery was reported at 2:50 p.m. Thursday in the 1100 block of Lowes Boulevard.
- An assault with bodily injury was reported at 5:55 p.m. Thursday in the 1000 block of Conner Court.
- An assault with bodily injury was reported at 7:40 p.m. Thursday in the 3500 block of Turner Avenue.
- Criminal mischief was reported at 9:30 p.m. Thursday in the 2900 block of East Central Texas Expressway.
- Possession of drug paraphernalia was reported at 10:07 p.m. Thursday at the intersection of Martin Luther King Jr. Boulevard and Zephyr Road.
Harker Heights
- A burglary of a residence was reported at 8 a.m. Thursday in the 1400 block of Summer Glenn Drive.


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In their own words…
"Welcome to Nottingham's only rooftop bar & restaurant with unique panaramic views that rival any of Europe's leading capital cities.
With its mesmerizing selection of continental wines, beers and spirits alongside simple, honest British food Salwater aims to please. Top it all of with an awesome weekly entertainment bill and you've got a venue where the feeling of summer never ends."
Continue reading to see reviews about Nottingham's Saltwater and leave your own


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Free Samples, Coupons and Competitions for Women in the UK – Get your Free Stuff Here.
August 6, 2015 | Videos
This dog is the cutest, but he's also very furry. You won't believe what this owner has to do to ensure his pet doesn't shed all over his house.


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Kate Moss VELVET HOUR by EDT SPRAY 3.4 OZ for WOMEN
Beautiful and distinctive, TrendToGo brings you another fine fragrance from Kate Moss ALL Fragrances are 100% Guaranteed Authentic. Add it to your cart now: KATE MOSS VELVET HOUR by Kate Moss EDT SPRAY 3.4 OZ for WOMEN Gender: Women's
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Online Training Programs DVDs, Videos, & more
All Categories A-Z
We're open 8:30 - 5:00 E.S.T. M-F 1-800-975-7640
About Online Training Programs
Online Regulatory Compliance training is becoming popular with facilities to deliver and train many of their employees because online Safety training can be used virtually anywhere, at any time. This is a very useful tool for companies with a number of locations, has hundreds of employees dispersed in different location or work odd shifts. Our online training system gives you and your employees unlimited access, as well as the best and most up to date safety information available. Our library is one of the largest libraries of full-motion video-based Safety and Regulatory Compliance courses in the industry, with over 115 titles to choose from, many of them in Spanish as well as English. You will have access to as many courses in the library as you like, with no set-up, library or subscription fees. There is a feature for employee training tracking. You have an option of four payment plans to choose from, including monthly "Pay-As-You-Go" and "All You Can Eat" (unlimited access).
OSHA's Position On Online Training Programs
Due to the emergence of online training popularity, more and more companies are making a move in that direction, in order to facilitate their training programs. Online Safety Training can afford multiple advantages. One, online training eliminates the need to provide a separate training area along with the necessary upkeep and storage of training videos, etc. The other main advantage is that training can be conducted from virtually any location, at any time. For example, the federally mandated hazardous waste training (for appropriate work areas) [OSHA 1910.120] may be conducted at any location that houses an Internet connection.
This can also be an exceptional benefit for companies that have more than one physical location or employees working at a number of different locations. This way, multiple sets of training materials or the transportation of training materials can be avoided. The use of online safety training can also provide the advantage of not having to continually update your company's training materials, as adjustments are made to OSHA regulations. Check out our huge inventory of available online training materials and see which ones are applicable to your organization. Whether it's Fire Safety Training [OSHA 1910.155] or any one of the other mandatory OSHA topics, our Online Safety Training subjects can become a valuable training resource.
These highly affordable training tools can be directly purchased on our website and should be considered as a cost-effective training option. Please feel free to give us a call with any questions that you may have. Let us assist you in meeting OSHA's mandatory training requirements.


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Building Energy Rating
Renewable Energy
Free Home Energy Manager App
Sustainable Energy Awards
Energy in Education
Primary School workshops
One Good Idea Project
Better Energy Communities
Grants Available
Energy Research Portal
Better Energy Homes
Electric Vehicles Grant Scheme
Energy Data Portal
Energy Show
Energy Awards
Energy use increased across all major sectors despite good progress on renewable energy and energy efficiency
23rd November 2016: The Sustainable Energy Authority of Ireland (SEAI) released Ireland's official national energy statistics today, which show significant growth in energy use across all sectors of the economy in 2015. The report, Energy in Ireland 1990-2015, highlights nearly 5% growth in energy use which is the first significant growth since 2010. In addition, energy-related CO2 emissions increased by 6%. This increase in energy use and CO2 emissions is in the context of strong economic growth in 2015.
Commenting on the new analysis of energy trends in Ireland, Jim Gannon, SEAI Chief Executive, said: "The publication of these energy figures is a timely and pertinent analysis of Ireland's energy usage following the conclusion of COP22 in Marrakech last week and Ireland's ratification of the Paris Agreement earlier in November. We are seeing good progress on renewable energy and energy efficiency, however, this needs to be further accelerated to keep pace with higher economic activity and demand for energy. The Government is already responding to this challenge with significant increased funding committed to energy efficiency and renewable heat in Budget 2017. This allows us to build on the progress to date and continue to decarbonise our energy system which will reduce costs, improve energy security and reduce environmental impact."
Jim Gannon continued: "In 2015 the average household emitted 5.5 tonnes of CO2of which 61% came from direct fuel use in the home and the remainder from electricity use. The transition to a sustainable, low-carbon energy system requires the participation of citizens and communities in both decision-making and action. It is vital that we have an informed debate about the choices for Ireland as we move to a low-carbon economy. SEAI is committed to the provision of timely, robust and transparent data to ensure that policy development, decision-making and our energy transition is based on high quality data and evidence."
The full report Energy in Ireland 1990-2015 and accompanying infographic can be downloaded below:
Energy in Ireland 1990 - 2015.pdf (size 2.5 MB)
Energy in Ireland Infographic 2015.pdf (size 134 KB)
For further information contact:
Luke McDonnell, Drury|Porter Novelli
085 7127243
About SEAI:
The Sustainable Energy Authority of Ireland (SEAI) has a mission to play a leading role in the transformation of Ireland to a society based on sustainable energy structures, technologies and practices. SEAI is partly financed by Ireland's EU Structural Funds Programme co-funded by the Irish Government and the European Union.
Head Office: Sustainable Energy Authority of Ireland, Wilton Park House, Wilton Place, Dublin 2. t: +353-1-8082100 f:+353--1-8082002
General Consumer enquires: Lo-Call 1850 376 666 email@example.com
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By using this website, you consent to the use of cookies in accordance with the SEAI Cookie Policy.
For more information on cookies see our cookie policy.


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Is your childs behaviour worrying you?
Parents face many challenges in raising their children to be safe, happy and well adjusted. It always hurts to see children struggle and no one ever wants to experience that. Most parents would move mountains to ensure their children are getting a good start in life.
How well is your child coping?
Frank Caruso and his passionate team have created Carusos Kids Calm & Behaviour to help your child maintain a healthy behaviour and temperament. This Australian made formula helps soothe and calm nerves and assists with the management of stress, tension, irritability and mild anxiety.
Suitable for children from two to teenage years, delicious tasting Carusos Kids Calm & Behaviour contains a special blend of French Marine Pine Bark, Magnesium, Vitamin C, Zinc and Silicon. It may assist behaviour, whilst providing key nutrients to help support stress, mild anxiety, nervousness, excitability, irritability, attention span, sleep problems, brain development and immunity.
Carusos Kids Calm & Behaviour has been developed to help your childs behaviour. French Marine Pine Bark may help supports healthy, good behaviour. This will help children to better focus their attention. Magnesium may also have a positive influence on behaviour. Whilst Zinc may help support a childs behaviour.
Stress and mild anxiety
Some children can become overwhelmed with stress and mild anxiety. So it is just as well Carusos Kids Calm & Behaviour contains Magnesium. This nutrient is used for relieving stress because stress increases the bodys need for Magnesium. As such, Magnesium has a calming effect on the body. Another reason why Magnesium is an important ingredient is because low Magnesium levels in the body increase ones sensitivity to stress. Low Magnesium levels may also lead to agitation and mild anxiety. Plus, low levels of Zinc are associated with behavioural and mild anxiety problems in children.
Excitability, irritability and sleeping problems
As Magnesium has calming effects, it also helps with the symptoms of excitability and irritability. And if your child has low levels of Magnesium they may be suffering from nervousness, agitation, mild anxiety, sleep problems, fatigue and tiredness.
Nervous system and brain
If your child has low levels of Magnesium in their body this may result in their decreased attention span. Magnesium is essential for regulating the central nervous system and calming it down. Whilst Zinc is involved in the functioning of the central nervous system and brain development.
Immune system
Carusos Kids Calm & Behaviour helps maintain and support the immune system.
Antioxidant power
Carusos Kids Calm & Behaviour is a powerful antioxidant formula. French Marine Pine Bark helps with antioxidant pathways involved in childrens behaviour. It also helps total antioxidant status in children. Vitamin C, Zinc and Silicon are also antioxidants.
Please follow dosage instructions depending on childs age or as advised by your Healthcare Professional.
Children 2-3 years: mix 1 scoop (1.25g) with approximately 100mL of your childs favourite drink (water, 100% Natural fruit juice etc..) and drink immediately, once a day.
Children 4-8 years: mix 2 scoops (2.5g) with approximately 200mL of your childs favourite drink (water, 100% Natural fruit juice etc..) and drink immediately, once a day.
Children 8 years and over: mix 4 scoops (5g) with approximately 200mL of your childs favourite drink (water, 100% Natural fruit juice etc..) and drink immediately, once a day.
Active Ingredients. Each 2.5g dose(2 scoops) of powder contains:
Pinus pinaster stem bark outer Oligopin (French Marine Pine Bark)20000mg (20g)
Magnesium Citrate 440.9mg
Equiv. to Magnesium 71.25mg
Magnesium Phosphate 344.9mg
Equiv. to Magnesium 71.25mg
Magnesium Aspartate Dihydrate
Equiv. to Magnesium 7.5mg
ETotal Magnesium 150mg
Silica Colloidal Anhydrous 247.5mg
Equiv. to Silicon 115.5mg 115.5mg
Ascorbic Acid (Vitamin C) 25mg
Zinc Oxide 9.33mg
Equiv. to Zinc 7.5mg
Contains Zinc which may be dangerous if taken in large amounts or for a long period of time. Always read the label. Use only as directed. If symptoms persist see your Healthcare Professional.
Vitamins supplements should not replace a balanced diet
Delivery Costs
Standard Parcels (under 3kg) over $149 = FREE!
Parcels under 3kg & under $149 = $7.95
Parcels over 3kg or bulky over $149 = $12.95
Parcels over 3kg or bulky under $149 = $16.95
We offer Express Shipping for parcels up to 5kg
Parcels under 1kg = $12.95
Parcels between 1-5kg = $19.95
- Express shipping is not available to PO Box addresses.
- Signature is required on delivery.
Your Discount Chemist use Australia Post to deliver standard parcels and Star Track for express shipping requests.
Delivery Time
Parcels arrive within 3-7 business days.
Express shipped parcels arrive within 1-3 business days (this may be slightly longer for remote country areas).
International Shipping
We offer fast flat rate express shipping all over the world based on 8 zones - these rates apply to all non-bulky parcels under 2kg*.
|Delivery Times*
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2-3 days
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3-4 days
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3-4 days
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3-5 days
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4-6 days
*All orders over 2kg and or bulky orders will be charged at different rates which will be quoted at checkout.
*Estimated delivery time in business days. Excludes time in customs if applicable and may be subject to delay due to causes outside of Australia Post's control.
Click here for more detailed information regarding shipping.


text: I've got an extra Freefly Movi M15 that has just been hangin aboot and while I could sell it and it will end up on ebay at some point or another if I can't find a worthy trade, I'll also update the thread with a price and some photos when that time comes but for now I'd be interested in trading for an Inspire Pro (would prefer a dual remote system but if you got a sweet single op setup we can chat or especially if you got a black edition) I've traded on "gentlemens handshakes" before and never had a bad experience I always prefer to chat on the phone first this way we can both determine a better judge of character. M15 is fully updated running flawlessly on 5.0.2 firmware (dubbed Akira; offers hyper stabilization, time lapse, hyper lapse and a couple other cool features) You get m15 with a couple batteries,2 sets of camera rails (new and old design) Rod mount, extra toad in the hole and a ninja star. No pelican case and I got no charger for it (I just used my standard rc charger)


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The Newsquest-owned Herald & Times Group has merged the picture desks of its three Glasgow-based newspapers, the Herald, Sunday Herald and Evening Times.
Picture desk staff from the newspapers will this week start work in the new centre, overseeing all non-text content across print and the web.
The desk will be run by former Evening Times picture editor John Young, who was appointed group multimedia editor in April.
The desk will also be responsible for the gathering and organisation of user-generated content submitted to the Herald Group's series of community websites.
In addition to the picture desk merger, a combined website for the Herald and Sunday Herald newspapers will be launched later in the year. The former deputy editor of the Sunday Herald, David Milne, has been appointed online editor for the new site.
[lnk|http://www.featuresexec.com/publications/info_features.php?pubid=1611|_self|The Herald]
[lnk|http://www.featuresexec.com/publications/info_features.php?pubid=1781|_self|Sunday Herald]
[lnk|http://www.featuresexec.com/publications/info_features.php?pubid=3266|_self|Evening Times]


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Click here for a printable version of my resume.
Richmond, Virginia USA
An experienced information technology professional that develops custom Web, mobile, and Apple TV applications, as well as the databases and APIs that they are based on.
• More than 20 years of Information Technology experience serving as a developer, software engineer, and consultant.
• Bachelor's Degree in Computer Science, with a concentration in Information Systems.
• Exceptional database-related skills, including design (data requirement analysis, data modeling, normalization), development (scripting, stored procedures, triggers, user-defined functions, and complex queries), and administration (performance tuning and optimization, security and authorization, backup and recovery, installation, and maintenance).
• Proven ability to document work, and to provide clear and professional technical evaluations and specifications.
• Ability to understand complex business requirements, and translate them into technical designs.
• Published author, with excellent written and oral communication skills.
Database Systems:
• Amazon Aurora
• SQL Server
• Oracle
• MySQL
• Airtable
• FileMaker
Programming Languages:
• Swift
• Xojo
• ColdFusion
• JavaScript
ERP & Accounting Systems:
• Lawson
• Solomon
Special Skills:
• Development of Apple TV apps using TVML, TVJS, TVMLKit, and Swift.
• Extensive experience with Amazon Web Services (AWS), including Elastic Compute Cloud (EC2), Relational Database Service (RDS), and S3 (Simple Storage Service).
• Design and development of specialized Web applications, including APIs, custom search engines, spiders / bots.
• Integration with payment processing systems, including PayPal, Authorize.net, and Stripe.
• Integration with FedEx and UPS shipping systems.
Custom Software Developer
Richmond, Virginia
May 2016 to Present
I specialize in developing custom software for businesses when there simply "isn't an app for that." That includes Web, mobile, and Apple TV applications - many of which are powered by databases and APIs.
Founder / Developer
Richmond, Virginia
May 2016 to Present
DailyOrbit is an alternative search engine that I originally developed in 1997. It had been relaunched to meet the needs of today's mobile-centric world.
Independent App Developer
Richmond, Virginia
June 2015 to May 2016
Developed custom apps for small businesses, as well as commercial apps.
• Developed an Apple TV app for Broadcastify, the world's largest source of public safety, aircraft, rail, and marine radio live audio streams.
• Developed ViewPoint, a Xojo-based Apple TV application server framework. ViewPoint is used to dynamically generate TVML and TVJS files that power Apple TV apps, as well as static assets such as image and audio files.
• Developed Keplr TV, an Apple TV app that provides access to classic science fiction movies.
• Developed The Unlikely Story, an Apple TV app that tells the story of artist William Girven, and his journey from homelessness to prominence.
• Developed an Apple TV app for Carbon Leaf, a Richmond, Virginia-based band.
• Developed Mandala Meditations, an Apple TV app that provides access to meditation videos.
• Developed an iPhone / iPad app that alerts first responders to emergencies and provides critical information (hazardous materials warnings, directions, etc) regarding the location involved.
• Developed ICD-10 Research, an iPhone / Apple Watch app for healthcare professionals to easily locate ICD-10 medical codes.
• Developed Full Disclosure, an iPhone app that provides access to the public disclosures that have been filed with the U.S. Securities and Exchange Commission (SEC).
• Developed Gov Opps, an iPhone app that helps users discover employment opportunities that are available with federal, state, and local government agencies across the United States.
Developer Evangelist
San Francisco, California
June 2015 to May 2016
As Developer Evangelist, I was tasked with helping developers use Airtable's API. Created technical content for Airtable's blog and community forum, hosted webinars, developed demo applications that use Airtable's API, assisting users, and more.
Independent Database Consultant
Simple Database Solutions
Midlothian, Virginia
April 2008 to June 2015
Worked as an independent consultant that specialized in the FileMaker platform. My core service was developing custom database solutions. I also provided FileMaker integration and training services. My clients ranged from small businesses to government agencies to Fortune 50 organizations, and included the Library of Congress, Siemens, CarMax, the University of Virginia, and many more.
Senior Developer
Target Marketing
Richmond, Virginia
November 2006 to April 2008
Designed and developed custom database applications for this value-added promotional products company. Applications developed included transactional fulfillment and inventory control systems, project and job management tools, as well as telemarketing-related solutions.
• Developed a custom fulfillment and inventory control system in FileMaker. The inventory system is transactional, with full support for commits, aborts, and rollbacks.
• Developed custom Web-based solutions using the FileMaker API for PHP.
• Integrated FileMaker-based applications with the company's SQL Server-based e-commerce system ("Nova") and accounting system ("Solomon").
• Maintained the company's multiple FileMaker Server instances. Responsibilities included server monitoring, backup scheduling, etc.
• Maintained legacy applications developed in FileMaker Pro 5 and 6.
• Managed large scale SQL Server 2000 to SQL Server 2005 upgrade project.
Independent Database Consultant
Tim Dietrich
Dulles, Virginia
August 2006 to November 2006
Worked as an independent database consultant to migrate a complex FileMaker Pro-based inventory management system from the FileMaker 7 platform to the FileMaker 8 platform. The project involved redesigning the application to take full advantage of the latest FileMaker technology and feature set, as well as consolidating the original multi-file solution into fewer physical files, and implementing a data separation model.
SQL Developer
Sprint / DHSoft Inc.
Herndon, Virginia
May 2006 to August 2006
Worked with a team of developers and business analysts to support databases that distribute targeted customer data to a number of CRM applications. This data is used to deliver and track the effectiveness of proactive, reactive, and cross-sell offers.
• Automated the load, validation, and processing of customer response data from external vendors.
• Developed SQL jobs to migrate data from production servers to development and testing environments.
• Worked with large SQL databases (millions of records), in a multi-database (SQL Server and Oracle) environment.
• Helped establish database standards and best practices for the SQL Server development team.
Database Consultant
Dulles, Virginia
May 2000 to April 2006
Served as a database consultant and developed custom applications for clients in a wide variety of industries. Services provided included database design, development and administration, web application development, and related services.
• Developed a lead generation and processing system for a marketing firm that services the mortgage industry. The database included detailed personal and financial information on more that 8 million homeowners.
• Developed a custom Web content management system for a prestigious private university.
• Developed a custom inventory management system for a jewelry wholesale company.
• Developed a custom business system for a skilled labor employment agency.
• Developed a custom telemarketing solution used to track sales, prospects, and employee compensation for a call center operation.
• Developed a system used by a large manufacturer of automotive products to manage their employee incentive program.
Senior Analyst
Bell Industries, Inc.
Richmond, Virginia
August 1999 to May 2000
Developed a comprehensive, integrated e-commerce strategy for this multi-regional, multi-industry corporation. Bell's primary business is Bell Tech.logix, a provider of integrated technology solutions for large and medium-sized organizations.
• Designed, developed, documented, and implemented various browser-based applications, including sales and management tools, reporting tools, etc. Applications were developed using FileMaker Pro, ColdFusion, Microsoft SQL Server, and DB2/400.
• Developed a browser-based reporting system that integrated with the corporate ERP system.
• Developed a data archiving system to provide authorized corporate users with access to historic sales and financial data previously stored in the corporation's mainframe system.
• Managed a team of web developers and designers, and established coding and documentation standards, workflow procedures, etc.
Software Engineer
Xperts, Inc.
Richmond, Virginia
February 1999 to August 1999
Developed custom database solutions for clients in various industries. Xperts is a Richmond-based computer consulting firm.
• Developed a browser-based project management tool for The Weather Channel.
• Developed a FileMaker-based product catalog for the lighting division of General Electric.
• Assisted Web development team with database-related issues, including modifications to database schemas and development of SQL scripts, stored procedures, queries, etc.
Web Developer / Integrator
Kent, Connecticut
September 1998 to February 1999
Developed e-commerce solutions and Web-based applications for this high profile computer products company. Outpost.com (formerly "Cyberian Outpost") is a global Internet retailer of computer products. Work included:
• Used Broadvision One-To-One application system to build dynamic Web pages that were personalized to correspond to a user's profile.
• Developed client-side Javascript to validate forms and enhance the user's overall experience.
• Developed complex SQL queries to integrate data from Oracle databases.
• Designed and developed a FileMaker / browser-based project management and bug tracking system for use by the development and creative services teams.
Bachelor of Science / Computer Science
Information Systems Concentration
Towson University
FileMaker Certified Developer
Versions 9, 10, 11, 12, 13
FileMaker, Inc.
Microsoft SQL Server Certification
Northern Virginia Community College
2017 ICD-10-CM Code Reference Guide for Mental, Behavioral and Neurodevelopmental Disorders: Codes F01 thru F99
July 2016
ICD-10-CM Code Reference Guide: Books 1 - 21
March 2015
FileMaker Pro 13 Absolute Beginner's Guide
Que Publishing
December 2014
The NeXT Computer
Association for Computing Machinery
ACM SIGSMALL/PC Notes, Volume 15 Issue 3
August 1989
Keplr TV
An Apple TV app that provides access to classic science fiction movies.
The Unlikely Story
An Apple TV app that tells the story of artist William Girven, and his journey from homelessness to prominence.
Carbon Leaf
An Apple TV app for Richmond, Virginia-based indie-rock band Carbon Leaf. The app includes videos of the band's live performances, interviews with band members, music, and more.
ICD-10 Research
An iPhone app that makes it easy for healthcare professionals to locate ICD-10-CM (diagnosis) codes.
Mandala Meditations
An Apple TV app that provides access to mandala animations set to music. The app is used as a means of relaxing mind and body.
Full Disclosure
An iPhone app that provides access to the public disclosures that have been filed with the U.S. Securities and Exchange Commission (SEC).
Gov Opps
An iPhone app that helps users discover employment opportunities that are available with federal, state, and local government agencies across the United States.
A class that maps commonly used FileMaker functions to the Swift programming language.
Two-factor authentication (2FA) for the FileMaker database platform.
A Xojo Web application that makes it easy to serve up files that are stored in FileMaker containers.
Framework for creating simple REST APIs for FileMaker databases.
A comprehensive auditing solution for the FileMaker database platform.
Generates barcodes and QR codes in FileMaker databases.
Displays event data that is stored in FileMaker databases in a calendar format.
Allows creation and modification of drawings directly from within FileMaker.
An HTML / text editor for use in FileMaker-based solutions.
Generates interactive maps based on addresses in FileMaker databases.
Captures signatures and stores them in FileMaker databases.
An award-winning sync framework for the FileMaker platform.
Publish data in FileMaker databases to the Web without writing any code.
Collects data from Wufoo online forms and stores it in FileMaker databases.
A Xojo-based framework for creating RESTful APIs for FileMaker databases.
A PHP-based solution for accepting credit card payments via the Stripe and storing them in a FileMaker database.
An extension to the FileMaker API for PHP.
A Xojo-based framework for creating RESTful APIs.
An alternative, open source framework for developing secure, public-facing, scalable Web sites and applications using Xojo.
FileMaker Excellence Award
2014 FBA Development of the Year
FileMaker, Inc. An Apple Subsidiary.
Xojo Developer Conference (XDC) 2016
October 2016
• Xojo's Role in the API Economy
• Powering Apple TV Apps With Xojo
REST Fest
September 2016
• APIs That Meet the Needs of Citizen Developers
• API Assistant
Testimonials are available here.
References are available upon request.
Email: firstname.lastname@example.com
GitHub: github.com/timdietrich


text:
Nassau County Evacuation
August 27, 2011 by USA Post
Nassau County Evacuation, For the first time Nassau County has ordered some North Coast residents whose properties are close to Manhasset Bay on Long Island Sound to evacuate at 5 pm Saturday.
The areas are mainly in the Port Washington peninsula, which juts north into LI Sound. Hurricane Irene is expected to make landfall on Sunday near Long Beach on the south coast with a population around 50,000.
Jon Kaiman, Supervisor of the Town of North Hempstead, said residents living in areas of storm surges before Manhasset Bay must go. Areas include Baxter Estates, Manorhaven, Manhasset Isle and parts of Port Washington North, overlooking the bay.
Kaiman said that residents who go "where water has flooded his home" in the past. Edward Mangano, Nassau County Executive, on Friday ordered the transfer, along with other evacuations. Unlike Long Beach, the northern coastal areas generally consist of single-family homes, not apartment buildings.
Newsday, the Long Island newspaper, delivered its sections have Sunday with the Sunday paper and said his website would be free. The New York Times said it would issue its Sunday paper.
In Great Neck, a residential area with about 45,000 people in North Henpstead, city officials took precautions. Park District of Great Neck removed floating docks at the marina at Steppingstone Park and said that near the outdoor pool Parkwood at noon. The employees were then to secure outdoor chairs and lounges.
Truck of the Long Island Power Authority (LIPA) and Verizon could see the preparation of emergency operations.
Meanwhile, many of the synagogues in Great Neck, said he would proceed with the regular Sabbath services. Temple Israel, a congregation of 930 conservative members, sent an email saying clergy member services will be held, including a bar mitzvah on Saturday. Rabbi Seth Adelson also published a commentary criticizing anyone could infer that Hurricane Irene, or any other natural phenomenon could be inferred as a sort of divine punishment.
"The weather and the destruction it can cause, it depends on the mood of God or, indeed, our behavior," Adelson wrote.
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