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Cell Reports, Volume 3, Issue 2, 350-358, 14 February 2013
Fungicidal Drugs Induce a Common Oxidative-Damage Cellular Death Pathway
Peter Belenky1, 2, 3, Diogo Camacho1, 2, 3 and James J. Collins1, 2, 3, 4, 5, ,
1 Howard Hughes Medical Institute, Boston University, Boston, MA 02215, USA
2 Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA
3 Center for BioDynamics, Boston University, Boston, MA 02215, USA
4 Boston University School of Medicine, Boston, MA 02118, USA
5 Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
Graphical Abstract
► Fungicide-dependent reactive oxygen species production leads to fungal cellular death ► The TCA, ETC, and RAS/PKA pathways are involved in fungicide-induced cellular death ► Antifungals elevate mitochondrial activity, the AMP/ATP ratio, and sugar production ► DNA damage plays an important role in fungicide-induced cellular death
- Amphotericin, miconazole, and ciclopirox are antifungal agents from three different drug classes that can effectively kill planktonic yeast, yet their complete fungicidal mechanisms are not fully understood. Here, we employ a systems biology approach to identify a common oxidative-damage cellular death pathway triggered by these representative fungicides in Candida albicans and Saccharomyces cerevisiae. This mechanism utilizes a signaling cascade involving the GTPases Ras1 and Ras2 and protein kinase A, and it culminates in death through the production of toxic reactive oxygen species in a tricarboxylic-acid-cycle- and respiratory-chain-dependent manner. We also show that the metabolome of C. albicans is altered by antifungal drug treatment, exhibiting a shift from fermentation to respiration, a jump in the AMP/ATP ratio, and elevated production of sugars; this coincides with elevated mitochondrial activity. Lastly, we demonstrate that DNA damage plays a critical role in antifungal-induced cellular death and that blocking DNA-repair mechanisms potentiates fungicidal activity.
A rapid rise in immunocompromised patients over the past five decades has led to increasing incidence of systemic fungal infections. Despite current treatment options, the morbidity and mortality rates associated with fungal infections, particularly those of Candida species, remain high (Ostrosky-Zeichner et al., 2010).
The polyene amphotericin B (AMB), introduced in the late 1950s, was the first widely used antifungal (AF) drug (Ostrosky-Zeichner et al., 2010). Due to its strong hydrophobicity, AMB penetrates the fungal membrane and binds to ergosterol, leading to membrane damage. Azoles, a second class of AFs, became available in the 1980s and act by inhibiting ergosterol biosynthesis to induce the accumulation of a toxic methylated sterol that stops cell growth (Ostrosky-Zeichner et al., 2010). Although azoles tend to be fungistatic, some azoles such as miconazole (MCZ) can be fungicidal under certain conditions and formulations (Thevissen et al., 2007). Unlike AMB and MCZ, the primary targets of the synthetic AF ciclopirox olamine (CIC) are not fully understood, though some evidence indicates that CIC acts by affecting DNA repair or directly inducing DNA damage (Leem et al., 2003).
Recent work with select AFs has indicated that AF-induced cellular death may follow apoptotic or necrotic pathways that involve the production of reactive oxygen species (ROS) (Phillips et al., 2003; Thevissen et al., 2007) and the modulation of central metabolism (González-Párraga et al., 2011; Yan et al., 2007). However, a comprehensive genetic and metabolic mechanism of AF-induced cellular death remains elusive.
Recent systems biology work from our lab, focused on bacterial cell death, identified a common mechanism of antibiotic action that involves a genetic and metabolic cascade that ultimately results in the formation of toxic ROS (Dwyer et al., 2007; Kohanski et al., 2007, Kohanski et al., 2008). Aspects of this mechanism have been harnessed to potentiate currently available antibiotics and combat the development of resistance (Dwyer et al., 2009; Kohanski et al., 2007, Kohanski et al., 2010; Lu and Collins, 2009). Similar approaches to AF mechanisms could provide important, meaningful insights and identify possible means to improve current treatments.
In this work, we apply a systems biology approach to identify mechanisms by which the aforementioned AFs—AMB, MCZ, and CIC—lead to fungal cellular death. We find that, despite their different primary modes of action, all three classes of fungicidal drugs induce a common oxidative-damage cellular death pathway in Saccharomyces cerevisiae and Candida albicans that involves alterations to cellular metabolism and respiration, culminating in the formation of lethal ROS.
Fungicide-Dependent ROS Production Leads to Fungal Cell Death
Based on previous work that supports the cidal role of ROS production in many modes of fungal death (Breitenbach et al., 2005; Henriquez et al., 2008), we hypothesized that ROS production is critical to AF-induced cellular death. We measured the formation of ROS following AF treatment in yeast using the dye 3′-(p-hydroxyphenyl) fluorescein (HPF), which is preferentially oxidized by intracellular hydroxyl radicals into a fluorescent product (Kohanski et al., 2007). We treated exponentially growing wild-type S. cerevisiae and C. albicans in synthetic dextrose complete (SDC) medium with the minimum concentration of fungicide required to achieve at least a 90% reduction in colony-forming units (CFU) after 3 hr of exposure. As a positive control, we also treated cells with H2O2, a potent inducer of hydroxyl radical formation (Perrone et al., 2008). After 1.5 hr of treatment, we found that all tested fungicidal drugs and H2O2 lead to dramatic induction of HPF fluorescence (Figure 1A, 1B, and S1), indicating that all tested fungicidal agents induce the formation of ROS. Conversely, fungistatic drugs added at concentrations 10-fold above the minimal inhibitory concentration or at the maximum soluble concentration did not lead to detectable ROS formation (Figures 1A, 1B, and S1).
To test whether the observed production of ROS contributes to AF-induced cellular death, we treated cells with thiourea, a potent scavenger of hydroxyl radicals in eukaryotic and prokaryotic cells (Kohanski et al., 2007; Touati et al., 1995). We found that exposing exponentially growing S. cerevisiae to 50 mM thiourea for 30 min prior to the addition of AF drugs considerably diminished the toxicity of all three AFs, reducing killing at 3 hr by ∼15-fold for AMB and CIC and by ∼10-fold for MCZ (Figure 1C). These results indicate that ROS production plays a critical role in cellular death following treatment by AMB, MCZ, and CIC.
Identifying a Common Transcriptional Response to AF Treatment
To build a comprehensive model of the yeast response to AFs, we measured changes in global gene expression of S. cerevisiae following treatment with AMB, MCZ, and CIC. To assess the effect of each AF treatment on gene expression, we performed a z-test between experiment and control samples, where the average and SD for the expression of each gene was calculated across a compendium of publically available expression data sets (see Extended Experimental Procedures). We compared the differential gene-expression profile of each treatment at each time point to the no-treatment control to identify the set of genes commonly perturbed by fungicide treatment (Figure 1D). This analysis revealed that 43 genes were commonly upregulated under AF treatment, while 79 genes were downregulated under the same conditions (Figure 1E). To investigate the biological pathways and processes in which these genes are involved, we ran a functional enrichment analysis on Gene Ontology (GO) terms associated with each one of these genes. The analysis was conducted with the Saccharomyces Genome Database's GO Term Finder tool using default parameters. The majority of the commonly downregulated genes are involved in protein synthesis, specifically, ribosomal biogenesis and tRNA synthesis. The 43 commonly upregulated genes fall into six general biological processes: the production of storage sugars, endocytosis, general stress response, osmolarity maintenance, central carbon metabolism, and the RAS/protein kinase A (PKA) signaling pathway (Figure 1E).
Our expression analysis identified the production of storage sugars as a key process upregulated following AF treatment. Specifically, genes involved in glycogen metabolism and the production of the storage sugar trehalose were robustly upregulated after the addition of fungicides (Figure 1E; Document S1. Tables S1–S3). Cellular production of trehalose and glycogen are energy-expensive pathways that consume significant amounts of ATP. Consistent with this, the activation of the trehalose pathway has been shown to increase ATP consumption, mitochondrial enzyme content, and respiration (Noubhani et al., 2009). Thus, the production of these sugars may contribute to increased mitochondrial activity and elevated ROS production.
TCA Cycle and Electron Transport Chain Play Critical Roles in AF-Induced Cell Death
To identify genes critical to AF action, we tested the AF sensitivity of single-gene knockouts identified through our common transcriptional analysis. In total, we tested the AF sensitivities of 81 single-gene knockouts (Document S1. Tables S1–S3) and found that 12 of them had increased resistance to all three AFs (AMB, MCZ, CIC).
Actively respiring, energy-producing mitochondria are the major source of ROS in yeast cells. However, S. cerevisiae respiratory activity is usually repressed under normal growth conditions in the presence of glucose (Hardie et al., 1999), though various stress responses have been shown to upregulate respiratory genes even in the presence of glucose (Gasch et al., 2000). We therefore hypothesized that treatment with fungicides induces a switch from normal fermentative growth to mitochondrial respiration.
To assess the role of mitochondrial respiration in AF-induced cellular death, we first analyzed the expression of mitochondrial genes in S. cerevisiae in response to AF treatment (Document S1. Tables S1–S3). Of particular interest, we found that the gene encoding the rate-limiting step of the tricarboxylic acid (TCA) cycle, citrate synthase-1 (CIT1), exhibited a slight increase in expression across all AF treatments. The induction of CIT1 is a hallmark of stress-induced respiration (Gasch et al., 2000). We found that deleting CIT1, CIT2, or CIT3 dramatically reduced yeast sensitivity to all three AFs when compared to the wild-type S. cerevisiae strain (Figures 2A and S2). To parse out the role of the various citrate syntheses in AF-induced cellular death, we created single and double deletions of CIT1 and CIT2 in the cit3 background. We found that deleting the remaining citrate synthases in this background provides additive resistance to AMB, with the triple mutant requiring more than 8-fold-higher drug concentrations to achieve the sensitivity of the single mutants (Figures 2C). Interestingly, a similar additive effect was not observed with MCZ or CIC, indicating that AMB toxicity is more sensitive to further changes in citrate metabolism than the other two drugs.
We also found that deleting succinate dehydrogenases (SDH1 or SDH2), enzymes that couple the oxidation of succinate to the transfer of electrons to the mitochondrial electron transport chain (ETC) (Chapman et al., 1992), decreased drug susceptibility. Additionally, we found that disrupting the TCA cycle at these two key points reduces the AF-dependent production of ROS (Figure 2B), indicating that this phenomenon involves the TCA cycle.
The TCA cycle produces NADH, which is then fed into the ETC to produce ATP. This activity also leads to the production of ROS as a byproduct of aerobic respiration. We therefore targeted the first committed steps of the ETC by deleting the intramitochondrial NADH dehydrogenase (NDI1) and the external NADH dehydrogenase (NDE1). The NDI1 and NDE1 deletions exhibited increased resistance to all three AF drugs and a concomitant reduction in AF-dependent ROS production (Figures 2A and 2B).
We next sought to assess the mitochondrial activity of AF-treated fungal cells. We utilized the MitoTracker Red dye, which enters the mitochondrial matrix by utilizing the proton motive force and thus labels metabolically active mitochondria in viable cells (Arita et al., 2006; Tomas et al., 2011). We incubated exponential phase S. cerevisiae in glucose-free synthetic complete (SC) media for 30 min and then switched them to SC containing 2% glucose or nonfermentable acetate for 1.5 hr. As expected, we detected approximately 5-fold more MitoTracker fluorescence in acetate-incubated cells (Figure 2D), indicating that the dye specifically labels cells with activated mitochondria. Adding CIC, AMB, or MCZ to glucose-incubated cells increased MitoTracker fluorescence by 10-fold, 60-fold, and 70-fold, respectively (Figure 2D). All three drugs induced considerably more mitochondrial activity than acetate, indicating that AF treatment has a greater impact on mitochondrial activity than normal respiratory metabolism. These results are consistent with our hypothesis that AF drugs induce a shift from fermentative growth to ROS-producing mitochondrial respiration.
RAS/PKA Pathway Is a Key Mediator of AF Toxicity
Having established that AFs induce mitochondrial-dependent ROS production, we next sought to ascertain the signaling events that lead to these metabolic changes. The RAS/PKA pathway, consisting of two GTPases (Ras1 and 2) and three PKA isoforms (Tpk1-3), has been shown to respond to cellular stress by inducing mitochondrial biogenesis and cellular death through the production of ROS via disordered mitochondrial respiration (Chevtzoff et al., 2010; Leadsham and Gourlay, 2010; Thevelein and de Winde, 1999). We found that aspects of this signaling pathway were upregulated in response to AFs (Document S1. Tables S1–S3). For example, TPK1 and TPK2 were robustly induced in response to AF treatment, and we found an increase in expression of other genes involved in the same pathway (Document S1. Tables S1–S3), namely, BMH2, CYR1, and SRV2. Further, we found that PDE2, the cyclic AMP phosphodiesterase that represses the RAS/PKA signaling, was significantly downregulated. Deleting PDE2 in the presence of activated RAS/PKA signaling has been shown to lead to the overproduction of ROS by dysfunctional mitochondria, resulting in cellular death (Leadsham and Gourlay, 2010). Together, these results indicate that the RAS/PKA signaling pathway is largely upregulated in response to AF treatment and may contribute to the production of ROS by mitochondria.
We tested the role of the RAS/PKA pathway in AF-induced cellular death by studying single-gene knockouts of the upstream and downstream portions of the pathway. We found that deleting either RAS1 or RAS2 reduced yeast susceptibility to all three AFs (Figures 2E and S2) and deleting the downstream effector kinases, TPK1, TPK2 and TPK3, also reduced or delayed killing. Additionally we found that deleting any one of the key members of the RAS/PKA signaling pathway reduced the drug-dependent buildup of ROS (Figure 2F). This finding suggests that RAS/PKA signaling is critical for the induction of mitochondrial ROS production in response to drug treatment.
Common Metabolic Changes Resulting from AF Treatment
Our results link AF treatment to distinct changes in intracellular metabolic activity as part of an induced common killing mechanism. To further explore the role of metabolism in AF-induced cellular death, we directly measured the effect of AF treatment on the intracellular metabolome of C. albicans. To do so, we utilized a platform for metabolite detection and relative quantification from Metabolon (Durham, NC, USA) (Evans et al., 2009). We treated exponentially growing C. albicans cells with AFs and collected samples for metabolomic analysis after 1.5 hr of treatment, a time point when we expected at least 90% of cells contributing to the metabolite pool to be irreversibly committed to the death pathway (Figure S3). We found that the three drug treatments (AMB, MCZ, and CIC) significantly changed (p ≤ 0.05) the relative abundance of between 155 and 213 metabolites when compared to the no-treatment controls (Data Set S1. Metabolomic Profile of C. albicans Treated with AFs, Related to Figure 3).
Glucose was the most highly induced metabolite, found to be greater than 600-fold more abundant in drug-treated cells (Figure 3B). Other carbohydrates, such as fructose and mannose, were also significantly more abundant in all of the treatment groups compared to the control, as was the disaccharide trehalose (Figure 3B). These results are consistent with our microarray data that identified the upregulation of polysaccharide biosynthesis as an important transcriptional response to AF treatment.
Our phenotypic analyses in S. cerevisiae suggested that cells may be switching from exclusively fermentative growth to mitochondrial respiration in response to AF treatment. Consistent with this result, we found that 2,3-butanediol and glycerol, two major fermentative waste products (González et al., 2000), were substantially reduced after AF treatment (Figure 3C). Furthermore, pyruvate levels were dramatically lower in drug-treated samples (Figure 3D), indicating possible consumption by the TCA cycle; of note, TCA cycle intermediates were also reduced (Data Set S1. Metabolomic Profile of C. albicans Treated with AFs, Related to Figure 3) in comparison to the untreated cells. These measurements, combined with our genetic data and measurements of mitochondrial activity and biogenesis, provide support for a common fungicidal mechanism of action that relies on reduced fermentation and induced ROS-producing mitochondrial respiration.
The dramatic elevation of intracellular sugars and the shift from fermentation to respiration suggests that the AF treatments may be increasing ATP consumption. Abundant nucleotides such as ATP are not accurately quantified using the metabolic platform we selected for this study. We therefore sought to measure ATP levels over time using high-performance liquid chromatography (HPLC). We analyzed the AMP/ATP levels in lysates collected from cells treated with the three AFs. We found that the AF treatments elevated the AMP/ATP ratio by between 4- and 24-fold (Figure 3E), through a large drop in ATP levels and a proportional rise in AMP levels. ATP levels are normally static but can change dramatically under severe stress conditions that induce necrosis (Henriquez et al., 2008; Osório et al., 2004). Our results suggest that fungicide-stimulated sugar production induces a necrosis-like rapid consumption of ATP.
DNA Repair Is a Critical Response to AF-Dependent ROS Production
ROS damage multiple cellular targets, including membranes, proteins, and DNA (Kohanski et al., 2007; Salmon et al., 2004). Recent work indicates that DNA repair, specifically double-strand break repair (DSBR), plays a critical role in C. albicans resistance to oxidative damage (Legrand et al., 2007, Legrand et al., 2008). We chose to analyze the role of DNA repair in AF-induced cellular death by testing the susceptibility of C. albicans strains with deletions of critical genes in nucleotide excision repair, mismatch repair, and DSBR. We used AMB, MCZ, and CIC at a range of concentrations and found that DSBR mutants were particularly susceptible to all three AFs when compared to wild-type (Figures 4A–4C). Specifically, we found that the minimal fungicidal concentrations for the DSBR mutants, rad50/rad50 and rad52/rad52, were reduced by as much as 10-fold compared to wild-type.
Next we utilized the TUNEL assay to quantify the relative abundance of double-strand breaks in C. albicans cells treated with AFs and H2O2. The TUNEL reagent fluorescently labels both double- and single-strand DNA breaks (Ribeiro et al., 2006). Treating cells with AFs and H2O2 elevated relative fluorescence as measured by flow cytometry (Figure 4D), indicating a considerable induction of DNA breaks.
We also assayed rad50 and rad52 knockouts in S. cerevisiae to test whether the role of DNA damage in AF toxicity is consistent with the results from C. albicans. We found that these mutants were considerably more susceptible to H2O2 and AFs than wild-type S. cerevisiae, although the differences were smaller than those seen with C. albicans. Together, these data provide support for the role of DNA damage as a factor contributing to a common mechanism of AF action and indicate that targeting DNA-repair mechanisms could be an effective means for potentiating fungicidal activity.
For additional details, please see Extended Results.
Caspofungin Induces ROS Production and Metabolic Changes Consistent with the Common Mechanism of Antifungal-Induced Cellular Death
The key metabolic changes observed after AF treatment may act as a possible fingerprint to test whether other unrelated AFs may be acting through a similar common mechanism. Caspofungin belongs to a new class of AFs, termed echinocandins, that function by inhibiting cell wall synthesis in C. albicans (Denning, 2003). To test if AFs from this class of cidal drugs could induce metabolic changes comparable to the common mechanism of AF-induced cellular death in C. albicans, we treated exponentially growing cells with caspofungin and conducted metabolic analyses. We found that the metabolic changes induced by caspofungin were similar to the changes induced by AMB, MCZ and CIC, respectively (Figure S4A–S4C). Specifically, we found an elevation of sugars and a reduction in fermentative byproducts. We also found that caspofungin treatment led to elevated ROS levels in C. albicans (Figure S4D). These data indicate that caspofungin acts via a similar common mechanism.
In this work, we utilized a systems biology approach to study the response of fungal cells to AF treatment. We found that despite divergent primary modes of action, fungicides from three distinct classes induce a common signaling and metabolic cascade that leads to ROS-dependent cellular death (Figure 4I). We propose that cellular changes and damage initiated through interaction with primary targets of AFs result in activation of a stress-like response that includes signaling through the RAS/PKA pathway. Through this and possibly other pathways, cells activate mitochondrial activity and shift from fermentation to respiration in response to AFs. This abrupt induction of mitochondrial activity leads to the overproduction of toxic ROS in a manner that depends on the TCA cycle and ETC. Additionally, we found that AF treatment leads to a buildup of monosaccharides and disaccharides, including glucose and trehalose. Both the import and synthesis of these sugars are energetically expensive, requiring the consumption of ATP and the production of AMP. It is likely that these metabolic changes lead to altered respiration and the overproduction of ROS by dysfunctional mitochondria, ultimately resulting in cell death (Figure 4I).
The common AF-dependent responses identified in this work have similarities to many fungal stress-response mechanisms (Gasch et al., 2000; Soufi et al., 2009), suggesting that AF agents may function by overactivating these pathways to induce cellular death. These similarities include a collapse of ATP levels (Osório et al., 2004), mitochondrial dysfunction that leads to the production of ROS (Breitenbach et al., 2005), and a central role played by the RAS/PKA pathway (Estruch, 2000). It is likely that AFs cause cellular damage that mirrors the effects of osmotic, heat, and alcohol stress, leading to the induction of stress-related pathways. Interestingly, the same responses that protect cells from mild environmental stress may in fact contribute to AF-dependent cellular death. AF insult to fungal cells may be sufficiently powerful such that the overactivation of these general stress responses does not protect the cell, but instead plays a net contributory role in cellular death through the consumption of ATP and the production of ROS.
In addition to similarities with known fungal stress pathways, the identified common mechanism of AF-induced fungal cellular death can also be compared to the previously identified common mechanism of antibiotic-mediated bacterial cellular death (Dwyer et al., 2007; Kohanski et al., 2007). Despite the complex and multilayered eukaryotic mechanism, robust similarities can be found between the two phenomena, indicating possible evolutionary conservation. In both prokaryotes and eukaryotes, cellular damage resulting from the primary drug-target interaction activates key metabolic regulators, such as RAS in yeast and ArcA in bacteria (Kohanski et al., 2008). These universal regulators modulate central metabolism through the induction of the TCA cycle and respiratory activity to produce toxic ROS.
The endpoint of the common antimicrobial mechanisms in both bacteria and yeast is cellular death through the production of toxic ROS. In both prokaryotes and eukaryotes, ROS induces double-strand DNA breaks (Dwyer et al., 2012; Foti et al., 2012) and other forms of cellular damage that lead to cellular death and are potentiated by blocking DNA repair. In yeast, we demonstrated that by blocking DSBR we could reduce the minimal fungicidal concentration of the tested AF drugs by as much as 10-fold. Similarly, deleting RecA, an Escherichia coli homolog of the yeast RAD genes, substantially enhances bactericidal antibiotic activity in bacteria (Kohanski et al., 2007). These findings suggest that inhibitors of DNA repair mechanisms may be robust potentiators of fungicides and bactericidal antibiotics.
The critical role of ROS in antimicrobial activity indicates that ROS generation may have evolved and been maintained in both prokaryotes and eukaryotes as a central mechanism to overcome environmental stress. Under various stress conditions, low-level ROS production can activate multiple protective responses including the upregulation of antioxidant enzymes and the induction of beneficial mutations (Belenky and Collins, 2011; Gems and Partridge, 2008; Kohanski et al., 2010). However, if ROS production goes above a certain threshold, it no longer serves a protective role and instead induces cellular death. Thus, it is likely that many lethal challenges, including fungicides, function by highjacking natural stress-response mechanisms to induce ROS production above this threshold.
Experimental Procedures
Fungal Strains and Media
S. cerevisiae strains used in this work were derivatives of BY4742, created as part of the Deletion Consortium (Winzeler et al., 1999). Deletion Consortium strains with reported phenotypes were verified by PCR. Stains PAB202 (BY4742 cit1Δ::kanMX4 cit3Δ::URA3), PAB205 (BY4742 cit3Δ::kanMX4 cit2Δ::LEU2), and PAB208 (PAB202 cit2Δ::LEU2) were created by direct transformation of PCR products as previously described (Brachmann et al., 1998). S. cerevisiae rad50 and rad52 knockouts were derivatives of MKP-0 and generously provided to us by Dr. Simone Moertl (Steininger et al., 2010). Wild-type C. albicans strain, SC5314, was used in metabolomic profiling and HPF measurements. C. albicans DNA repair mutants used in this work were derivatives of DKCa39, generously provided by Dr. David T. Kirkpatrick. A complete list of DKCa strains used in this study is provided in Document S1. Tables S1–S3. SDC media and synthetic complete media with 2% acetate (pH 6.5) were prepared as previously described (Burke et al., 2000; Wickerham, 1946).
Fungicidal Killing and Fluorescent Dye Assays
Overnight yeast cultures were diluted into the indicated media and grown to an optical density 600 of 0.2, at which point the AFs were added. CFU were measured by plating six serial dilutions onto YPD agar plates. A more detailed description is included in Extended Experimental Procedures.
S. cerevisiae Microarrays
Yeast cells were incubated in 25 ml SDC and grown in 250 ml flasks at 30°C and 300 rpm. AFs were added at an OD600 of 0.2. Cells were harvested after treatment and their RNA was isolated and processed as previously described (Schmitt et al., 1990). The complete microarray analysis procedures are described in Extended Experimental Procedures.
Metabolomic Profiling
C. albicans cells were lysed and assayed by Metabolon as previously described (Shakoury-Elizeh et al., 2010). ATP and AMP were extracted as previously described (Walther et al., 2010) and analyzed by HPLC. This procedure is more fully described in Extended Experimental Procedures.
Antifungal Drugs
AF drugs were resuspended at 250 times the working concentration in DMSO (CIC was solubilized in ethanol) and frozen at 80°C in one-time-use aliquots. DMSO was added to cells at 0.4% as the no-treatment control. To account for observed potency differences between different lots of AF drugs, each set of experiments was performed using frozen stocks from the same lot. Each AF drug was titrated against S. cerevisiae and C. albicans in order to identify the minimal fungicidal concentration. This drug concentration was used for the phenotypic assays in order to achieve maximal sensitivity for resistant mutants.
Fungicidal Killing and Fluorescent Dye Assays
Overnight yeast cultures were diluted into the indicated media and grown to an OD600 of 0.2, at which point the AFs were added. Colony forming units (CFU) were measured by plating six serial dilutions onto YPD agar plates. Cells treated with MCZ were washed before serial dilution to stop growth inhibition associated with high concentrations of MCZ. CFU were counted after 48 hours of incubation on agar plates. When indicated, thiourea (Fluka) was added 30 min prior to the addition of AFs. HPF (10 μM) (Invitrogen) and MitoTracker (1 μM) (Invitrogen) dyes were added to PBS-washed cells at the indicated time points and incubated for 30 min prior to flow cytometry measurements, which were taken on the FACSCalibur (Becton Dickinson). All experiments described in this section were conducted in 0.5 ml of SDC in 24-well plates incubated at 900 rpm and 37°C for C. albicans and 30°C and 300 rpm for S. cerevisiae. TUNEL assays were conducted as previously described (Ribeiro et al., 2006) and are described in detail in the TUNEL section of the Extended Experimental Procedures.
High-Throughput 96-Well Screen
To identify genes critical to AF action, we chose to test the AF sensitivity of single-gene knockouts identified through our common transcriptional analysis. In the first pass-through, we identified 56 testable genes. This set was later expanded to include targets identified through phenotypic analysis and metabolomic profiling, for a total of 84 target genes (Document S1. Tables S1–S3). We tested the initial 56 targets using a rapid high-throughput 96-well assay with AMB and MCZ to identify single-gene knockouts with elevated resistance to AFs (Document S1. Tables S1–S3). Knockouts that were more resistant to at least one of the AFs were tested individually using the 24-well colony forming (CF) assay. Target genes selected after the initial 56 strains were assayed individually using the 24-well CF assay.
The first 56 S. cerevisiae strains were acquired from the deletion library and stored in 96-well plates in replicates of three. To start the assay, the plates were defrosted and 10 μl of stored cells were inoculated into 100 μl of fresh synthetic dextrose complete (SDC) media for 16 hr. These overnight cultures were diluted into a fresh 96-well plate with 100 μl of SDC to achieve an OD600 of approximately 0.2. Colony forming units (CFU) in each well were measured at the start of the assay and after 8 hr of incubation. The AF susceptibility of each strain was compared to that of wild-type. Strains with significant susceptibility differences from wild-type were assayed in 0.5 ml of SDC media using the 24-well plate CF assay described above.
Metabolomic Profiling
C. albicans cells were incubated in 100 ml of SDC in 250 ml flasks at 37°C and 300 rpm. Cells were exposed to AFs at an OD600 of 0.2, and cellular pellets from 100 ml of media were collected after 1.5 hr of treatment. Cells were lysed and assayed by Metabolon Inc. (Durham, USA) as previously described (Evans et al., 2009; Shakoury-Elizeh et al., 2010).
ATP and AMP Measurements
To quantitate the cellular AMP/ATP ratio, C. albicans cells were incubated in 25 ml of SDC in 250 ml flasks at 37°C and 300 rpm. AF drugs were added at an OD600 of 0.2, and 2 ml of cells were collected every 10 min for the first 90 min of incubation. Cells were lysed and nucleotides were extracted using boiling buffered methanol as previously described (Walther et al., 2010). Lysates were desiccated and resuspended in 60 ml of water prior to HPLC analysis. Nucleotides were separated isocratically in 100 mM sodium phosphate pH 6.5 on a ZORBAX SIL SAX 70Å 5um, 4.6 × 150mm column (Agilent, Santa Clara CA).
S. cerevisiae Microarrays
Yeast cells were incubated in 25 ml of SDC and grown in 250ml flasks at 30°C and 300 rpm. AFs were added at an OD600 of 0.2.
Yeast cells were incubated in 25 ml of SDC and grown in 250 ml flasks at 30°C and 300 rpm. Antifungals were added at an OD600 of 0.2. The rate of yeast killing by MCZ and CIC, respectively, was reduced by the transition from the 0.5 ml culture to 25 ml cultures. Rather than increasing drug concentration to match 0.5 ml killing levels, CIC and MCZ samples were collected after extending incubation times to achieve killing levels comparable to the 0.5 ml experiments. RNA from untreated cells was collected at 0, 0.5, 1 and 2 hr after treatment. RNA from AMB-treated cells was collected at 0.5, 1 and 2 hr after treatment. RNA from MCZ-treated cells was collected at 2, 5 and 8 hr after treatment. Cellular RNA was isolated and processed as previously described (Schmitt et al., 1990).
The effect of AF treatment on S. cerevisiae global gene expression was assayed using Affymetrix yeast microarrays (Affymetrix GeneChip Yeast Genome 2.0 array). The collected microarray data were added to a compendium of publicly available microarrays (obtained from the Gene Expression Omnibus) for a total of 536 chips. The set of expression profiles was normalized as a batch with RMA Express (Bolstad et al., 2003). The SD of the expression of each gene was calculated across the entire compendium of expression profiles, allowing us to calculate a z-scale difference between a treatment and a control condition (no-treatment) using the formula:
This allowed us to determine gene expression changes in units of standard deviation, which is a form of the z-test. For each of the time points in each treatment, the z-score difference was converted into p-values and the sets of up- and downregulated genes (p < 0.05) were identified. The set of differentially expressed genes was merged across all time points, resulting in a global set of differentially expressed genes in AF-treated cells as compared to untreated cells. A common set of genes differentially expressed in response to all three antifungal drug treatments was determined by the intersection of the three distinct gene sets (Figures 1D and 1E).
TUNEL Assay
DNA strand breaks were fluorescently labeled with TUNEL reagent from the "In Situ Cell Death Detection Kit," Roche (Mannheim, Germany). After 2 hr of treatment 5 ml of OD 0.2 cells were fixed with 3.7% formaldehyde for 30 min at room temperature, and then washed with digestion buffer consisting of 10 mM Mes pH 6.5 and 1 M sorbitol. Cell were digested for 45 min at 30°C with 2 U of Yeast Lytic Enzyme, MP Biomedicals, (Irvine, CA) in 50 μl of digestion buffer. Cells were then washed in PBS with 1 M sorbitol and resuspended in permeabilization solution consisting of 0.1% Triton X-100 and 0.1% sodium citrate and 1 M sorbitol for 10 min at room temperature. Cell were again washed in PBS with 1 M sorbitol and labeled for 60 min with 10 μl of TUNEL reaction mixture. Cells were then again washed in PBS with 1 M sorbitol and resuspended in sheath fluid. Mean relative florescence was measured by flow cytometry on the FACSCalibur (Becton Dickinson).
We thank David T. Kirkpatrick for the set of DKCa strains, Simone Moertl for the MKP-0 strains, Merck & Co. for providing caspofungin, Katie Vignola and Tim Hyde at Metabolon for their help with metabolomic profiling, and Norman Gerry (Coriell Institute) for processing the microarrays. This work was supported by the Howard Hughes Medical Institute and a National Institutes of Health Director's Pioneer Award (DP1 OD003644).
Accession Numbers
The compendium containing 536 microarrays has been deposited to the Many Microbe Microarrays Database database (http://m3d.bu.edu/) at http://m3d.bu.edu/datasets/Belenky_scerevisiae_compendium.txt.tar.gz.
Supplemental Information
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Breitenbach et al., 2005 (2005). The actin cytoskeleton, RAS-cAMP signaling and mitochondrial ROS in yeast apoptosis. Trends Cell Biol. 15, 637–639. Abstract | Full Text | PDF (72 kb) | CrossRef | PubMed
Burke et al., 2000 (2000). Methods in Yeast Genetics. (Cold Spring Harbor, NY: Cold Spring Harbor Press). PubMed
Chapman et al., 1992 (1992). SDH1, the gene encoding the succinate dehydrogenase flavoprotein subunit from Saccharomyces cerevisiae. Gene 118, 131–136. CrossRef | PubMed
Chevtzoff et al., 2010 (2010). Reactive oxygen species-mediated regulation of mitochondrial biogenesis in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 285, 1733–1742. CrossRef | PubMed
Dwyer et al., 2007 (2007). Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli. Mol. Syst. Biol. 3, 91. PubMed
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Estruch, 2000 (2000). Stress-controlled transcription factors, stress-induced genes and stress tolerance in budding yeast. FEMS Microbiol. Rev. 24, 469–486. CrossRef | PubMed
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González et al., 2000 (2000). Characterization of a (2R,3R)-2,3-butanediol dehydrogenase as the Saccharomyces cerevisiae YAL060W gene product. Disruption and induction of the gene. J. Biol. Chem. 275, 35876–35885. CrossRef | PubMed
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Publication Information
Received: July 12, 2012
Revised: November 28, 2012
Accepted: December 18, 2012
Published: February 14, 2013


text:
Tour Our Gardens
Western Pennsylvania Conservancy Community Gardens
Our community gardens are located in 20 counties throughout the region. The listings below provide information for each location.. To view the gardens, click on their links below. You can find gardens in your area by clicking here. If you are interested in volunteering in the flower gardens this year, please contact Lynn McGuire-Olzak at (412) 586-2324 or email@example.com.
City of Pittsburgh Gardens
Allegheny County Gardens
Statewide Gardens
AliquippaAltoonaBensonBrownsvilleButlerConnellsvilleRoute 119 and Pittsburgh Street EbensburgCenter Street & S. Locust Street - Ghost Town Trail ErieFarrellFranklinGreensburgGreenvilleMain Street and College Avenue Main Street and South Front Street Grove CityHarrisburg7th Street Extension and Forster Street HermitageHollidaysburgHuntingdon | IndianaRoute 286 and Indian Springs Road JohnstownBedford Street and Johnstown Expressway - Off Ramp Bedford Street and Johnstown Expressway - On Ramp Meadville1216 South Main St - Second District Elementary School Route 322 Triangle - Mary B. DeArment Memorial Park MercerMonessenNanty GloChestnut Street & 2nd Street - Ghost Town Trail New KensingtonOil CityEast Front Street and Wilson Avenue Main Street & Halyday Run Road ShippenvilleSlippery RockRoute 173 & East Cooper Street State CollegeCollege Avenue & University Drive Tyrone11th Street and Pennsylvania Avenue 15th Street and Lincoln Avenue WashingtonWaynesburg |

subset: commoncrawl


text:
Bronco Profile
The chaplain is in the House
With the way things have gone recently in Congress, looking to the heavens for some help and guidance might seem like a very good idea. In fact, that's what Pat Conroy, S.J., M.Div. '83 is there to do.
Winter 2014
Potions and poisons
Medical sleuth B. Joseph Guglielmo '73, dean of the nation's top school of pharmacy, on the challenges of treating patients. And a health care system.
Fall 2013
The artful baker
Connecting with amateur baker turned pie-making guru Kate McDermott '76.
Fall 2013
Miller's Tale
To tell the story of Bob Miller '67 is to tell the coming-of-age tale of Las Vegas itself. And it's the chronicle of a man who served a decade as governor of Nevada. Quite a journey for the son of an illegal bookie from Chicago.
Spring/Summer 2013
Blood. Sweat. Tears. Repeat.
Nina Acosta '82 was a tough enough cop to pass the test for the LAPD's SWAT team. Then she learned the hard way about gender discrimination. So how did she do on Survivor?
Spring/Summer 2013
Range of motion
Misha Patel Bechtolsheim '05 studied business and dance at SCU but assumed they were mutually exclusive. She soon learned otherwise.
Spring/Summer 2013
The play's the thing
Kurds, Arabs, countrymen: Shakespeare Iraq brings the Bard to Ashland like you've never heard him. And you can thank Peter Friedrich '91.
Winter 2013
A valedictorian of Olympic proportions
Kelly Crowley '99 medaled twice in cycling at the 2012 Paralympics—and won gold in Athens in swimming in '04 and was valedictorian of SCU.
Can you stand the heat?
It took months of space flight for the Curiosity rover to reach Mars. And, to survive the heat of entry, it took a shield that a team led by Robin Beck '77 designed.
Fall 2012
The stunt woman
As fans of The Hunger Games know, Tara Macken '08 goes down fighting.
Summer 2012
Winter 2014
Table of contents
Rise up, my love
There are the sanctuaries built for worship—and that carry beauty and grace for all to see. Then there are the improvised places of faith, perhaps more subtle in how they speak to the wonder worked there.
The chaplain is in the House
With the way things have gone recently in Congress, looking to the heavens for some help and guidance might seem like a very good idea. In fact, that's what Pat Conroy, S.J., M.Div. '83 is there to do.
Welcome to Citizenville
Who published the one book on government in 2013 that conservative firebrand Newt Gingrich told all true believers that they should read? Well, the author is now lieutenant governor of California. Before that, he was mayor of San Francisco. That's right: It's Gavin Newsom '89.
Mission Matters
Women's soccer wins the West Coast Conference championship.
Patent trolls, beware
The White House has brought on SCU's Colleen Chien, a leading expert in patent law, as senior advisor.
A sight of innocence
George Souliotes went to prison for three life sentences after he was convicted of arson and murder. Twenty years later, he's out—after the Northern California Innocence Project proved he didn't do it.


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China Front and Center in New Jon Huntsman Documentary
But his campaign fizzled before it could become an issue.
Skip to content, or skip to search.
Skip to content, or skip to search.
But his campaign fizzled before it could become an issue.
Breitbart claims to have sources.
The Huntsman family biz is reportedly on the block.
This is what they do when you "wax philosophical."
Campaign debts include a $356,000 bill for an ad-maker.
We watched 50-something hours of debates, so you don't have to.
The candidate's concession speech showed why he won't be the GOP's standard-bearer against Obama.
After putting all his chips on New Hampshire, Huntsman folds.
Perry was in a Palmetto State of Mind, Newt Gingrich was channeling Reagan, and Jon Huntsman talked about ... you guessed it! China!
Mitt is projected to finish with a double-digit victory.
Michael Steele made an awkward joke today.
Winning support in New Hampshire, but in need of a fat check from his dad to really hurt Romney.
Paul's The Revolution is top book among Huntsman's Facebook supporters.
The market decided long ago that he needed to win New Hampshire.


text: Welcome to Xander! The comic ran from August, 2010 to August, 2012, and ended with 125 color illustrated pages. You can read the entirety here, and at some point soon we'll publish a printed volume if you'd like one. Since the comic ended the site has been adjusted to display the first page, so read on from here! Contact the writer at firstname.lastname@example.com.


text:
Listing Items 289-297 Of 297
1943 WW2 Victory Farm Volunteer US Crop Corps Vintage Food Poster
1943 Victory Garden US Crop Corps WWII Military Poster PRINTED BY: US Government Printing Office AGE: dated 1943, WWII era CONDITION: A, Linen Mounted. Machine folded as war posters were, to send out in envelopes. The folds are unobtrusive unless scrutinized closely. beautiful! This poster is a call for children... -
1940's Red Cross Vintage WW2 Poster with African American Child
1940 WWII Junior Red Cross Vintage Military Poster PRINTED BY: Litho in USA AGE: c. 1940's WW2 Era CONDITION: A, Linen Mounted, beautiful condition! This horizontal WWII Red Cross art deco poster depicts children in front of a large globe map of the world. They are advertising a Junior Red... -
1940's Norman Rockwell Original Vintage Boy Scout Poster Mansfield NH
1940's Norman Rockwell Vintage Poster AGE: c. 1940's ARTIST: Norman Rockwell, printed signature copyright B & B, (Brown & Bigelow). It has a printer's mark lower Left edge of poster which reads: 4501 Brown & Bigelow, St. Paul, Minnesota. CONDITION: A, Linen Mounted, absolutely beautiful condition. This is an original... -
1938 Tuberculosis TB WPA Era Vintage Health Poster by LLoyd Coe
1938 TB Tuberculosis Lloyd Coe Vintage Christmas Seals Poster This is a pre WW2 WWII era American medical or US health poster in wonderful A condition, linen mounted. It features deco art work, WPA style graphics of a young mother lighting a candle in the window, with her two children... -
1918 For Every Fighter A Women Worker WW1 Vintage YWCA Poster
1918 WWI Womens YWCA Antique American Military Poster PRINTED BY: United States Litho Company AGE: c 1918 WWI Era lithograph. ARTIST: Ernest Hamlin Baker, graphic designer, poster artist, illustrator CONDITION: B+, Linen Mounted, border patch, unobtrusive unless scrutinized very closely, very bright and clean, displays beautifully! NOTE: "Back Our Second... -
1918 YWCA WW1 Back Our Girls Over There Vintage Poster Underwood
1918 WWI YWCA American Telephone Operator Poster AGE: WWI era, 1918 lithograph. ARTIST: Clarence Frederick Underwood, (Clarence F. Underwood) Graphic designer, artist, illustrator, well known for his beautiful women and postcard art CONDITION: A, Linen Mounted This is a beautiful original early Clarence Underwood military & telecommunications world war one... -
1918 He Can Win WW1 Vintage Red Cross Poster by Dan Smith
1918 WWI Red Cross Dan Smith Vintage Poster PRINTED BY: Designed and printed by Thomsen-Ellis Co. Baltimore and New York NYC AGE: WW1 era c. 1918 lithograph. ARTIST: Dan Smith, graphic designer, poster artist, illustrator CONDITION: B, Linen Mounted, restored tear at top with no paper loss, unobtrusive unless scrutinized... -
1958 Original Joseph Binder Red Cross Flags Vintage Poster
1958 Joseph Binder Junior Red Cross Art Deco Vintage Advertising Poster Printed by: The American National Red Cross, Poster 615, (Rev. 5-58). It has a printer's union label, dated 1958. This antique poster features an American flag and Red cross flag and sparse and simple art deco graphic design. Artist:... -
1917 US Navy Reuterdahl Vintage WW1 Poster by Reuterdahl
1917 WWI US Navy Reuterdahl Original Military Advertising Poster PRINTED BY: Latham Litho & Printing Co. of Brooklyn NY AGE: c. 1917 World War WWI One era lithograph. ARTIST: H. Reuterdahl, graphic designer, artist, illustrator CONDITION: B+, Linen Mounted, left top corner restored tear, minor wrinkles, although we list this...


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Avalon Organics Glistening Shampoo Ylang Ylang -- 11 fl oz
Really does what it says
This shampoo really does what it says it should do-- it makes my hair really shine. It also seems to have helped with improving my hair's elasticity. I have not had as much breakage or fallout when combing my hair since using this shampoo.
February 13, 2013
Avalon Organics hair products are wonderful!
Great buy/great results. Simple and natural.
September 23, 2011
I have a full head of hair and this shampoo leaves it looking FLAT.
I would recommend instead: Avalon Organics Lemon Clarifying Shampoo with Shea Butter. It smells great and leaves hair clean and full-bodied.
August 3, 2011
Not crazy about it
Despite already a being a fan of Avalon products, I wasn't crazy about the Ylang ylang scent. Love ylang ylang but wish the sent were stronger. Still have sitting in my shower months and months later.
June 26, 2011
New Favorite Shampoo
I've tried many shampoos and my "Go To Brand" is now Avalon Organics. The Glistening Shampoo has become my new favorite and I interchange it with the Biotin B-Complex Thickening Shampoo and Conditioner. The Ylang Ylang in the Glistening Shampoo gives it a very nice smell and it leaves my gray/white hair soft, shiny and yes......glistening. Since adding this new product to my hair care needs, I've received many comments on how nice it looks.
May 29, 2011


text:
Submit Review
Campground Search
204637 User submitted reviews online!!
Location :
Bastrop, Texas
Bastrop RV Park
[ 7/10 ]
Nice quiet park. Pool was nice, could have been cleaner, but the park was nice. The management was wonderful and helpful. They even have a big ice machine by the pool for filling up your ice chest, free! Haven't seen that too often. Stores nearby, Wal-Mart and HEB. If you are here in the summer, make sure your canopy faces east, the sun was pretty nasty in the afternoon. Didn't have a resort feel because we saw only 2 families having fun. The other visitors stayed inside their RV the whole time we were here. I would go back again for sure.
Canyon Lake, Texas
Rio Raft and Resort
[ 5/10 ]
The pool was very big and nice and the water was very clean. No bath houses, just a pot in a closet with an outdoor sink. Not even enough for the tent campers, so if you don't have your own bathroom, forget it. Last row didn't have cable tv, but they didn't tell us that, so we were all set-up before we realized there was no cable in that row. It is a nice park otherwise. Shade available. There is a Super S grocery nearby.


text:
- Make This for Dinner: Steven's Wife Michelle's Lentil Soup
- Flash Frozen: 10 Tasty Reasons to Stock Up On Frozen Fruits and Veggies
- The New York International Olive Oil Competition Returns to New York for its Second Amazing Year
- 5 Super-Healthy Seeds To Eat Now!
- Brand New at Fairway: Hot Tamales and Extraordinary Empanadas!
Steve Jenkins
joined the Fairway family in January 1980. He was an out-of-work stage actor...
Read More >>Carlon Colker, MD
is the Chief Executive Officer and Medical Director of Peak Wellness, Inc.
Read More >>Hannah Howard
Hannah has been working with food since the age of 16, when she spent her summer scooping gelato...
Read More >>Blogroll
Tag Archives: press
Break Out of your Rib-Eye Rut: Master Butcher Ray Venezia Shows Us Lesser Known, Super Flavorful Cuts of Beef
We're all for breaking out a filet for a big night, and we're mighty proud of our strips and tenderloins. But with a whole glorious butcher counter before you, it'd be a shame to stick to the familiar favorites. Our … Continue reading
Posted in Hannah Howard
Tagged beef, butcher, fairway, fairway market, flatiron, hanger, london broil, meat, meat cuts, press, ray venezia, tri-tip
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Thanks for the Shout-Out, CNBC!
We know how uniquely awesome Fairway is. We know our customers know. Still–it's nice to hear! Especially from Jim Cramer, Melissa Lee, and David Faber, hosts of CNBC's "Squawk on the Street." Squawk on the Street airs live from the … Continue reading
Posted in Hannah Howard, Uncategorized
Tagged cnbc, fairway market, jim cramer, press, television
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text:
Girl's Camouflage Clothing
Having a baby girl? The Camo Shop carries many choices: dresses, overalls, gift set outfits in the Realtree camo pattern. Girls camo clothing have either bows or pink trim. Shop for baby showers or everyday clothing.
Kids Camoflauge Sweat Jacket
$29.95 -
Childs Camo Long Bubble Overalls w/Tail Bow & Emblem
$29.95 -
Childs Camouflage Dress w/Panty
$21.95 -
Camo Overall Skirt with Emblem
Camouflage Long Overall w/ Daddys Little Deer Emblem Kids
$29.95 -
Mossy Oak Camo School Supply Pack
$24.99 -
Girls Hoodie Pink Camo Cutie
Regular Price: $26.00
Now only: $24.00
Mossy Oak Sweat Jacket Pink Trim
Boys 4 or 6 Pocket Camo Pants
$24.95 -
Girls Hoodie - Pink Camo Cutie
$26.00Out of stock


text:
Do you have a question? Chat with Us Live
All of our in-stock items ship the same day you order if your order is placed by 6 p.m. ET. Engrave & Ship today items will be shipped out the same day if personalization is approved by 3pm ET. Delivery to the 48 contiguous states takes 2-5 business days. Delivery to Hawaii, Alaska and Puerto Rico takes 5-7 business days. We will not deliver to a Post Office box.
We stand behind our products, period. If you need to return an unused stock product, we'll be happy to credit your account, exchange the item or give you a merchandise refund if returned within 30 days from receipt of your order.
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Loved them.
By Barb
from Turlock, CA
About Me Executive Director
Best Uses
Comments about Baudville Lapel Pin - Heart Hand:
We plan to use them as a table favor for our 20 annversary celebration of our agency. The hand is similar to the hands in our logo.
Want this item customized with your logo or organization's name?


text:
Friday, August 28, 2009
© Copyright 2014
Gwinnett Daily Post
LAWRENCEVILLE - For months - as a church faced the development of a waste transfer station next door, as a county budget crisis unfolded and a trash hauling plan went to court - Sabrina Smith and her neighbors and friends began asking questions.
The answers, she said, never seemed to come. But she and about 250 others began to get some feedback Thursday during a town hall meeting she organized with residents. "I just want to know answers to very simple questions," the Lawrenceville woman said at the meeting's opening. With a panel of residents and volunteers and a crowd that included four mayors, the sheriff and a commissioner, residents sounded off on issues from the controversial closing of a library to the possibility of losing access to water from Lake Lanier. "It's important that Gwinnett continues to move forward," developer George Thorndyke, a panelist, said. "We are stalled and we have no direction to move ahead." While the county recently tapped leaders to create a citizens study panel to investigate the budget, organizer Debbie Dooley said people can't wait six months for answers. People suggested the end of duplication of services with the school board, staggering library hours to keep more open and working on solutions other than new roads to help with traffic. "I think we have an opportunity to improve quality of life in Gwinnett County and make sure the services we provide are the ones the citizens want," said Mike Levengood, one of the leaders of the Engage Gwinnett program. The first meeting for that group will be held from 6:30 to 8:30 p.m. Sept. 9. Elsie Post, of Suwanee, said she planned to attend more meetings. "There were actual answers," she said, referring to Commissioner Mike Beaudreau's responses to many of the concerns voiced Thursday. "It was refreshing to ascertain that we are on the same page." James Wall, a Dacula man angered over the decision to close his library branch, was more skeptical, saying he had little hope for a change. "It didn't hurt," though, to attend, he said.More like this story
- Town hall on county woes set ( August 25, 2009 )
- 3 garbage contracts dumped ( February 12, 2008 )
- Trash lawsuit: County working on plan
Commissioner taking ideas at meetings ( February 21, 2009 ) - 20 talk trash at hearing
Residents, providers discuss sanitation plan ( December 11, 2007 ) - Controversial computer lab motion nixed ( October 22, 2009 )


text:
Gain 360-degree support for all product-related processes – from the first product idea through manufacturing and service. With our product development software, you can manage products throughout their entire product lifecycle.
Want to learn more about SAP for Industrial Machinery & Components?
Find more SAP solution information. Solutions ›
*All implementation results are for informational purposes only and the examples provided while based on actual SAP customers' experiences do not represent commitments or guarantees by SAP and/or its partners. Actual pricing, costs, and implementation results may vary, based on customer-specific requirements and needs. The only warranties for SAP products and services are those that are set forth in the express warranty statements accompanying such products and services, if any. Nothing herein should be construed as constituting an additional warranty.


text:
Saint Justin of Rome
Priest. Martyred for giving Christian burial to the bodies of martyrs.
Additional Information
- Book of Saints, by the Monks of Ramsgate
- Catholic Online
- Dictionary of Saints, by John Delaney
- Our Sunday Visitor's Encyclopedia of Saints
MLA Citation
- "Saint Justin of Rome". Saints.SQPN.com. 3 December 2013. Web. 16 March 2014. <>


text:
Plaintiffs seek a declaration that DOMA § 3 ... violates the [US] Constitution, as applied to plaintiffs and those similarly situated, by refusing to recognize their lawful marriages for purposes of conferring family-based immigration waivers and benefits. As a direct and proximate result of the violations of the Constitution alleged herein, plaintiffs have been denied, and will continue to be denied, legal protections and benefits under the [INA] that would be available to them were they spouses of different sexes.
This week 132 members of the US House of Representatives [official website] filed an amicus brief [JURIST report] arguing for DOMA to be overturned as unconstitutional. The brief was filed in the appeal of Karen Golinski v. Office of Personnel Management, the landmark case in which the US District Court for the Northern District of California [official website] ruled that DOMA is unconstitutional [JURIST report]. Last month 10 US senators filed their own amicus brief in the case, arguing in the opposite [JURIST report] that the federal government had a legitimate interest in creating a uniform federal definition of marriage to "[avoid] massive legal uncertainty." That filing came the week after the House Bipartisan Legal Advisory Group (BLAG) submitted its brief defending DOMA [JURIST report], arguing that laws relating to sexual orientation should be evaluated with a rational basis [Cornell LII backgrounder] test and that the government's denial of federal benefits to lawfully married same-sex couples is a rational means to preserving a single federal definition of marriage and preserving federal funds. The BLAG DOMA defense group of attorneys [JURIST report] was established by House Speaker John Boehner (R-OH) [official website] on a 3-2 BLAG vote in March of last year after the DOJ announced that it would no longer defend the constitutionality of Section 3 on order from President Obama, after he announced he would continue to fight for the repeal of DOMA [JURIST report].


text: Aerobotix, a FANUC Authorized Integrator, provides turn-key robotic coating systems and other automated process equipment to clients within the aerospace, military, and general manufacturing industries. From simple pick-and-place robots to fully engineered and integrated multi-robot systems, we can take care of all of your robotic and automated coating needs. Our other services include research and development, process development, systems and process 3D modeling, simulation of operations, and offline robot programming. Aerobotix is the premier supplier of robotic coating systems for the application of unique and difficult coatings. This might range from equipment used for painting Line-X™ in a truck bed, sealing a window, painting camouflage on a Humvee, or applying the specialized coatings to the world's most advanced stealth aircraft. Our equipment can be found working with full-up aircraft, large piece parts, engine inlets, missiles, ground vehicles and UAV's.


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Neither one of my grandma's could cook very good but I have great memoriesof both of them.
As a Very Best Baker, you can print coupons, chat in the Forums, plus rate, review and save recipes!
Remember me on this computer
Become a Very Best Baker to print coupons, share recipes and chat in the Forums.


text:
Watch Teach for America's New DREAM Act Video
by Jamilah King on June 25 2013, 3:09PM
Worlds colliding.
Topics: /NOW
Watch Teach for America's New DREAM Act Video
by Jamilah King on June 25 2013, 3:09PM
Worlds colliding.
Topics: /NOW
L.A. May Send Hundreds of Foreign Teachers Back to Their Home Countries
by Jorge Rivas on December 12 2012, 12:26PM
Foreign teachers who have been teaching in L.A. schools for more than six years are being forced home.
Topics: Immigration, /NOW
Viola Davis Welcomes Protestors Who Disrupted 'Won't Back Down' Premiere
by Jorge Rivas on September 28 2012, 11:45AM
An estimated 50 parents and teachers demonstrated outside the "Won't Back Down" film premiere in New York Sunday.
Topics: /NOW, Schools & Youth
So Who Actually Won the Chicago Teachers Strike?
by Jorge Rivas on September 19 2012, 1:35PM
Chicago public school teachers voted on Tuesday to end their strike and resume classes Wednesday in the third-largest U.S. school district in the Nation.
Topics: /NOW, Schools & Youth
Los Angeles Leads the Way on Data-Driven Reform With Teacher Scores
by Julianne Hing on April 13 2011, 1:28PM
The city's schools are increasingly the frontline in the battle over business-style reforms for public education.
Topics: Schools & Youth
Yale Cuts Teaching Program, Funds College for New Haven Kids
by Julianne Hing on November 18 2010, 11:57AM
Many saw it as an important avenue for Yale to support New Haven's public education system.
Topics: Schools & Youth
Is Arizona Targeting Teachers through Unfair English Standards?
by Michelle Chen on September 9 2010, 10:03AM
The Justice Department's taking the state's "English only" policy to court.
Topics: Politics, Schools & Youth
Louisiana's Trafficked Filipino Teachers Take Recruiter to Court
by Julianne Hing on August 13 2010, 2:51PM
Faced intimidation and exorbitant fees, according to suit.
Topics: Global Affairs, Schools & Youth
Senate Passes $10 Billion Teachers' Fund
by Julianne Hing on August 5 2010, 3:19PM
Congress' last minute aid package helps school districts crippled by recession.
Topics: Schools & Youth
War Spending Bill Won't Pay for Teachers' Jobs
by Julianne Hing on July 23 2010, 4:01PM
The White House has successfully intimidated the Senate into passing the bill without $10 billion for teachers' jobs and other domestic spending.
Topics: Schools & Youth
White House: Let Food Stamps Pay for Teacher Jobs
by Julianne Hing on July 19 2010, 8:05AM
More madness in the administration's zeal for its testing-centered education reform.
Topics: Schools & Youth
Obama's High-Stakes Teacher Bashing
by Julianne Hing on July 8 2010, 4:14PM
The White House's outsized reaction to House Democrats' effort to help states pay educators makes no sense—unless understood as crass, anti-teacher politics.
Topics: Schools & Youth


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Restaurants in Albuquerque, Santa Fe and Northern New Mexico, searchable by cuisine and location, with ratings and reviews. Get educated before you eat! Charlie's Front and Back DoorFront Door hours:
Mon-Sat: 11am-2pm, 5-9pm
Sun 11am-2pm
Back Door hours:
Mon-Sat: 11am-9pm
Sun: 11am-8pm (hours can change without notice) Full review: I liked it both ways (10/23/2008) This place is a dive. And we mean that in a good way—unpretentious and broken-in like a favorite pair of scuffed boots. The cozily dim restaurant (front) and bar (back) are separated by a kitchen that services both rooms with Charlie's signature New Mexican-American fare. Roll up your sleeves for delicious sour cream-chicken enchiladas and enormous, juicy burgers; then order a round of Bud Light for your blue-collar buddies. (Alibi Staff, August 11, 2010) Community Reviews |
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text:
Hosted at dailymotion.com This video is provided and hosted from a 3rd party server. Our search functionality is helping you discover publicly available websites & their content. Footytube as a search engine does not host or upload this material and is not responsible for the content.
Community Opinion & Comments
How did the teams perform?
Share your thoughts
Page 1 of 6
wasn't an impressive performance at all. Rooney is showing sgins of age. Valencia is the spark of life on ManU. Don't expect them to carry the cup this season
expected a dominant performance and it was there. valencia's goal was brilliant. the rotation from mcarthy was poor
ANTONIO VALENCIA :D He's Great :D Got him on my Theatre of Dreams MUFC Shirt :D Great player, he can be very importtant to MUFC :D
Yeah personally I don't understand it, I mean I think resting players giving up on a game is stupid anyway, but I don't understand how its advantageous for them, because in my eyes burnley now have the confidence boost from their brilliantly played game against arsenal. Also Man u's defence is so weakened atm surely it would be an opportune time to try and steal 3 points that were before considered unattainable?? look at what fullham did today, they took them appart
maybe should have put more past the keeper as that had ten changes and was basically their reserve side
I'm sure rooney would like to be more clinical than he is currently. He scores great goals but rarely scores with half-chances. Normal service resumed for Man Utd, which was not the hardest task given the starting line-up. Pathetic mentality from Mick McCarthy, Spoils the EPL spirit.
despite losing 3-0 marcus hahnemann had a great game in goal. and aside from the stupid handball by zubar wolves did decent on defense, especially considering most of the regular starters were out. the scoreline could have been much worse. awesome display from united though.
Wow. I could have beat the Wolves this game. And, is it just me or is Foster better than this Kuczkyck (spell?) guy??? Man, it was like Man U was just running attacking drills out there... jeez. Although, that last goal.... wow!
i don't think so. when kuz-o makes an error goals aren't the result. also, i think he has better presence and confidence in the box.
- 8 votes
- 7 votes
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3 - 0
W. Rooney PEN (30)
N. Vidic(43)
L. Valencia(66)
D. Gibson (69)
N. Vidic(43)
L. Valencia(66)
D. Gibson (69)
S. Castillo (41)


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50% Off Solid Chip Original Pages
Pantone had created new versions of their popular Solid Chips Books in January, 2005. The new books had larger Pantone chips pages, measuring 5.5" x 9.25", allowing for 25% larger chips of each color and larger type sizes for important descriptive information. The colors, color formulations and page arrangements have remained the same. The new binders were larger, but have the same three-ring size accommodating both Original size and New replacement pages. Pantone has enhanced and replaced the Pantone PMS Pages in May, 2010. The new enhancements feature 224 new colors and chromatic arrangement of all 1,341 solid colors.
For your choice and convenience, Hyatt's maintains the world's largest inventory of both Original, PMS and PLUS pages. Many of the Original pages are available at 50% off overstock discount prices.


text:
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Product Code: 05-5140
Oceanic ION 3 Scuba Mask - Designed for Women and Smaller Faces
A three-window mask featuring Seamless Panoramic Lens technology to increase visibility while underwater. No more blind spots! Designed to fit women and smaller faces, this mask comes with Oceanic's Neoprene Comfort Strap and easy-adjust swiveling buckles.


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Evaluation of experimental behaviour of normal and high strength reinforced concrete walls in two-way action
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Title | Evaluation of experimental behaviour of normal and high strength reinforced concrete walls in two-way action |
Author | Doh, Jeung-Hwan; Fragomeni, Salvatore; Loo, Yew-Chaye |
Editor | Yew-Chaye Loo, Sanaul H. Chowdhury, Sam Fragomeni |
Year Published | 2002 |
Publisher | Balkema Publishers |
Peer Reviewed | Yes |
Published | Yes |
Publication Title | Advances in Mehcanics of Structures and Materials |
Research Centre | Centre for Infrastructure Engineering and Management |
Faculty | Faculty of Engineering and Information Technology |
Publication Type | Conference Publications (Full Written Paper - Refereed) |
Please use this identifier to cite this record: http://hdl.handle.net/10072/1523
Griffith University copyright notice
Copyright in individual works within the repository belongs to their authors or publishers. You may make a print or digital copy of a work for your personal non-commercial use. All other rights are reserved, except for fair dealings or other user rights granted by the copyright laws of your country.


text:
October 29, 2013 at 11:51AM Verizon's Galaxy S 4 can now surf more LTE bands after Android 4.3 update
October 15, 2013 at 11:11PM Verizon's LTE boosted in 'major markets' by new frequency, hits 80 Mbps down (update)
August 13, 2013 at 3:08PM Amazon aims to unify push notifications across app platforms with SNS service
June 28, 2013 at 11:30AM T-Mobile to acquire 10MHz of LTE spectrum from US Cellular in $308 million deal
April 13, 2013 at 6:25AM Telus reportedly in talks to buy Mobilicity, spectrum likely to be the prize
March 26, 2013 at 1:45PM Existing iPhone 5 handsets cannot have AWS HSPA+ support enabled, but Apple is shipping the new A1428 carrier-unlocked on April 12th
March 18, 2013 at 9:00AM T-Mobile's LTE ambitions get real: network expansion, the BlackBerry Z10 and an OTA update for the Galaxy Note II
December 25, 2012 at 12:42PM Video-streaming Xmas cheer restored for Netflix users as AWS outage gets fixed for affected devices
December 24, 2012 at 8:17PM AWS outage brings Netflix down for some devices on Christmas Eve (update: back up!)
December 18, 2012 at 10:31PM AT&T scores WCS spectrum victory, covers 608 markets and 82 percent of Americans
October 5, 2012 at 1:18PM Likely HTC Windows Phone 8X with AT&T-friendly LTE surfaces at the FCC, just in time
September 28, 2012 at 8:41PM Samsung Galaxy Note II variants for AT&T, T-Mobile, Verizon possibly caught at the FCC
September 27, 2012 at 7:11AM FCC chairman green-lights AT&T's use of WCS spectrum for LTE with proposed order
August 28, 2012 at 6:06PM Verizon and Leap Wireless announce $120 million spectrum swap in wake of FCC approval
August 23, 2012 at 6:43PM FCC approves Verizon deal to buy cable company spectrum, asks for concessions
August 21, 2012 at 10:37PM Amazon launches Glacier archiving service, a cheap way to put your files on ice
August 16, 2012 at 11:15AM DOJ, FCC clear Verizon buyout of cable companies' spectrum, require giving up some airwaves (update: not quite for the FCC)
August 9, 2012 at 2:25AM T-Mobile USA Q2 2012 results show net customer losses of 205k, progress on LTE and AWS
August 2, 2012 at 10:05AM AT&T agrees to acquire NextWave for $600 million, gobbles up WCS and AWS spectrum
June 30, 2012 at 12:14AM Don't worry, you're not the only one: Netflix is currently down, admins hard at work (Update: back up)
June 12, 2012 at 5:37AM Samsung Galaxy Note with T-Mobile-ready 3G swings by the FCC (update: demo units?)
May 18, 2012 at 8:14PM Samsung Galaxy S III for T-Mobile hits FCC, brings future-proofed HSPA+ for good measure
May 14, 2012 at 9:04PM Alliance for Broadband Competition forms to sway opinion against Verizon's AWS acquisition
May 10, 2012 at 2:46AM T-Mobile reports customer growth for Q1 2012, tries not to think about Q4 2011
April 15, 2012 at 3:45AM Canonical's AWSOME API bridges OpenStack and Amazon clouds, Ubuntu has its head in both
March 29, 2012 at 11:48PM Verizon's CEO has a plan for wireless pay-TV, if the government will allow it
March 6, 2012 at 12:36PM T-Mobile, Sprint and DirecTV file with FCC to halt Verizon's AWS acquisition
February 23, 2012 at 2:18AM T-Mobile lost more customers in Q4, will launch LTE in 2013 with AWS spectrum from AT&T
February 22, 2012 at 10:54AM T-Mobile wants FCC to block Verizon's spectrum deals with cable industry
January 29, 2012 at 11:31PM T-Mobile keen to help iPhone users, plans to offer new procedures for unlocked phones


text: Current 80 times Most Times Hitting Oneself In Head With Table Tennis Paddle While Balancing On A Skateboard Embed <iframe width="560" height="345" src="http://recordsetter.com/embedvideo/4566" frameborder="0" allowfullscreen></iframe> Loading... Sign up for RecordSetter Sign up Your name: Your e-mail address: Choose a password: I am 14 or older By clicking "create account", you agree that you've read and understand the Terms of Use Already a member? × Why not? We think only a record holder can know what you go through to set a record, so we only let people that have already set world records downvote other people's records. What can I do? Go set a record! If you can think of it, you can set a new world record for it. × points Follow Record Unfollow Record Break This Record Rate it: Legendary Funny Cute Creative Gross Impressive Tags: most, table tennis, head, paddle, consecutive, hitting, skateboard Post Comment Post to Facebook Follow replies All Comments for this Record Category Under review comments Most Times Hitting Oneself In Head With Table Tennis Paddle While Balancing On A Skateboard You can press ESC to close this dialog or click close. Related Records Longest Fingerboard Rail Grind 110 inches Longest Stationary "Yeah Right" On Skateboards 01:16.75 min/sec Longest Stationary One-Footed "Yeah Right" On Skateboards 02:04.21 min/sec Longest Time To Hold A Musical Note While Riding A Skateboard And Balancing Pool Noodle On Head 00:19.44 min/sec Fastest Time For A Five-Year-Old To Ride A Toy Skateboard 50 Feet 00:07.20 min/sec Longest Skateboard Trick Combo In A Self-Recorded Video 31 tricks Longest Time Balancing A Skateboard On Feet While Performing A Shoulder Stand 09:01.30 min/sec Longest Self-Filmed Skateboard Hang Ten 01:10.14 min/sec Longest Time Balancing Skateboard On Chin While Seated 04:46.94 min/sec Longest Time Riding On A Skateboard In Primo And Balancing On A Rola Bola 00:40.23 min/sec Longest Overhead Basketball Shot Using A Skateboard 25 feet Most 180 Turns On A Skateboard In 30 Seconds 61 times Fastest Five-Yard Primo Walk 00:09.97 min/sec Longest One-Foot Nose Manual 182 feet Longest "Baby Freeze" On A Skateboard 00:51.15 min/sec


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The Caroline Pryce Walker Conquer Childhood Cancer Act of 2008 is an earmark specifically designed to increase pediatric cancer research and create a national registry for kids with cancer. It was never funded in 2009, which is one of the main reasons Clyde residents Warren and Wendy Brown went to Washington.
Watch the attached video to see Jonathan Walsh's full report on what the Browns found out about how many of your tax dollars are -- or are not -- being allocated to childhood cancer research.
Also, click the attached link for Jonathan's story about the Browns' trip to Washington.


text:
When I profiled John Barth in 2008, he described, in detail, his writing routine for the past half century. Barth said that, whether in Chestertown or Florida—where he and his wife, Shelly, spend their winters—he writes every day. After breakfast, he does stretching exercises to warm up "body and spirit," he said. Then, he goes to his desk and puts in wax earplugs, a habit from when his children were little. He reviews a printout of the previous day's effort, usually two to four pages, uncaps his Parker 51 pen—purchased at an English stationery store that advertised itself as the original site of "Mr. Pumblechook's Premises" from Dickens's Great Expectations—and begins to write. His first draft is always composed on loose-leaf paper in a binder purchased during freshman orientation in 1947. Every one of Barth's books has been written in this binder, with (since 1964) the same Parker pen. "That's the kind of anal-retentive I am," he noted. "But it's a happy routine."
These days, it isn't so happy. As Barth states in the current issue of Granta, the U.K. literary magazine, the Parker pen has gone dry for the past two years. "Although I've still gone to my workroom every weekday morning for the hours between breakfast and lunch, as I've done for decades, and faithfully re-enacted my muse-inviting ritual," he writes, "I find that I've written ... nothing."
For an iconic author with a steady output that's never wavered, that's a sobering assessment. When I emailed Barth about it, he pointed out that his essay is titled "The End?" and the question mark is more hopeful than desparate. And he recently told an NBC affiliate in Miami that he expects the next piece of writing will come "when the Muse sees fit to unhibernate. Remember that Sophocles is said to have written his `Oedipus at Colonus' at age 90. I'm a mere (and still-healthy, and patient) 81-year old."
Barth has a book of collected essays, Final Fridays, scheduled to come out later this year.
[photo: the great Dave Colwell]


text:
- Dora the Explorer (5)
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So many ways to celebrate Halloween!
Geo Halloween CostumeHomemade Team Umizoomi Halloween costumes for the Mighty Math Powers crowd |
Milli Halloween CostumeMeasuring from Milli's outfit, this will be an extremely fun Halloween! |
Bot Halloween CostumeNothing's out of reach when your child heads out for Halloween in Bot's costume! |
Dora's Fairy Princess WandYour little ones can create magical moments with this fairy princess wand |
Tasha CostumeTake Tasha trick-or-treating! |
Uniqua CostumeYour little Uniqua fan will feel pretty in pink in this costume. |
Tyrone CostumeAwesome antlers announce that Tyrone is making the rounds this Halloween! |
Nick Jr. Jack-o'-LanternsLight up Halloween night with your kid's favorite characters! |
Tolee Face PaintingTurn your preschooler into Kai-lan's koala pal, Tolee! |


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Lion and Netbiosd
Ok, I know this is a little old and since im playing around with little snitch and have exactly the same questions as some of you, the answer appears to be that plusnet is the polish devision of British telecom. Im still trying to figure out what netbios is connecting to and why? Perhaps its polling for DNS servers but unlikely.
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Yeah, I have the same issue.
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@Marc Wilson
Seems you are not interested in this problem. So why do you answer?
I´m using Little Snitch and got a message today that netbiosd tried to connect to 10.37.129.255 on port 137 (netbiosd-ns).Netbiosd is part of the BSD system. The first line of the description says: "netbiosd is responsible for interacting with NetBIOS networks".
So far I´m not sure if one needs netbiosd or not.
Like (0)
My imac is trying to connect to the same ip address (10.37.129.255). I denied the connect via Little Snitch and we'll see what effect it has.
Like (0)
my Littlesnitch alert me three times:
1- On 14-gen-2013, netbiosd tried to establish a connection to user-pc on port 139 (netbios-ssn). The request was denied via connection alert.
2-On 14-gen-2013, netbiosd tried to establish a connection to user-pc on port 445 (microsoft-ds). The request was denied via connection alert.
3-On 06-feb-2013, netbiosd tried to establish a connection to 192.168.0.255 on port 137 (netbios-ns). The request was denied automatically during login.
I blocked any connection.
Like (0)
Same here. Blocked here too. Apple installed this, Apple needs to drop the word on what this is. So, Apple?
Marc, before you even bother, troll elsewhere. I don't care.
Like (0)
LittleSnitch flagged it as trying to connect to another computer on my Wi-fi network. Said computer is a Windows machine, and once I blocked netbiosd I can no longer see that computer in Finder. Not sure if this helps anyone, but my guess is that netbiosd is meant to help Apple computers talk to Windows machines.
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Network Basic Input/Output System.
NetBIOS is a communication protocol, sort of a languace that computeres talk to each other in. What disturbs me is that at least some communications seem to go directly out through this process and not the respective processes that initiated tem.
That means NetBIOSd (the process not protocoll) appears to have devolved (from a firewall/net security point of view) to be a method of bypassing your firewall, it seems that certain applications can ask NetBIOS to ping locations on it's behalf... I do not know whether they are actually able to send data or not but according to the specification of the protocol itself they should be able to. Even if they cannot, at the very least they can 'know' that they have been firewalled by using NetBIOS to ping the location other behalf and seeing if it unreacheable too.
Due to the fact that NetBIOS is a Unix/BSD method of achieving said way, and was probably left in to make the Network Utility backend and not much else, I am inclined to block it. If any application whatsoever can make a connection via this daemon without the connection being traced back to that application, then a malicious application/user can most probably take advantage of it to contact a remote network once seeded/send out important user information/do any host of malicious things, all whilst looking like a humble part of the OSX system.
This is very much the problem that I used to have in Windows with Services all being hosted as multiple svchost processes... very much destroying your ability to destroy them as you see fit in the event of a virus threat... not to mention COM events, which did just this and allowed applications to ask windows to do things on it's behalf. Many a good virus shut your system down using COM (as in locked you out) the moment they got hands on elevated priviledges.
Like (1)
Thanks for that great info.
everytime I see (microsoft-ds) I block it forever - keeps showing different port - and each time I get (netbios-ssn) or (netbios-ns) I block until quit - just in case.
Everything works fine so I am going to assume its some kind of "sniffer" (introduced as a "useful" tool)
From now on I shall block all permanently - see how it goes - I will report back if it gives problems.
Little Snitch is one GREAT APP - very happy buyer!
Like (0)
I just encountered this today, but in my case Netbios wanted to contact forum.techhive.com!!! Safari was open but with no tabs loaded, and no other browsers were open. Why the %$*& does my OS want to connect to techhive's forum? I am not a user of Techhive's forum, and never visit it delivberately. I may have ended up there after a google search at some point yesterday (more than 12 hours ago - why is Netbios doing it's thing now?!).
Like (0)
Stumbled across this thread and am intrigued. I have little snitch installed and also allow Netbios on my local LAN to talk to Windows machines/shares. I permantly block it inbound.
However, really weird thing happened just 30 minutes ago. I am on holiday in Portugal, using Hotel Wifi, standard Open Wifi with proxy sever asking you for username and password. Booted Mac up and Netbiosd tried to connect 3 times (I blocked it once every time to see how many times it tried) to:
18.104.22.168 - port 445
Which is a Ministry of Defence IP address, I used RIPE to look it up.
This kind of freaked me out, as I am an I.T. consultant who is very aware of any suspicious traffic.
My Mac has never done this before in the previous 3 years I have had it and I have travelled a bit with it.
Is there any way I can dig into the NetBiosd process to see what it was trying to do talking to a MOD IP address??
Like (0)
I just got the same message as I was booting up Parallels. Is it that Windows or Parallels is trying to set up communication between my Mac OS and Windows?
Like (0)
Graham I am getting the same netbiosd request as you (microsoft-ds). I would be very interested to hear the result of your block all action in this matter. As I am inclined to do the same but shall wait for your research. Thanks
Like (0)
downtownbarber Cleveland
I am having a problem with connecting to the internet on my Mac mini, ATV2 & iPhone 5. All on one network though, my iPhone is connecting to my home and other networks just fine. I ran netstat in network utility and there's an active established internet connection to an ip address with .microsoft-ds on the end, this is what led me here. I have no idea how to handle this and find it difficult to follow some of the comments. If you wouldn't mind helping me I'd appreciate that and will post whatever you'd need to help me with this.? Before everything stopped working I was able to upload files to Pogoplug cloud but couldn't get my magicjack to work or connect to safari. That was very strange but now nothing connects, at least on my side. According to network utility there is a connection and it looks like it's working great but I can't do anything. This is starting to worry me, I've done all the troubleshooting steps recommended by Apple. Right now I'm making a backup of the mini and plan on doing a full wipe if I don't get it resolved through the support community. All my software is up to date running 10.9. The network I'm on is an open network at my work. The router or gateway is "AMBIT Broadband" and the provider is Time Warner.
Hope to hear back
Like (0)


text:
Nutritional Info
- Servings Per Recipe: 16
- Amount Per Serving
- Calories: 40.7
- Total Fat: 0.0 g
- Cholesterol: 0.0 mg
- Sodium: 27.8 mg
- Total Carbs: 10.7 g
- Dietary Fiber: 0.5 g
- Protein: 0.1 g
View full nutritional breakdown of Christmas Punch calories by ingredient
Christmas Punch
Submitted by: PRINCESSAMYIntroduction
Holiday punch Holiday punchNumber of Servings: 16
64 oz 100% Cranberry juice
2 liter Diet Sprite Zero
1 cup Cranberries optional
2 limes
Pour cranberry juice and sprite into punch bowl. Then chop cranberries and slice limes. Add fruit to punch. Cover and chill. Add ice when ready to serve.
Number of Servings: 16
Recipe submitted by SparkPeople user PRINCESSAMY.
Number of Servings: 16
Recipe submitted by SparkPeople user PRINCESSAMY.


text:
E3 2011: More Back to the Future Could Be on the Way
Telltale said it's open to continuing the series.
June 8, 2011
During its E3 update, Telltale said it would be open to continuing the Back to the Future games beyond Episode 5, which is coming later this year. Design director Dave Grossman said that Universal has been a "wonderful partner," and if they and the fans of the game would support more episodes, then Telltale would be happy to make more.
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text:
nessus-xmlrpc is a Ruby library for the Nessus XML-RPC interface. You can use it to start, stop, pause, and resume scans. It lets you watch progress and status of scans, download reports, etc. It comes with an example command line program that shows how easy it is to interact with the Nessus scanner.
Collax Business Server is an all-in-one Linux server for small- and medium-sized businesses. It delivers all the important network services within a heterogeneous business environment for communication, infrastructure, compliance, groupware, and storage, all in a reliable and secure way which is easy to manage. It also provides essential security functions such as firewalling and virus and spam filtering, to protect against hacker attacks, viruses, and unsolicited email messages.
A tool that makes it easier to resolve TCP/IP connection errors.