Biofilm-forming Candida species cause infections that can be difficult to eradicate, possibly because of antifungal drug tolerance mechanisms specific to biofilms. In spite of decades of research, the connection between biofilm and drug tolerance is not fully understood.
We used Saccharomyces cerevisiae as a model for drug susceptibility of yeast biofilms. Confocal laser scanning microscopy showed that S. cerevisiae and C. glabrata form similarly structured biofilms and that the viable cell numbers were significantly reduced by treatment of mature biofilms with amphotericin B but not voriconazole, flucytosine, or caspofungin. We showed that metabolic activity in yeast biofilm cells decreased with time, as visualized by FUN-1 staining, and mature, 48-hour biofilms contained cells with slow metabolism and limited growth. Time-kill studies showed that in exponentially growing planktonic cells, voriconazole had limited antifungal activity, flucytosine was fungistatic, caspofungin and amphotericin B were fungicidal. In growth-arrested cells, only amphotericin B had antifungal activity. Confocal microscopy and colony count viability assays revealed that the response of growing biofilms to antifungal drugs was similar to the response of exponentially growing planktonic cells. The response in mature biofilm was similar to that of non-growing planktonic cells. These results confirmed the importance of growth phase on drug efficacy.
We showed that in vitro susceptibility to antifungal drugs was independent of biofilm or planktonic growth mode. Instead, drug tolerance was a consequence of growth arrest achievable by both planktonic and biofilm populations. Our results suggest that efficient strategies for treatment of yeast biofilm might be developed by targeting of non-dividing cells.
Nosocomial fungal infections are a major problem for immune compromised patients with a severe underlying disease . Fungi can cause infections by colonizing mucosal surfaces in the oral cavity, airways, wounds and the gastrointestinal tract . Fungi can also adhere to invasive medical devices and cause severe septicemia upon detachment . The hallmarks of biofilms are surface attachment and production of an extracellular matrix (ECM) . Failure to eradicate microbial infections is often attributed to the unique lifestyle of cells in biofilms and it is widely accepted that cells in a biofilm possess antimicrobial tolerance mechanisms that are distinct from their planktonic counterparts .
Drugs currently being used to treat systemic mycoses belong to four major classes. The azoles target cytochrome P450 and inhibit cell membrane ergosterol biosynthesis, resulting in accumulation of toxic ergosterol intermediates . Azoles have poor efficacy against Candida species other than C. albicans, such as C. glabrata. The number of nosocomial blood isolates of these non-susceptible Candida species has increased in the past decades, possibly because of the selection that frequent azole use impose . The echinocandins inhibit 1,3-β-glucan synthases, resulting in a reduction in cell wall 1,3-β-glucan , and the polyenes target ergosterol and cause pore formation in the fungal cell membrane . The fourth class is the antimetabolite flucytosine. Flucytosine is deaminated upon uptake in susceptible cells and converted to 5-fluorouridine triphosphate, which is incorporated into RNA, inhibiting protein synthesis . Flucytosine can also be converted to 5-fluorodeoxyuridine monophosphate which acts on thymidylate synthase to inhibit DNA synthesis . Despite the pronounced diversity in antifungal mechanism of action and chemical structure, most antifungal agents are inactive against fungal biofilms .
Several mechanisms have been suggested to be responsible for drug tolerance of yeast biofilms. One of them is the ECM layer that contains β-1,3 glucans and extracellular DNA ,. Treatment of biofilm cells with glucanases or DNase result in increased efficacy of antifungal agents, which indicate a role of ECM on antifungal drug tolerance ,. However, it has been shown that antifungal susceptibility is independent of amount of matrix produced and antifungal drugs can diffuse through the matrix layer in inhibitory concentrations ,. The ECM, in combination with the nutrient-limited environment that results from a large number of microbial cells, might induce expression of genes that help cells cope with stressful conditions. Altered gene expression could involve differential regulation of general stress-response genes that affect drug tolerance. For example, efflux pumps are reported to be upregulated in young and intermediate , biofilms in Candida species. However, efflux pump knockout mutants remain drug resistant , and up-regulation is lost in mature biofilms ,. Furthermore, since polyenes and echinocandins are not a substrate of any known efflux pumps , efflux pumps are not responsible for biofilm-mediated tolerance to these drug classes. None of the suggested tolerance mechanisms are solely responsible for the multidrug tolerance associated with biofilm, and it might be a combination of several individual mechanisms that cause multidrug tolerance in yeast biofilms.
Candida is the most frequent cause of fungal infections and extensive research has been performed with this organism to investigate regulation of biofilm formation and antifungal drug recalcitrance . However, due to a limited repertoire of genetic and molecular techniques available for some Candida species, the knowledge about yeast biofilm regulation and drug tolerance is incomplete. The genetic tractability of another fungus, Saccharomyces cerevisiae, has made it a model organism for the study of fundamental issues in fungal biology . Transition from yeast to filamentous morphology is correlated to virulence in Candida albicans and key signaling pathways controlling this process is conserved in S. cerevisiae. Candida glabrata is phylogenetically more closely related to S. cerevisiae than to other Candida species  and they have homologous cell-surface adhesins . C. glabrata and S. cerevisiae furthermore form biofilms as haploids with similar biofilm architecture: thin layer of biofilm cells with yeast morphotype surrounded by a low density of ECM ,. S. cerevisiae is therefore relevant for the study of C. albicans virulence and C. glabrata biofilm. S. cerevisiae has previously been used as a model organism to study yeast biofilm development and regulation by taking advantage of the molecular tools available for this organism -. However, much less effort has been made to investigate the response of S. cerevisiae biofilm cells to antifungal treatment ,,. S. cerevisiae has the potential to cause human infections  and its ability to adhere to plastic surfaces , makes it a relevant organism for the study of yeast biofilm tolerance towards antifungal agents.
Fungal and bacterial research report 1000-fold higher tolerance level of mature biofilms compared to proliferating planktonic populations ,. Research in bacteria has shown that the tolerance phenotype is similar between biofilm and planktonic cells when cultivated for equally long time in identical medium -. This indicates that tolerance mechanisms are not biofilm-specific and that planktonic cells can achieve the same level of tolerance. To address if growth arrest is also relevant for drug tolerance in yeast biofilm, we have compared susceptibility of common antifungals in biofilms and planktonic cells cultivated under similar conditions. We used in vitro biofilms of S. cerevisiae and C. glabrata cultures to investigate antifungal tolerance to drugs from each of the major antifungal drug classes used for systemic treatment of human pathogenic fungal infections: the polyene amphotericin B (AmB), the azole voriconazole (VOR), the antimetabolite flucytosine (5FC), and the echinocandin caspofungin (CAS). We found that the ability of biofilms to survive antifungal treatment was dependent on the mode of action of the antifungal agent and the growth state of the yeast cells.
We initially determined antifungal drug susceptibilities of exponentially growing planktonic S. cerevisiae and C. glabrata cells towards four antifungal compounds, AmB, VOR, 5FC, and CAS. Drug susceptibilities of S. cerevisiae cells were similar to C. glabrata as determined by minimal inhibitory concentrations (MIC), except for VOR that was 4 μg/ml against C. glabrata, and 1 μg/ml against S. cerevisiae (Table 1).
VOR: voriconazole, 5FC: flucytosine, CAS: caspofungin, AmB: amphotericin B.
Yeast biofilm architecture and antifungal drug sensitivity was investigated using confocal laser scanning microscopy (CLSM). Mature GFP-tagged biofilm cells were challenged with an antifungal agent for 24 hours and stained with propidium iodide (PI) to identify dead cells. S. cerevisiae biofilms contained a thin layer of cells (approximately 30 μm) with a few dead cells distributed throughout the biofilm. Biofilms treated with VOR, 5FC, or CAS had the same architecture and mixture of living and dead cells as untreated control cells (Figure 1), showing that the drugs were inactive against yeast biofilms. AmB was the only tested drug with anti-biofilm activity, killing most cells after 24 hours (Figure 1). The small subpopulation of cells that survived AmB treatment was randomly distributed in the biofilms.
Mature S. cerevisiae and C. glabrata biofilms have similar sensitivity to antifungal drugs. Antifungal drug activity against 48-hour biofilms was visualized by confocal laser scanning microscopy. GFP-tagged S. cerevisiae was stained with propidium iodide (PI, 1 μM) to mark living (green) and dead (red) cells. C. glabrata was stained with Syto9 (3 μM) and PI (1 μM) to mark living (green) and dead (red/yellow) cells. Biofilm cells were treated for 24 hours with the indicated antifungal agents. VOR: voriconazole (10 μg/ml for S. cerevisiae and 50 μg/ml for C. glabrata), 5FC: flucytosine (80 μg/ml), CAS: caspofungin (10 μg/ml for S. cerevisiae and 5 μg/ml for C. glabrata), and AmB: amphotericin B (10 μg/ml for S. cerevisiae and 20 μg/ml for C. glabrata).
To determine if results from the S. cerevisiae biofilm model applied to drug susceptibility in a pathogenic yeast, we investigated the antifungal drug susceptibility of C. glabrata biofilms. C. glabrata was cultivated under conditions similar to S. cerevisiae cultures and developed a thin layer of biofilm cells (approximately 25 μm). After 48 hours, mature biofilms were challenged with an antifungal drug for 24 hours and stained with Syto9 and PI to visualize living and dead cells. Results were similar to those obtained with S. cerevisiae. Most C. glabrata biofilm cells exposed to VOR, 5FC or CAS showed living cells with a few dead cells distributed in the biofilm, similar to the appearance of the untreated control cells (Figure 1). AmB treatment killed most cells with a small, surviving subpopulation randomly distributed in the biofilm. These results suggested that S. cerevisiae could be used as a model organism to study antifungal tolerance in biofilms of the pathogenic C. glabrata.
Planktonic microbial cells cultivated in a closed system take up nutrients from the environment and enters a stationary growth state with decreased metabolic activity when nutrients become limited. To investigate if the metabolic activity of biofilms at 48 hours was reduced compared to a 4 hour biofilm, we measured metabolic activity using FUN-1 staining. FUN-1 permeabilizes the plasma membrane and biochemical processing of the dye by an unknown pathway identifies metabolically active cells with intravacuolar structures . Most cells in a 4-hour biofilm showed high metabolic activity as estimated by staining intensity of the vacuole (red color, Figure 2), but staining decreased with biofilm incubation time. After 48 hours, only a small subpopulation of cells in the biofilms showed FUN-1 staining of vacuoles indicating a lower or different metabolic activity in mature biofilm than that found in young 4 hour biofilm (Figure 2).
Metabolic activity of yeast cells in biofilms decreases with incubation time. S. cerevisiae cells stained with FUN-1 (10 μM) after incubation of biofilm for 4 hours and 48 hours respectively. Metabolically active cells produce red cylindrical intravacuolar structures.
We hypothesized that the limited metabolic activity and resulting lack of growth observed in cells in mature biofilms may be an important cause of the low antifungal activity of the drugs tested. We measured therefore the killing kinetics of the antifungals using an exponentially growing planktonic population and a growth-arrested planktonic population. Untreated control cells proliferated with a doubling time of 1.5 hours in the exponential growth phase for the first 8 hours of incubation (Figure 3A). The density of cells exposed to VOR increased at the same rate as the untreated sample for the first 7 hours of incubation. Subsequently, the azole drug inhibited growth, resulting in a decrease in viability after 24 hours. After two hours of exposure to 5FC, the growth of exponential phase populations was inhibited and cells remained at the same viability and density for 24 hours showing that 5FC had fungistatic activity. CAS had an inhibitory effect on exponential growth within the first hours of exposure and a consistent killing rate throughout the experiment that resulted in a 10-fold reduction in colony forming units (CFUs) after 24 hours compared to the initial population. Challenging cultures with AmB rapidly decreased the viable population, reaching the lower detection limit for CFUs after 5 hours.
Antifungal drug susceptibility in exponentially growing and growth arrested planktonic cells. (A) Killing kinetics of exponentially growing planktonic S. cerevisiae cultivated in 2% glucose exposed to 5 times the MIC of the indicated antifungal drugs. (B) Killing kinetics of growth-arrested planktonic S. cerevisiae cultivated in carbon-depleted minimal medium exposed to 5 times MIC of the indicated antifungal drugs. VOR: voriconazole (5 μg/ml), 5FC: flucytosine (40 μg/ml), CAS: caspofungin (5 μg/ml), AmB: amphotericin B (5 μg/ml). n = 3, error bars show standard deviations.
We next investigated how growth arrest affects susceptibility to antifungal agents by incubating cells in carbon-depleted medium. Starting cell density was similar to the starting density used for time-kill studies on the exponential growing populations to eliminate cell numbers from affecting comparisons between the two experiments. Growth-arrested S. cerevisiae exposed to VOR, 5FC or CAS had viability similar to untreated control cells, showing that the drugs had no activity against non-growing cells in stationary phase (Figure 3B). Cells exposed to AmB were killed, but the killing rate was lower than the rate observed for exponentially growing cells. The lower detection limit for CFUs was not reached in the first 8 hours of drug exposure, but only after 24 hours.
The antifungal activity of 5FC, CAS and AmB against exponentially growing planktonic S. cerevisiae, but not against growth-arrested cells, suggested that drug activity depended on cell growth. To test if growth-dependent drug activity also applied to cells in biofilms, the viability of a growing S. cerevisiae biofilm was quantified as CFUs and visualized with CLSM. Quantification and visualization assays were conducted after 24 hours of drug treatment. The number of cells in untreated 4-hour biofilms increased 8-fold over 24 hours, as determined by CFUs (Figure 4). Growth in 4-hour biofilms exposed to VOR or 5FC was inhibited compared to untreated control biofilms. Cell numbers determined by CFUs increased 3-fold with VOR treatment and 1.3-fold with 5FC treatment. In contrast, 80% of biofilm cells exposed to CAS were killed, with surviving cells sporadically distributed in the biofilm (Figure 4). AmB had a fungicidal effect on young biofilms, killing 99.7% of cells during a 24-hour exposure. Biofilms treated with AmB still contained minor surviving subpopulations (Figure 4).
Antifungal drugs are active against growing biofilms. (A) Quantitation of biofilm cell viability. Biofilms were grown for 4 hours and viability determined as CFUs. Biofilms were then treated with 10 times the MIC of the indicated antifungal drug for 24 hours and CFUs were measured. Shown is log change in CFUs after 24 hours treatment. n = 3, error bars are standard deviations. Statistical significance between treated and untreated samples was evaluated with Student's t-test. *P < 0.01. (B) GFP-tagged S. cerevisiae in 4-hour biofilms treated for 24 hours with the indicated antifungal agents. VOR: voriconazole (10 μg/ml), 5FC: flucytosine (80 μg/ml), CAS: caspofungin (10 μg/ml), and AmB: amphotericin B (10 μg/ml). Cells were visualized as described in the legend to Figure 1.
We observed that most drug classes tested in this study were inactive against mature biofilms (Figure 1) and planktonic cells that are arrested for growth (Figure 3), whereas growing biofilm and planktonic cells were susceptible to both AmB, VOR, 5FC, and CAS. These data suggest that the physiological state of the cell in response to ceased proliferation, rather than a biofilm-specific response mediate drug tolerance in yeast biofilms.
To determine the effect of growth arrest on drug tolerance, we investigated differences in antifungal drug susceptibility between stationary cultures of cells grown planktonically or in biofilms for 48 hours. S. cerevisiae biofilms were cultivated on flat polystyrene surfaces. For planktonic control populations, S. cerevisiae was cultivated in glass tubes . An isogenic biofilm-deficient flo11 knockout mutant was included as a negative control for biofilm formation . The average inoculum before drug challenge was 1.6 × 107 CFU/ml for biofilm cells, 1.8 × 107 for planktonic cells and 7.5 × 106 for flo11, minimizing the influence of different cell densities on drug susceptibility between the cultivation assays.
All three cultures, biofilm, flo11 control, and planktonic, were challenged for 24 hours with antifungal agents. No significant effects on CFUs were seen after treatment with VOR, 5FC, or CAS (Figure 5), indicating that growth arrested S. cerevisiae was not susceptible to any of the drugs under any of the three growth conditions. Only treatment with AmB significantly decreased population sizes (P < 0.01, Student's t-test). Exposure to AmB killed 95-98% of the yeast populations regardless of growth condition (Figure 5). However, in all three growth conditions, a subpopulation of 2-5% of cells survived drug treatment, so AmB was unable to eradicate the entire S. cerevisiae population.
Stationary S. cerevisiae cells grown in biofilms and planktonic cultures have similar drug tolerance. Cultures were grown for 48 hours and viability was measured as CFUs. Cells were treated with 10 times MIC of the indicated antifungal drug, or left untreated as a control, and CFUs were measured after 24 hours. Shown is log change in viability. White bar: wild type S. cerevisiae Σ1278b grown as biofilms on polystyrene surfaces. Grey bar: wild type grown planktonically in glass flasks. Black bar: flo11 knockout mutant grown planktonically in glass flasks. VOR: voriconazole (10 μg/ml), 5FC: flucytosine (80 μg/ml), CAS: caspofungin (10 μg/ml), AmB: amphotericin B (10 μg/ml). n = 3, error bars show standard deviation. Statistical significance between treated and untreated samples was evaluated with Student's t-test. *P < 0.01.
In the current study, we found that antifungal drug efficacy against S. cerevisiae biofilm was dependent on cell growth. Only growing S. cerevisiae cells were susceptible to growth inhibition by the fungistatic drugs VOR and 5FC, and killing by CAS. However, S. cerevisiae cells in both growing and stationary state were efficiently killed by AmB. We further observed that the effects of antifungals were independent of biofilm or planktonic modes of growth.
Multidrug tolerance mechanisms in biofilms are suggested to include production of an ECM and a densely packed microbial structure that shields cells, preventing antimicrobials from reaching their targets. Cell-surface proteins in the Flo family are responsible for S. cerevisiae adhesion and ECM production . Flo11p is the only flocculation protein expressed in the Σ1278b S. cerevisiae strain and it is essential for biofilm formation in liquid medium ,-. We showed that a flo11 mutant has an antifungal tolerance phenotype that is similar to mature yeast biofilms (Figure 5). This finding suggests that cell-cell adhesion and Flo11p-dependent matrix production are not obstacles for cell penetration by antifungal drugs in S. cerevisiae biofilm. This observation is in contrast to the drug sequestering role of matrix β-1,3 glucan in Candida biofilms ,, and might reflect differences in matrix composition between Σ1278b S. cerevisiae and Candida biofilms. The ECM layer is, however, not the sole contributor to the drug tolerant phenotype because systemic antifungals can penetrate the ECM of Candida species biofilms at concentrations that exceed the MIC values, and no correlation is observed between the amount of matrix produced and drug susceptibility ,. In agreement with this, our results show similar drug susceptibility between S. cerevisiae and C. glabrata biofilms, suggesting another important contributor to the observed antifungal drug tolerance phenotype.
Heterogeneous microbial biofilms often contain large subpopulations with low metabolic activity ,. We showed that the metabolic activity of most cells in yeast biofilms decreased as the biofilm matured (Figure 2) and we observed no increase in cell density in mature biofilms (Figure 5A). Therefore, large fractions of cells in mature biofilms are likely in a stationary state. Even though antimicrobial agents have diverse modes of action, most are dependent on active growth to kill cells , which we confirmed in the present study.
The drug 5FC has fungistatic activity against Candida species . We found a similar fungistatic activity against proliferating S. cerevisiae Σ1278b planktonic and biofilm populations (Figure 3A and Figure 4). However, 5FC was inactive against mature biofilms and growth arrested planktonic cells (Figures 1, 3B and 5). This result is not surprising since fungistatic drugs do not kill cells but only inhibit proliferation. Therefore, the viability of high-density, nongrowing microbial populations such as mature biofilms or stationary phase planktonic cells is expected to be unaffected by treatment with fungistatic drugs.
Echinocandins have fungicidal activity against Candida species  and we found that CAS killed 90% of exponential growing planktonic S. cerevisiae cells (Figure 3A) and 80% of proliferating biofilm cells (Figure 4). Despite the ability of CAS to kill exponentially growing yeast cells, CAS had no activity against mature biofilms (Figures 1 and 5). CAS inhibits 1,3-β-glucan synthase, which disrupt the yeast cell wall and result in osmotic stress and cell lysis . However, the synthase is most active in growing cells ,, so CAS is unable to kill growth-arrested cells  as we observed in this study (Figure 3B) including the cells in mature biofilms.
The polyene AmB was the only drug tested in this study with activity against cells in mature biofilms (Figures 1 and 5) and growth arrested planktonic cells (Figure 3B). AmB binds to ergosterol in the cell membrane and form pores that increase the permeability of electrolytes and small molecules. Pore formation results in loss of membrane potential and eventually cell lysis . Since ion diffusion and lysis are passive processes, cell metabolism is not required for AmB to kill cells. Consistent with this mechanism, AmB killed both growing and non-growing yeast cells (Figure 3). However, although AmB killed cells in non-proliferating, low-density yeast populations, AmB-tolerant subpopulations were observed in stationary state planktonic and biofilm populations (Figure 5).
Azole drugs are fungistatic against C. albicans, but less active against other Candida species, which show a clear increase in cell density after azole treatment ,. The poor efficacy of the azole drug VOR against S. cerevisiae cells within the first 7 hours of exposure (Figure 3A) might be because S. cerevisiae and the closely related C. glabrata share a mechanism that makes them intrinsically resistant to azoles.
Bacterial cells grown as biofilms or grown to stationary state as planktonic cells have similar drug-tolerance phenotypes ,,,; we have in the present study extended this phenotypic similarity to include yeast. Our results indicated that the biofilm mode of growth itself does not result in antifungal tolerance. Rather, the lack of cell division and the physiological state of stationary phase cells is responsible for the drug-tolerant phenotype.
A combination of factors is probably responsible for the multidrug tolerance of cells in biofilms. However, as long as biofilm populations contain non-proliferating cells, some of the most commonly used antimicrobials will have reduced efficacy. Biofilm tolerance to drugs is conditional and depends on the mode of action of the tested drugs, as well as cell physiology and environment . We showed that standard laboratory yeast biofilm models and methods can determine cell culture conditions under which antifungal drugs are effective or ineffective. Our findings imply that biofilm tolerance phenotypes might be caused by the large number of stationary cells within mature biofilms rather than specific biofilm mechanisms. Our data therefore suggest that future research on novel drugs and treatments should focus on strategies that are effective against stationary non-growing cells, rather than attempting to develop specific anti-biofilm treatments. The results obtained in this study are based on in vitro experiments and relies on the value of S. cerevisiae as a model organism for the pathogenic fungi. Our results indicate that S. cerevisiae and C. glabrata biofilms have similar antifungal sensitivity, but the results should also be verified in an in vivo model.
S. cerevisiae Σ1278b YS-11 (MAT a can1Δ::STE2p-spHIS5 lyp1Δ::STE3p-LEU2 his3::HisG leu2Δ ura3Δ) was used as reference strain (a gift from the Boone Laboratory, University of Toronto). A flo11 mutant that does not form biofilm was obtained from the Σ1278b gene deletion library . C. glabrata (ATCC 90030) was obtained from the American Type Culture Collection. A strain expressing green fluorescent protein (GFP) was constructed by expressing the GFP gene from the TEF1 promoter. The TEF1 promoter was PCR amplified from pSP-GM2  with primers TEF-F: 5'-CGTGCGAUGCCGCACACACCATAGCTTC and TEF-R: 5'-ACGTATCGCUGTGAGTCGTATTACGGATCCTTG. GFP was amplified from pJBA27a  with primers GFP-F: 5'-AGCGATACGUAGCATGCGTAAAGGAGAAGAA and GFP-R: 5'-CACGCGAUTATTTGTATAGTTCATCCATGCC. The GFP and TEF1 DNA fragments were simultaneously fused and cloned into a digested vector with USER (uracil-specific excision reagent) technology as previously described ,. In short, the vector pXI-2  was digested with Asi SI and nicking enzyme Nb.BsmI. Ten μl of digested vector was mixed with 5 μl of each DNA fragment, 1 μl USER enzyme and 1.5 μl mili-Q water. The mix was incubated for 25 minutes at 35°C followed by 25 minutes at 25°C. Subsequently, the reaction mixture was used directly to transform competent Escherichia coli cells (DHα5). The resulting plasmid was denoted pRKB5. The TEF1p-GFP fragment was inserted in chromosome XI position (91,575..92,744) of the reference strain using a high-efficiency transformation protocol  and transformants selected on synthetic complete agar medium that did not contain uracil.
All experiments were performed in synthetic complete medium (0.67% yeast nitrogen base supplemented with glucose and amino acids) , which is the standard medium for the study of S. cerevisiae biofilms . A 0.2% (w/v) glucose concentration was used in all biofilm experiments. Yeast extract peptone dextrose (YPD)  agar plates were used for colony counting. Antifungals VOR, 5FC, AmB and CAS were from Sigma-Aldrich. All antifungals were dissolved in DMSO in 5 mg/ml stock solutions and stored at -20°C. All experiments were performed in triplicate.
Minimal inhibitory concentrations (MIC) were determined as previously described  with modifications. In short, two-fold dilution series of antifungal drugs were prepared in fresh synthetic complete medium with 2% glucose (w/v) and distributed into 96-well microtiter plate. Synthetic RPMI medium is recommended for MIC assays by EUCAST, but S. cerevisiae grows poorly in RPMI and MIC was therefore determined in synthetic complete medium. Visibly turbid overnight cultures were diluted to OD600 0.1 in fresh medium and transferred to microtiter plates containing aliquots of serially diluted antifungal drug. Plates were statically incubated at 30°C for 24 h and absorbance was measured with a microplate spectrophotometer (BioTek PowerWave 340). Growth inhibition of ≥ 50% was determined as MIC for CAS, VOR and 5FC and ≥ 90% growth inhibition was determined as MIC for AmB as recommended by EUCAST .
Visibly turbid cultures were diluted to OD600 0.1. After 2 hours at 30°C, cells were transferred to biofilm chambers (Technical University of Denmark) with a polyvinyl chloride (PVC) coverslip surface (Rinzl, Electron Microscopy Sciences). After 4 or 48 hours static incubation at 30°C, medium was removed from biofilm chambers and centrifuged and antifungal drug was added to the supernatants at 10 times the MIC. Spent medium with drug was introduced to biofilm cultures followed by 24 hours at 30°C. Chromosomally integrated GFP and 3 μM Syto9 (Invitrogen) were used to visualize live cells and 1 μM propidium iodide (Sigma-Aldrich) was used to stain dead cells. CLSM was performed with a Zeiss LSM710 microscope equipped with excitation lasers at 488 nm and 514 nm. Imaging used an EC Plan-Neofluar 40x/1.30 Oil lens.
Preparation of cell cultures and CLSM imaging was as described above except a Plan-Apochromat 63x/1.40 Oil DIC M27 objective was used. Metabolically active cells were distinguished from inactive cells with 10 μM FUN-1 as described by the manufacturer (Molecular Probes, Probes for yeast viability, MP 07009). Cells were considered metabolic active if they produced red cylindrical intravacuolar structures ,.
Overnight cultures were diluted to OD600 0.01 in fresh synthetic medium. Yeast cultures were grown to exponential phase in baffled shake flasks at 30°C and samples were distributed to test tubes for exposure to antifungal drugs at 5 times the MIC before incubation at 30°C with aeration. Samples were extracted at indicated time-points. CFUs were determined by plating serial dilutions on YPD agar.
Visibly turbid cultures were diluted to OD600 0.1 in synthetic medium and grown in baffled shake flasks for 2 hours. Culture samples were distributed to glass tubes for planktonic cells and polystyrene microtiter plates for biofilms and incubated statically at 30°C. After 4 or 48 hours, cells were challenged with antifungal drug at 10 times the MIC, added in spent medium, for 24 hours. Viable cells were determined by counting CFUs on YPD agar. Biofilm cells were washed twice in saline and CFU was determined.
Unpaired Student's t-test was used for statistical analysis. P < 0.01 was considered significant. All statistical calculations were performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA.
We thank Charlie Boone and Owen Ryan (University of Toronto) for the Σ1278b strains. We also thank Tomas Strucko (Technical University of Denmark) for GFP construct strategy design. This work was supported by the Danish Agency for Science Technology and Innovation is (FTP 10-084027).
RB, BR and AF designed the experiments. RB performed the experiments. RB, BR and AF analyzed the data. RB, BR and AF wrote the manuscript. All authors read and approved the final manuscript.


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When it comes to purchasing room furniture, leather-based is definitely a good choice. Not only does it look great with many designs, but its extremely long lasting (its the perfect material for a home with children or animals) and its ultra-simple to clean, as well. The downside of leather furniture? It may have a much higher price tag than material, microfiber or faux leather Mccullers End Table with Storage. This reclining loveseat remains a financial budget-friendly choice due to 1 guru technique: its seating area is upholstered with leather, as the attributes are upholstered with increased affordable fake leather-based. Which means you get the appear and feel of the complete leather-based loveseat with no hefty cost. Use the lever on the loveseats equip to relax and relax on its higher-density foam filling, and consider all the money held on. This specific loveseat comes with an additional reward: expert set up will come in many locations of the nation for an additional charge.
This couch is caused the bell-formed contour along with the higher armrest from the cushioning. Additionally, the top is supported by obstructed part edges. The thighs, however, have became a member of the fake wood wrapping. The sofa provides a comfy and lavish feeling. This is the immediate consequence of extremely flexible cushioning within the rayon furniture. You can sit down effortlessly for the moment to relax. Each one of the shades provided with this sofa shows its noteworthy element, Flexible design. The red-colored tone would be ideal for both subjects with mild and dark shades. That remains constant for different shades that it offers too. The couch is located in space. Whether or not its Thanksgiving holiday or perhaps a huge loved ones reunion, you can rely on it. Race-watching yet still time falling apart in your couch together with your buddies is really a genuine option.
The Lawson might be known as perfect American sofa design. It's comfy and straightforward: The boxy form is large and characteristically has three back again cushions as well as three chair soft cushions. The classic Lawson also offers A taller back and container-formed soft cushions that have welts in the sides, as do the rear pillows. Well suited for cuddling and sleeping, this couch style was created for Jones Lawson, a united states copper mineral tycoon in the turn of the hundred years. He wanted a settee which was quite different from the picky Victorian designs that were common in those days. Current versions of the Lawson may also incorporate metal or wood included in the hands. Most importantly, this couch design is the one that will change its vibe using the fabric you select for that furniture. The form is really versatile that it can be glamorous, magnificent, commercial, casual or official depending on your material choice.
The term this is actually the comfort and sophisticated appear. This set includes a tufted back rest and nail head accent that gives it the subtle look. Its gray color contributes to its impressive appear. It can match up to 8 inches mattress. That on your own lets you know how comfy the established can be. Notably, it doesn't include a bed mattress. So you may need to purchase one. The trundle includes a castor to allow easy access. You may even pull the trundle for people to take a seat on. Simply to mention, it should take putting together, however its super easy to put together.
Even though this reclining loveseat has a heftier price tag, its classic design, options for customization and automated reclining program make it worth the dough. Its handcrafted wood body is cushioned with lower blend cushions for a softer feel than youd receive from high-density foam soft cushions, and also the furniture will come in a whopping 72 color and material combinations. The supply of so many choices makes it additional simple to pair this lying loveseat with different patterns, supplies, highlights and decorations designs. Plus, for those who have children or animals (or each!), you have the option to choose a more durable, stain-resistant material. One client discussed the Sunbrella canvas material has organized well towards her pets and children. This lying loveseat may have a classic, old-school look, nevertheless its constructed-in technology is not. Not only does it have an automatic reclining program (that provides you with a complete lie down, with your foot rest completely similar towards the seat), but it features a Universal serial bus interface for implementing or simply getting your iPad, apple iphone, Amazon kindle or laptop.
This couch is caused the bell-shaped shape along with the upper armrest from the cushion. Additionally, the top is based on obstructed part edges. The legs, nevertheless, have became a member of the fake wooden wrapping. The sofa provides a comfortable and extravagant sensation. This is the immediate consequence of very flexible cushioning within the polyester furniture. You can sit down easily for now to unwind. Each of the shades offered with this couch demonstrates its significant component, Versatile style. The red tone could be ideal for each topics with light and darkish shades. That continues to be constant for different shades it provides too. The sofa is located in room. Whether or not its Thanksgiving or perhaps a large family gathering, you can trust it. Race-watching while at the same time falling apart on your sofa with your buddies is really a genuine choice.
Copyright © Gravity 2 Drawer Nightstand By South Shore in End Tables All right reserved.


Behind those wrinkled eyes are answers to all our questions. Yet he looks on silently, for he thinks that we must find answers of our own. A fibre glass sculpture with many interpretations by Hiralal Rajasthani.


For lack of a topic, someone say "word block" as I dig right into it. We are all unique persons and special in our own way but isn't it funny how we just sometimes imagine that we are more important than other people when put in certain similar situations. Like you deserve better than the other person or somehow you should be pulled out from that scenario first, like you're too cool for it. A special treatment kind of thing. With that thought, all your common courtesy becomes too common and you let it out the window all together, I mean both the common and the courtesy.
Parrot Talk: We are unique and special in our own way but we're all still parrots when faced with the same predicaments.
Your fuel tank is blinking *empty* and it's bumper to bumper. There's a fat chance you could stall.
Traffic has got nothing on you, you're just bumping music and enjoying it but of course cursing a lil bit here and there.
Lets assume those are 6 different people, each frustrated due to their own reason. However unique we all are, we've have been subjected to this situation together. The ones driving their sleek, normal or beat up cars, to the ones using public transport. Maybe the ones in their cars at least have their own space, air conditioning, good music unlike the ones in public transport. But still we're all facing the same problem.
There's a chance that person 1. could think that person 2. as per the reasons provided above, is having a field day as they wait for the traffic as they clear off. For a moment there person 1. puts themselves on a pedestal, up there and all over sudden it's them against the world. If they could shove the cars to pave some way for themselves, they would. This is you, if you see it fit to just overtake all the other "patiently parked" cars and squeeze in somewhere as the far front and feel good about yourself.
Another crazy scenario, you enter the bank and you find the queue has nicely and stagnantly coiled itself like a well fed anaconda, did you just see what I did there?. Now this is me, I have just come to pay my rent because my landlord has this account at National Bank. Am telling you it's like that bank is cursed, whether you go in the morning, tea time, lunch time, the long lines never miss. The rent has to be paid and as much as I think that I have to go back to the office or I have other errands to run, I have to snail through the line. Sometimes I wish I knew someone so that I can make that payment in a jiffy or I knew one the people at the front of the queue, so that I could sneak my way through. I also feel overly special sometimes, maybe almost all the time. Like why the hell are there long lines in the first place, if only I had a say over it.
It's amazing how the world brings you back to your feet, when your head is floating up in the sky. Such situations remind us that we're all unique but there are certain things that put us in the same calibre with the rest regardless.


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Home > Health & Fitness > Vitamin/Mineral Chart > Why Do You Need a THC Free CBD?
Why Do You Need a THC Free CBD?
By Living Better on March 16, 2021
Cannabis sativa L., an incredibly useful species of plant that includes both hemp and marijuana varieties, has been gaining acceptance and growing in popularity in the United States, where previously all forms of this plant were banned. There are many reasons to return cannabis to its status as a beneficial herb – in addition to its industrial uses (as hemp fiber, oil, and seed,) cannabis has a number of medicinal uses that have gone ignored as governments sought to regulate its more intoxicating compound, THC.
About THC and CBD
The two most well-known cannabis compounds, due to their medicinal (and recreational) uses, are THC (tetrahydrocannabinol) and CBD (cannabidiol.) THC stimulates appetite, elevates mood, reduces the perception of pain, and produces a euphoric "high" (which can impair cognition and motor function for a time.) CBD lowers anxiety, reduces inflammation and neuropathic pain, and can improve sleep. It also has a dramatic effect on reducing seizures in certain rare childhood seizure disorders. While both compounds can be found in all cannabis plants, THC is more concentrated in the marijuana plant, and CBD is more concentrated in hemp. Because the two compounds have distinct effects, there is a demand for a THC free CBD.
The Farm Bill of 2018 gave hemp and hemp products, including CBD, legal status in the U.S. Currently, "hemp" is defined as cannabis plants and their derivatives containing less than 0.3% THC. The laws continue to shift in favor of the usefulness of hemp, and cannabis in general. A proposal in the Senate would raise the level of THC allowable in hemp plants to 1% (reducing the requirement on farmers to destroy plants, which don't recognize the arbitrary limits set on them.) Also, the number of states allowing medicinal or full recreational use of cannabis continues to grow.
At the federal level, and in states where marijuana and THC is currently illegal, the sale, possession, and use of products containing more than 0.3% THC is against the law. In some states, products containing THC are allowed only when prescribed for a medical reason. In some others, all marijuana products are legal. However, CBD products that do not contain THC are legal in all 50 states.
Drug Testing for Work or Sports
CBD provides a number of benefits useful for working people and athletes, where the perception-altering effects of THC would be unwelcome. A broad-spectrum CBD, which contains other cannabinoids and terpenes that work in concert to produce anti-inflammatory and neural-protective effects in the body, is considered the best way to receive the benefits of CBD. However, while CBD does not trigger a positive drug screening, some other cannabis compounds like cannabinol (CBN) and THC will.
Athletes and others who want the pain-relieving, anti-inflammatory, and anxiety-reducing effects of CBD without risking a failed drug test need broad-spectrum CBD that THC free. Bluebird Botanicals makes such a product.
High on Life (Not THC)
Non-psychoactive cannabinoids have a growing body of research that supports the health effects of daily use. CBD and related compounds can lower inflammation, which is a factor in a wide range of diseases including arthritis, autoimmune disorders, allergies, chronic pain, and cancer. It acts as an antioxidant to protect against oxidative stress (another factor in many progressive disorders.) It reduces anxiety and can improve sleep. With all of these benefits, there is good reason to seek a CBD that can be used daily without the intoxicating effects of THC.
CBD compounds that do not contain psychoactive substances are also safe for children. To date, there is one FDA-approved medicine containing CBD that is intended for children, Epidiolex, which treats a severe seizure disorder. While research in this area is still lacking, it is possible that CBD may have other uses for childhood disorders characterized by inflammation, anxiety, and/or poor sleep.
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The 2017 National FFA Band, Chorus and Talent applications are available on www.ffa.org/convention (scroll down to the band, chorus and talent section). Members will need to log in using their user name and password to access each of the applications.
Applications are due on Friday, July 7, 2017. State Staff and applicants will be notified of each application's status by Friday, August 11, 2017.


CULN 150 is an introduction to the fundamental concepts, skills, and techniques of basic baking. Special emphasis is placed on the study of ingredient functions, product identification, weights, measures, and proper use and maintenance of bakeshop tools and equipment. Students are assigned to stations each day and are required to apply the basic baking concepts and techniques in preparing items such as quick breads, yeast breads, rolled-in dough, pâte à choux, pies, cakes, cookies, puddings and pastry creams.


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Amanda Voisard
Home › Advocate Magazine › Spring 2015 › Sticking it Out
Sticking it Out
GUEST ESSAY Colorado Plateau Advocate magazine, Spring 2015
In a West transformed by climate change, there will be boomers, busters, and stickers. Which are you?
BY WILLIAM DEBUYS
"We aren't going anywhere."
You hear that statement, in various forms, at Hopi and Zuni, up on the Big Rez, and down at Cibecue and Ajo. You hear it over in Colorado's Ute country and among the New Mexico Pueblos and the Paiutes of Utah. It most often comes up when people have been rebuffed by some incarnation of Authority but refuse to give in. It expresses equal parts patience, resistance, and a long, deep view of time.
Wallace Stegner never wrote much about Indian people. The omission is conspicuous in the brilliant, cumulative portrayal of the West that was his life's work. One of Stegner's most memorable formulations painted the white settlement of the West as moving forward on parallel tracks, one dominated by "boomers" and "busters" and the other by "stickers." Stegner viewed the first group, to which his father tragically belonged, with a mixture of compassion and contempt. It included hustlers and hucksters, promoters of get-rich-quick schemes in agriculture and commerce, speculators in mining, railroads, and real estate, bloviating politicians, drum-beating journalists, and legions of others committed to deceiving themselves and others about the possibilities of the West.
The second group, the stickers, consisted of people who came to the West to make a home, not a killing, and who were clear-eyed enough to see the limitations and fierce realities of the actual land.
Stegner argued that the best thing for the stickers would have been adoption of John Wesley Powell's plan for western settlement. Major Powell, in whom Stegner found a kind of intellectualized, replacement father, advocated environmental restraint, using land only for the purposes to which it was best adapted and distributing it in ways that avoided great concentrations of wealth. Stegner thought that Powell and the stickers, had they been allowed, might have built a West more in harmony with the environment, and more democratic and broadly prosperous, too.
If Stegner, who died in 1993, were alive today, maybe he would finally include native people in his favored group. After all, they are the original stickers.
The problem of nativeness touches us all, and it touches the land, too. Outsiders, at least those who consciously try to connect to where they live, face the challenge of sinking roots in their chosen place, which means, to borrow a phrase from Wes Jackson, becoming native to it. Even natives sometimes have to work to stay native, not in the sense of birthright, but in the sense of feeding the connection that binds them to a particular patch of the planet. George Eliot wrote, "If we had a keen vision and feeling of all ordinary human life, it would be like hearing the grass grow and the squirrel's heart beat, and we should die of that roar which lies on the other side of silence." She's right, of course, but short of frying our minds, if we want to become native or stay native to our chosen places, we are pretty much obliged to live alertly in them, striving to absorb their beauty and complexity.
These things matter because what Earth most needs today is people who are loyal to it.
The boomers and busters haven't gone away. They've got tar sands, desert real estate, and bad politics to sell us, along with ten thousand new consumer products we don't need. If they haven't already, sooner or later they will train their crosshairs on every mesa, canyon, and creekbed in the region, as their brethren are doing all over the world. Under such an onslaught, every locality on Earth needs natives, whether by birth or adoption, who will rally to its defense.
I don't mean to set up a simple paradigm of good guys and bad guys. Although the world produces a few certifiable devils and saints, the rest of us, 99.99% of humanity, live among the gray shades of the moral universe: imperfectionsЯus. One thing we can probably count on, though, is that by aligning ourselves with the good of the land, we take a step in the right direction.
Unfortunately, climate change won't make things easier. As the Southwest gets hotter and drier, it will become harder to live in.
Some people will leave because they don't like the new conditions; others will leave because they can't make ends meet. Future realities are liable to be hard on everybody. The silver lining, however, is that those who manage to stay and stick, if only hanging on by their fingernails, will make excellent neighbors. Even as problems mount, however, the bloodless math of demographics guarantees that more people will continue to move to the region, swelling its population and burdening it more. Some will come in spite of the challenges (things might be worse where they hail from); others will come unconscious of them.
The stickers will learn (in the unlikely case that they have not already figured it out) that making a commitment to the land, like any act of love, guarantees embattlement, sorrow, and, if a person lives long enough, bereavement. Nothing stays the same forever, and climate change, even within the candle-flame span of a human life, will accelerate the general pace of transformation. Advocate readers, however, start with an advantage: they already love the desert. That's good. There is going to be a lot more of it.
On the other hand, we also love free-flowing water—who doesn't?—and, sadly, there will be considerably less of that. In the power game of regional economics, thirst consistently trumps love. No less powerful will be the desperation of cities and agricultural districts fighting to stay alive. Groundwater won't provide much relief. Most of the region's aquifers are already depleted; others soon will be. Competition for renewable rain-and-snow-fed surface water will ratchet upward. No river or stream, no matter how strong its protection under in-stream flow agreements, federal reserved rights, or other doctrines, will be safe from raiding, and the raids will be smart, well-financed, and unrelenting. It's often been said that, where conservation of land and water is concerned, every victory is temporary and every defeat permanent. Nowhere is this truer than along the waterways of the Southwest.
And the drying up of rivers and streams is hardly the only thing we have to worry about.
The list of threats is nearly endless: energy development, urban sprawl, ATVs, habitat loss, forest mismanagement, strip mines, uranium tailings, vandalism, road building, pipelines and powerlines, overgrazing, pollution. Good grief! Sometimes all you want to do is get back in bed, pull up the covers, and bury your head in pillows. But that would be a bad choice. Ed Abbey got it right. He said, "Be as I am –a reluctant enthusiast... a part-time crusader, a half-hearted fanatic. Save the other half of yourselves and your lives for pleasure and adventure. It is not enough to fight for the land; it is even more important to enjoy it. While you can. While it's still here. So get out there and hunt and fish and mess around with your friends, ramble out yonder and explore the forests, climb the mountains, bag the peaks, run the rivers, breathe deep of that yet sweet and lucid air, sit quietly for a while and contemplate the precious stillness, the lovely, mysterious, and awesome space."
We who love the Colorado Plateau and the lands that surround it are among the most fortunate people on the planet. We live immersed in beauty. We get to drink it through all our senses whenever we reach out and touch the redrocks, even when we do so from far away through the miracle of imagination.
Nowhere else is the profligate grandeur of this blue planet more liberally and lavishly revealed.
Walk in beauty, you betcha. Walk in it all you can, as often as you can. And rally to defend it the same as you would if your kid or your mother were in danger.
Remember: we aren't going anywhere.
Guest writer William deBuys is the author of eight books, most recently The Last Unicorn: A Search for One of Earth's Rarest Creatures. He lives in northern New Mexico.
Read more Advocate stories ›
EDITOR'S NOTE: The views expressed by Advocate contributors are solely their own and do not necessarily represent the views of the Grand Canyon Trust.
Advocate Spring 2015 - Spring 2015 Issue Sidebar
Advocate Spring 2015 - Sticking it Out Dead Horse
Dead Horse State Park.
Advocate Spring 2015 - Sticking it Out Commitment Quote
"...making a commitment to the land, like any act of love, guarantees embattlement, sorrow, and, if a person lives long enough, bereavement."
Advocate Spring 2015 - Sticking it Out Spring Water
Advocate Spring 2015 - Sticking it Out Solar Panels
Advocate Spring 2015 - Sticking it Out Kids Running
Advocate Spring 2015 - Sticking it Out Rig
Advocate Spring 2015 - Sticking it Out Boomers Quote
"The boomers and busters haven't gone away. They've got tar sands, desert real estate, and bad politics to sell us, along with ten thousand new consumer products we don't need. If they haven't already, sooner or later they will train their crosshairs on every mesa, canyon, and creekbed in the region..."
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Advocate Spring 2015 - Desecration Row (Teaser Cartoon)
John Sherffius and American Rivers
Advocate Spring 2015 - Desecration Row Feature
Grand Canyon Under Siege
BY KEVIN FEDARKO
What happens at the Grand Canyon—good or bad—will reverberate throughout the rest of the nation's parks. READ ›
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text:
Q: Display users logged in data I would like to display the users data from the table, so once the user has logged in to the website, text shows on the page saying "Welcome (firstname) (lastname)."
The columns for firstname is "Firstname" and lastname is "Lastname".
I have tried many different methods, but all of them seem to return errors and I cannot get it to work. If anyone can help, that would be great.
Here is my code:

<title>Landing page</title>
<link rel="stylesheet" type="text/css" href="css.css">

<?php
/* ESTABLISH FIRST YOUR CONNECTION TO YOUR DATABASE */
$con = new mysqli("localhost", "root", "", "user_login"); /* REPLACE NECESSARY DATA */

if (mysqli_connect_errno()) {
printf("Connect failed: %s\n", mysqli_connect_error());
exit();
}

if($stmt = $con->prepare("SELECT Username, Password FROM users WHERE Username = ? AND Password = ?")){
$stmt->bind_param("ss",$_POST["username"],$_POST["password"]);
$stmt->execute();
$stmt->store_result();
$checklog = $stmt->num_rows;

if($checklog > 0){
  /* HERE IS YOUR CODE WITH SUCCESSFUL LOGIN */
  session_start();
  $_SESSION["logged_in"] = "YES";
  echo "
You are now logged in
";
  echo "
Link to protected file
";
  echo "
Link to protected file #2
";

}
else {
  /* HERE IS YOUR CODE WITH UNSUCCESSFUL LOGIN */
  session_start();
  $_SESSION["logged_in"] = "NO";
  echo "
You are NOT logged in
";
  echo "
Link to protected file
";
  echo "
Link to protected file #2
";
}

$stmt->close();
}

?>

Public Page

Logout

What is the mistake i am doing and how can i fix it ?

A: You can do $stmt->fetch(PDO::FETCH_ASSOC)
And store your Result in the $result
$result = $stmt->fetch(PDO::FETCH_ASSOC)
$Firstname = $result["Firstname"];

As you might have only one matching row, I would recommend you have to have your condition like $checklog==1
So, You shall have something like this
$checklog = $stmt->num_rows;
if($checklog == 1)  // Allows only for one match
{
session_start();
$_SESSION["logged_in"] = "YES";
$result = $stmt->fetch(PDO::FETCH_ASSOC);  // Fetch the row into $result
$Firstname = $result["Firstname"]; // This is your First Name
$Lastname = $result["Lastname"]; // This is your Last Name
}

You can have the First Name and Last Name in your session like this
$_SESSION["FirstName"] = $result["Firstname"];
$_SESSION["LastName"] = $result["Lastname"];

Update :
Here's the cool and entirely updated version that is hand made just for you :)
It took my cool 10 minutes to construct it :)
<?php
$conn = new PDO("mysql:host=localhost;dbname=user_login",'root','');
$name = $_POST["username"];
$pass = $_POST["password"];
$sql = "SELECT * FROM users WHERE Username = ? AND Password  = ?";
$q = $conn->prepare($sql);
$q->execute(array($name,$pass));
$count = $q->rowCount();
if ($count==1)
{
$q->setFetchMode(PDO::FETCH_BOTH);
while($row = $q->fetch())
{
echo $row['FirstName']; //Just to show you output
session_start();
$_SESSION["FirstName"]  = $row['FirstName'];
}
}
?>

A: try this..
<?php   if($stmt = $con->prepare("SELECT firstname,lastname,Username, Password FROM users WHERE Username = ? AND Password = ?")){
$stmt->bind_param("ss",$_POST["username"],$_POST["password"]);
$stmt->execute();
//$stmt->store_result();
//$checklog = $stmt->num_rows;
$results = $stmt->fetch(PDO::FETCH_ASSOC);
 $_SESSION['firstname'] = $results['firstname'];
 $_SESSION['lastname'] = $results['lastname'];

print_r($_SESSION);

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text:
TheBiteTour > Best Places to Visit > The 14 Highest Mountains in the World
The 14 Highest Mountains in the World
The 20 Most Famous and Visited Monuments in the World
Have you ever wondered which are the highest peaks on Earth? It is a question children often ask but it also fascinates adults. When we think about that, the first names that come to mind are Everest and K2. And then? Then, we don't know.
To make things right and learn the highest points on the planet, here's a list of the 14 highest mountains in the world!
14. Shishapangma
13. Gasherbrum II
12. Broad Peak (K3)
11. Gasherbrum I
10. Annapurna
9. Nanga Parbat
8. Manaslu
7. Dhaulagiri
6. Cho Oyu
5. Makalu
4. Lhotse
3. Kangchenjunga
1. Everest
Credit: Dirk Groeger from Munich, Germany
Height: 8,027 m
Geographic Location: China
First Climb: May 2, 1964, by Hsu Ching, Chang Chun-yen, Wang Fuzhou, Chen San, Cheng Tien-liang, Wu Tsung-yue, Sodnam Doji, Migmar Trashi, Doji, and Yonten.
First Winter Climb: January 14, 2005, by Piotr Morawski and Simone Moro
For years, the mountain was inaccessible due to Chinese restrictions. In 2011, when the restrictions were finally lifted, all records were broken, with only 10 hours needed to get to the top. The feat is attributable to the Swiss mountaineer Ueli Steck.
Credit: www.adventureconsultants.com
Geographical Location: Pakistan and China
First Climb: July 7, 1956, by Fritz Moravec, Josef Larch, and Hans Willenpart
First Winter Climb: February 2, 2011, by Simone Moro, Denis Urubko, and Cory Richards
This mountain is considered one of the best in the world to start climbing all the "8,000", or the 14 highest peaks in the world. It's because it has a traced path that allows you to get almost to the top in a Jeep, thus allowing anyone to climb (and favoring any rescue). In winter, the ice is so solid that the mountain is the safest of the "8,000".
Credit: Aapkamajid
First Climb: June 9, 1957, by Fritz Wintersteller, Marcus Schmuck, and Kurt
First Winter Climb: March 5, 2013, by Maciej Berbeka, Adam Bielecki, Tomasz Kowalski, and Artur Małek
It is called Broad Peak because the summit is very wide. It is a very difficult mountain for climbing, although not very high because the snow is always very soft and the risk of avalanche is very high.
First Climb: July 5, 1958, by Andrew Kauffman and Pete Schoening
First Winter Climb: March 9, 2012, by Adam Bielecki and Janusz Gołąb
Gasherbrum means "splendid peak". It was first named K5 in 1856 by Thomas George Montgomerie when he first spotted the peaks of the Karakorum from more than 200 km away. He actually named five: K1, K2, K3, K4, and K5 where the K stood for Karakoram. Today, K1 is known as Masherbrum, K3 as Broad Peak, K4 as Gasherbrum II, and K5 as Gasherbrum I.
Geographical Location: Nepal
First Climb: June 3, 1950, by Maurice Herzog and Louis Lachenal
First Winter Climb: February 3, 1987, by Jerzy Kukuczka and Artur Hajzer
Every excursion, even the smallest one, starts from the city of Pokhara. A splendid citadel surrounded by green hills that are reflected in a mountain lake. Due to hiking, this city has become very touristy and there are many travel agencies that will help you make a trekking route suitable for you.
Credit: Guilhem Vellut (*_*) on Flickr
Height: 8,125m
Geographical Location: Pakistan
First Climb: July 3, 1953, by Hermann Buhl
First Winter Climb: Feb 26, 2016, by Simone Moro, Muhammad Ali Sadpara, and Alex Txiko
The Italian mountaineer Moro conquered the summit of Nanga Parbat in winter, making history. In fact, he is the first-ever to climb the summit in winter. There were other climbers with him, but Moro was the first to plant the tricolor flag. In fact, the climb is recent since it was completed in 2016.
Credit: Pratapgrg
First Climb: May 9, 1956, by Toshio Imanishi and Gyalzen Norbu
First Winter Climb: January 12, 1984, by Maciej Berbeka and Ryszard Gajewski
Although lower than the previous ones, Mount Manaslu is "famous" for its avalanches which took the lives of several climbers over the years. The tragedy that touched everyone's heart is the one from 1972 when 15 mountaineers died. Unfortunately, this repeated in 2012, when 13 mountaineers died, of which 2 were Italians. No wonder, Manaslu remains the most difficult peak for many climbers and the one that touches the soul with the tragedies it caused.
Credit: Mountain Planet
First Climb: May 13, 1960, by Kurt Diemberger, Peter Diener, Nawang Dorje, Nima Dorje, Ernst Forrer, and Albin Schelbert
First Winer Climb: January 21, 1985 by Andrzej Czok and Jerzy Kukuczka
In 2008, a group of climbers spotted great footprints on these peaks and, in Japan, rumors that they belonged to the Yeti started spreading. Japanese newspapers reported that huge footprints of the Yeti had been found at 4,800 meters on Mount Dhaulagiri. This lead to weeks-long expeditions to find the Yeti, but, as it was expected, it wasn't found.
Credit: Cho Oyu, view from Gokyo
Geographical Location: Nepal and China
First Climb: October 19, 1954, by Joseph Joechler, Pasang Dawa Lama, and Herbert Tichy
First Winter Climb: February 12, 1985, by Maciej Berbeka and Maciej Pawlikowski
In September 2014, the flag of the Four Moors was placed on the summit. It was the mountaineer Max Caria, a true Sardinian, who managed to conquer the summit of Cho Oyu.
Credit: Craig Taylor
First Climb: May 15, 1955, by Jean Couzy and Lionel Terray
First Winter Climb: February 9, 2009, by Simone Moro and Denis Urubko
Once again the man of the records, Simone Moro, has marked the history of mountaineering by climbing Makalu, the 5th highest mountain in the world.
First Climb: May 18, 1956, by Fritz Luchsinger and Ernst Reiss
First Winter Climb: December 31, 1988, by Krzysztof Wielicki
The climb was done using six fields. First, climbing on the Khumbu gravel and then on the northwest side that leads to the summit of Lhotse.
Credit: Carsten.nebel
Geographical Location: Nepal and India
First Climb: May 21, 1955, by George Band and Joe Brown
First Winter Climb: January 11, 1986, by Krzysztof Wielicki and Jerzy Kukuczka
Kangchenjunga was considered the highest peak in the world until Everest and K2 were climbed when it was found to be lower than both of them. Since then, Kangchenjunga has become the scene of the exploits of the best mountaineers from around the world even though the mountain is still relatively unknown.
Credit: Kuno Lechner
First Climb: July 31, 1954, by Achille Compagnoni and Lino Lacedelli
First Winter Climb: the mountain has never been climbed in the winter period
It is called the mountain of the Italians exactly because two Italians were the first to climb it. They also inspired the film "La Montagna degli Italiani" (The Mountain of the Italians).
Credit: Rdevany at the English Wikipedia
First Climb: May 29, 1953, by Edmund Hillary and Tenzing Norgay
First Winter Climb: February 17, 1980, by Krzysztof Wielicki and Leszek Cichy
Everest is part of the Himalayas chain. Before succeeding in climb it, several attempts were made between the first and second world war. The choice of the side to climb was mandatory since the southern side was closed for political reasons.
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Learn How to Cook Nigerian Food Recipe From PweshKitchen - Nigerian, African and Intercontinental Food..
1. Wash your Cocoa Yam then set aside.
2. Wash your meat and Stock Fish then set aside.
3. Salt your fish and soak in hot water for about 15 minutes.
4. Pound your Ashanti Pepper (Uziza seed).
5. Boil your Cocoa Yam for about 30 minutes and pound.
Tip: Pound your Cocoa Yam immediately after boiling because if it gets cold it becomes more difficult to pound.
1. Boil your washed meat, stock fish and dry fish with seasoning cube, salt and pepper for about 20 minutes.
2. Add the quantity of water you want for the soup to the boiled meat and allow to boil for about 15 minutes.
3. Add your red oil to the pot of boiled meat, cover the pot and allow to boil for another 10 minutes.
4. Add your Ashanti Pepper (Uziza seed), Crayfish, Dry pepper and Seasoning cube and Salt. Allow to cook for 15 minutes.
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I know, long dry spells and then two posts in one day. Bedtime tonight was just too ridiculous not to share. Paul: I want to tell you guys a story. Stymie cut a big gash in his head at work Saturday. Levi: DID HE DIE?!?! Me: No, you bloodthirsty child. P: Well, I didn't see… Continue reading Bedlam … er, bedtime.
He's five, this boy. He's pretty excited about it. He climbed into bed with me this morning and, as light snuck in under my just-cracked eyelid, chirped, "Did you FORGET TO TELL ME SOMETHING TODAY?!?" Happy birthday, buddy. Five is a nice number.


Aberlour Distillery is located in Speyside, in a small town of the same name. This spot is where the River Lour empties into the River Spey, at the foot of Ben Rinnes -- a local mountain.
One of the most interesting things about the history of Aberlour, is the confusion around the date of their founding. Some sources claim 1826, others say 1879.
So which is it? Well, both, actually. In the same town where the production distillery now sits, there was a small distillery of the exact same name: Aberlour.
The location was then purchased and demolished. So... the "current" Aberlour was established there in 1879 by James Fleming, who worked at the Dailuiane Distillery for ten years to learn the business. Note that the official bottle labels confirm the 1879 date as well.
That being said, the first production run didn't drip off the new stills until 1880. The distillery has changed hands a few times since then, most notably when the Cambell & Sons bought them in 1945. The current owner -- Pernod Ricard -- received it as a part of their purchase of Campbell in 2001.
In terms of production, most of Aberlour's single malt releases are aged in ex-Bourbon barrels. The 10YO is the youngest, but they release across a wide range of ages up to 30YO.
What does Aberlour taste like?
The Flavor Spiral™ shows the most common flavours that you'll taste in Aberlour Scotch. It's based on all Aberlour drinks in our large database and gives you a chance to taste Aberlour before actually tasting it.
Aberlour specialized in producing Scotch Whiskies for the French market for decades. Some say that's why their single malt has a distinct, Cognac-like character to this day.
It's now my absolute pleasure to introduce a Whisky some of may know very well, Aberlour A'Bunadh. Just to stress again, that is pronounced "Abina." And A'Bunadh is Gaelic, and it means "origin." What do we mean when we say "origin?" Well, Aberlour has been here since 1879 and our aim was to produce a Whisky that would be as close to the original spirit of Aberlour. Matured exclusively in Sherry casks, as would have happened in the late 19th century. Non-chill filtered, to give that beautiful mouthfeel. And most importantly, a cask strength. So I recommend you treat A'Bunadh with the greatest of respect, that you tread carefully when trying A'Bunadh for the very first time. This is a remarkable Whisky. It's intense, it's robust, it's exotic. So let's delve in and see what we get. And first of all, look at that colour, cask strength, open first fill [SP], all that also Sherry casked, a beautiful ruby auburn colour coming through.
Now, it has a cask strength and I would normally add a fair bit of water to this one. But let us try it first of all without water. Let's just take in the percentage first. This is badge number 24, and we're at 60.2% alcohol. And some of you are thinking what age is it. Age isn't important, it's not important at all to A'Bunadh. What's important is quality and the exceptional quality of those Sherry casks that are used in maturing the versatile and fruity Aberlour spirit. Enough talking.
Wonderful, wonderful, wonderful. Coming through, those Sherry notes, dark, dusty, fruity. And I'm even going to venture into the palate at 60.2%. Explodes in the mouth. There are so many layers to this, the oak, the Sherry, the dark fruits, chocolate praline. Let's add some water to it and open it up. Now at 60%, I would recommend adding a great deal of water. You know, some master blenders will nose and taste their Whisky at 20% alcohol. That's a good guide, adding the same amount of water as in the glass, but some spirits will need less. But A'Bunadh certainly needs to be brought alive by some addition of Whisky. So I've probably taken it down to about 35% alcohol or 30% alcohol here, so let's delve back in. You could get lost in there for a very long time, nosing and appreciating. And there comes through those spices, the nutmeg, the cinnamon, chocolate praline, raisins, floral, fruity, dark Sherry. Let's try it.
Magnificent, absolutely magnificent. What a wonderful finish. This is a Whisky you want to get to know intimately, this is a Whisky you want to be your best friend. It's magnificent, the oak remaining in the mouth, the dark fruit, great long finish. Everything A'Bunadh promises it delivers.


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There are two ways one can group all the strategies for Sudoku: by difficulty and by family. Difficulty is rather subjective but necessary, for example, when selecting the order in which strategies are tried in the solver. Some strategies will always be easier for some people to spot than others, but I believe I have chosen an ordering which is not too controversial. So the main documentation has a side menu organised by difficulty.
With chaining strategies, there is definitely a theme going through them. This theme is all about bi-value (only two candidates left in the same cell) and bi-location (only two occurrences of a particular candidate left in the same unit) pairs and the incredible number of deductions one can make from them. You will find, if you read through this group, that earlier strategies become part of a more general theory as the theme develops. Thus, for example, Remote Pairs are a subset of XY-Chains; that is, XY-Chains is a more general approach of which Remote Pairs are a specific instance. Do read the introductory articles Introducing Chains and Links and Weak and Strong Links.
Exotic strategies do overlap with chaining ones, but they have a peculiar flavour of their own and some wonderful, if obscure, logic. They are definitely worth presenting as a demonstration of people's ingenuity but you will only need to have recourse to them on the extreme puzzles.
There are naturally special strategies for Jigsaw and Killers because of their differences. These are now included for the first time on this site.
This strategy list is by no means complete. Many strategies can be further extended and we do not have a complete theory of all Sudoku puzzles. If you are interested in the concepts behind creation and grading, there is a PDF document here called Sudoku Creation and Grading. With the community's help I hope to extend the documentation here.
For those people wondering why "Escargot" cannot be solved by the solver, there is an article on this special Sudoku here. This is an early 'ultimate puzzle' but this crown has been usurped by the puzzle created by Arto Inkala, which is also in the example list.
Interesting Sudokus for other reasons.
Article: The 17 Clue 'Proof' - my take on the January 2012 computerised exhaustive search proof.
Article: The Chaos Within Sudoku - A Richter Scale on a paper by Maria Ercsey-Ravasz and Zoltan Toroczkai at the Faculty of Physics, Babes-Bolyai University, Romania. Very interesting paper.
My response to Crooks Algorithm is here.
I'm pleased to include on this web site the Sudoku Song (MP3 file) by Peter Levy (official web site here). Peter wrote and recorded this song a couple of years ago and managed to capture the essence of the Sudoku craze to great acclaim.
This page was last modified on 23-January-2019.


Can you believe 16 months have passed? I have wrapped up my contract with Children's Wish Foundation International and reopened my doors at red berry.
While working with CWFI, I spearheaded all design, communications, and marketing to consistently articulate and fulfill their mission. I was truly privileged to help them make a difference in the lives of critically ill children.
Now, I look forward to a new year filled with new opportunities! Stay tuned for updates to my portfolio, as well as pending projects. Happy 2016!
Here are my two best secrets for developing an effective brand. Many organizations do not understand the power of a strong brand identity. But the only way to reach your customer in today's media-saturated marketplace is with clarity and consistency of message.
I am delighted to announce a brand launch for Georgia Statewide Afterschool Network (GSAN), a program of Voices for Georgia's Children, and red berry's first project through Catchafire.org. Catchafire matches professionals who want to volunteer their skills with nonprofits and social enterprises who need their help. I believe in employing my creative expertise to further important causes!
Brand identity is the perception of your product or service that distinguishes it from the competition. Know where to begin? Grab a cup of coffee. Part 1 of your homework is to define your brand.


Join their all-female troupe at Corn Exchange Newbury for a bicycle-powered production of Shakespeare's 'The Tempest' like no other. In usual HandleBards style, expect riotous amounts of energy, a fair old whack of chaos, and a great deal of laughter.
Tickets for this performance will be available to purchase from the Corn Exchange website, here.


text:
Альберт Иванович Соколов:
 Соколов, Альберт Иванович (1922—2004) — советский футболист, нападающий.
 Соколов, Альберт Иванович (1928—2001) — советский партийный деятель, председатель Смоленского горисполкома.

redpajama_set_name: RedPajamaWikipedia


text:
Thoroughly enjoyed this cruise.
Serenade of the Seas Cruise Review by drjimlewis
Cabin Type: Superior Ocean View Stateroom with Balcony
This cruise was back-to-back with Norwegian fjords. We chose it because we have done the Caribbean and Mediterranian, and wanted something different.
The itinerary was supposed to be Stockholm, Estonia, St Petersburg, and Helsinki, but the weather was bad in Stockholm, so we went to Estonia, then spent two nights in St Petersburg instead of the one we were scheduled for, and then went to Helsinki. I know some folks were disappointed, but we were happy to spend more time in St. Petersburg as we have both spent a lot of time in Sweden.
Seranade is a little smaller than the larger ocean-going ships, as it goes up through the fjords of Norway, and I expect a larger ship may not be able to navigate some of those narrow passages. We actually enjoyed the smaller ship. The big ones can be a bit hectic.
Food and service were excellent, as usual for RC. We had two excellent waiters in the main dining room, and very much enjoyed dining with them. Their names were Pablo and Vyacheslav. Pablo was from the Dominican Republic and Slav was from the Ukraine.
Excellent cruise and highly recommended.
drjimlewis's Full Rating Summary
Superior Ocean View Stateroom with Balcony
Cabin D2 9624
Nice cabin. It was quiet, had a great view, and was pretty standard with king bed, sofa, shower, table with chair for writing, working at computer. Both 220 volt and 110 volt outlets. Hair dryer included. No coffee pot, but we carried a small pot that made two cups of hot water and carried Folgers singles so we could have coffee immediately in the morning.
Port is very convenient and accessible.
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Old Town of Tallinn
Walking tour. About 2 miles total, a bit tedious on cobblestones, but I'm 75 and it was manageable. We enjoyed the Russian Orthodox church, seeing the old town, doing a bit of shopping, and hearing about the history of Estonia.
View All 189 Old Town of Tallinn Reviews
Enjoyed this tour a lot.
Finnish Home Visit
Visited a farm where they raise Arabian horses. The hostess prepared lunch for us. Seafood, salad, and desert. On the way to the farm, we stopped briefly at a small town for a bio break, had a chance to shop a bit, and then went on.
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This port was canceled because the weather was so bad that we could not get in. Note that you can't leave the rating blank, so I gave it a 1. Stockholm is a nice town, which I have spent a lot of time in.
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drjimlewis


Jeanette Hough, one of our 3 Republican-endorsed members of the Fairfax County School Board, has resigned her At-Large seat because her husband has been assigned to a job overseas.
There will be a special election to fill her seat on Tuesday, August 29th. Whoever wins will serve the remainder of her term, which expires January 2020.
School Board Member Elizabeth Schultz has called this our "Scalia seat". The FCSB has a 9-3 Democrat majority. From bloated budgets to raising their own pay to imposing transgender ideology on Fairfax County schoolchildren, they have acted recklessly. We took 2 seats in the 2015 elections, changing the dynamics on the school board by forcing some reforms and making the Democrats think twice before ramming their agenda items through. If we lose this special election, we will lose much of the ground we gained in the last 18 months. If we win, we will have a third responsible voice on the board and strengthen our chances of taking more seats in the 2019 regular elections.
The Fairfax County Republican Committee has endorsed Chris Grisafe, a Coast Guard veteran who will be a champion for students, parents, and taxpayers.
Turnout will be extremely low – possibly as low as the single digits. It's critical that we get out the vote on August 29th!
It's time to vote – Click here for critical election day info!