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The pH-dependence of the ability of Bgl2p to form fibrils was studied using synthetic peptides with potential amyloidogenic determinants (PADs) predicted in the Bgl2p sequence. Three PADs, FTIFVGV, SWNVLVA and NAFS, were selected on the basis of combination of computational algorithms. Peptides AEGFTIFVGV, VDSWNVLVAG and VMANAFSYWQ, containing these PADs, were synthesized. It was demonstrated that these peptides had an ability to fibrillate at pH values from 3.2 to 5.0. The PAD-containing peptides, except for VDSWNVLVAG, could fibrillate also at pH values from pH 5.0 to 7.6. We supposed that the ability of Bgl2p to form fibrils most likely depended on the coordination of fibrillation activity of the PAD-containing areas and Bgl2p could fibrillate at mild acid and neutral pH values and lose the ability to fibrillate with the increasing of pH values. It was demonstrated that Bgl2p was able to fibrillate at pH value 5.0, to form fibrils of various morphology at neutral pH values and lost the fibrillation ability at pH value 7.6. The results obtained allowed us to suggest a new simple approach for the isolation of Bgl2p from Saccharomyces cerevisiae cell wall.
Amyloids are fibrillar aggregates of proteins consisting of long β-sheets in which β-strands are located perpendicular to the fibril axis (cross-β-structure).1 Initially, amyloids attracted researchers' attention because of the fact that many human and animal diseases, including Alzheimer and Parkinson disease, are associated with formation of amyloid deposits called "amyloid plaques" in various tissues.2 However, over the last decade a diversity of proteins have been shown to fulfill their role in vivo in the form of amyloid fibrils.3-6 Amyloids were found to be highly widespread at the cell surface of various microorganisms, including fungi.7-11 Hydrophobins,3,9 repellents,10 and adhesins11 have been shown to form amyloids on fungal surfaces and many secreted cell wall proteins of Ustilago maydis are predicted to be amyloids.12 The investigation of amyloid proteins of microorganisms is of special importance for understanding of the potential of microbe amyloids to cause harm to human and animal health, particularly by constituting the "nucleus" of amyloid deposits in macroorganisms.13
Recently, we studied the cell wall (CW) of Saccharomyces cerevisiae yeast, a microorganism of great significance for industry, medicine and pharmacology, in order to identify amyloid-forming proteins. We showed that the major, conserved and thermostable cell wall protein14,15 glucantransferase Bgl2p, described for a wide range of yeast species, formed the amyloid aggregates.16 The amyloid-like characteristics of Bgl2p include fibrillar morphology of the aggregates revealed in Bgl2p preparations using transmission electron microscopy, as well as interaction of Bgl2p-containing CW with Congo Red (CR), giving strong green birefringence, and β-sheet-rich secondary structure shown by circular dichroism analysis and thioflavin T (ThT) fluorescence.16 Whether there were conditions, in which Bgl2p did not show the ability to fibrillate, had been unknown by the time we started this work.
In view of the importance of understanding the mechanism of amyloid fibril formation for biotechnology, biology and medicine, it is necessary to investigate factors and conditions which define the existence of proteins in a soluble or an amyloid form.17 Also it was suggested that the key elements of the fibril formation may be common to different proteins and simple model systems could help to clarify many general aspects of this process.18 In the present work, the Bgl2p fibrillation process was investigated more thoroughly using Bgl2p synthetic peptides as model systems. We focused on the pH dependence of the fibrillation processes because of the facts that the pH value has a strong impact on the amyloid fibrillation tendency and the structure of amyloid fibrils depends on the pH at which they are prepared.18,19
This approach allowed us to identify trends in the ability of Bgl2p, isolated from cell walls by heating, to fibrillate at different pH values, based on which we offered a new method of Bgl2p extraction from yeast CW.
The Bgl2p sequence was analyzed using six computational algorithms (FoldAmyloid, TANGO, AGGRESCAN, PASTA, WALTZ and DHPRED), and the obtained results were compared. The underlying principles of these algorithms can be found in the articles dedicated to these issues.20-28 The results of the analysis are presented in Figure 1. Several potential amyloidogenic determinants (PADs) were predicted at least by four or even by five methods out of six. The predicted PADs were TALFFTAS (аа 12-19), FTIFVGV (аа 83-89), NAFS (аа 190-193) and GVNVIVFEA (аа 268-276). The rest of the protein sequence was presented by areas, which none of the programs used predicted as potentially amyloid ones, as well as by those that were predicted to be potentially amyloid, but by less than four of the programs. Several sequences were predicted by FoldAmyloid and AGGRESCAN. It should be noted that the comparison of predicted aggregation propensity results by different methods and experimental data obtained in vivo the programs FoldAmyloid and AGGRESCAN demonstrated better results than in the case of the TANGO, PASTA and WALTZ algorithms.17 To verify whether the predicted PADs of Bgl2p really had the pronounced propensity to form amyloid structures, we synthesized PAD-containing peptides with a length of 10 aa.
The peptides were synthesized with blocked (acetylated and amidated) and non-blocked termini. Hereinafter "NB" and "B" at the end of peptides mean "non-blocked" and "blocked" termini, respectively. The first predicted PAD (аa 12-19) was discarded, since it fell into the Bgl2p post-translationally processed N-terminal cell wall transport signal region, which was absent in Bgl2p from CW. Unfortunately, the peptides which contained PAD (aa 268-276) turned out to be insoluble in all solvents tested, therefore this PAD was also not studied further. The control peptide NDVRSVVADI (aa 141-150 "B") from the region which was predicted to be non-amyloid according to our bioinformatic analysis, was synthesized as well. Because several sequences were predicted by FoldAmyloid and AGGRESCAN, we decided to synthesize peptides VDSWNVLVAG (aa 166-175 "B" and "NB") with one of such sequences, SWNVLVA (aa 168-174).
In further experiments the soluble PAD-containing peptides AEGFTIFVGV (aa 80-89 "B") and VMANAFSYWQ (aa 187-196 "B" and "NB"), the peptides VDSWNVLVAG (aa 166-175 "B" and "NB") and the non-amyloidogenic control peptide NDVRSVVADI (aa 141-150 "B") were examined.
In the first step of our work the ability of the peptides to form fibrils was determined using the amyloid-specific fluorescent probe ThT.29 To avoid presence of charges on termini of the peptides investigated (to keep their state close to the one inside the Bgl2p molecule) measurements of ThT fluorescence of peptides with blocked (acetylated and amidated) termini were made. Peptides AEGFTIFVGV (aa 80-89 "B") and VMANAFSYWQ (aa 187-196 "B"), containing PADs of different length, fibrillated at all pH values investigated (3.2–7.6) after half an hour of shaking (Table 1). Peptide VDSWNVLVAG (aa 166-175 "B") did not fibrillate during this time frame (half an hour), but it fibrillated at pH 3.2, 3.9 and 4.5 after shaking during 1.0, 1.5 and 14.0 h consequently (Table 2). Fibrils of this peptide at pH value 5.0 were visualized with ThT using fluorescent microscopy (Fig. 2A). Thus, peptide VDSWNVLVAG (aa 166-175 "B") fibrillated at pH values from 3.2 to 5.0 and it can also be viewed as а PAD-containing peptide. At pH ranging from 6.2 to 7.6 the fibrillation of this peptide was not detected (data not shown). The non-amyloidogenic control peptide NDVRSVVADI (aa 141-150 "B") did not induce the characteristic ThT fluorescence at pH values ranging from 3.2 to 7.6 even after shaking for 14 h (data not shown).
Fibrils of peptide VDSWNVLVAG (aa 166-175 "NB") at pH value 5.0 were visualized with ThT in microscopy (Fig. 2B). Some additional experimental data in support of the amyloid properties of the investigated peptides with PADs is given in Supplementary Information section (Figs. S1–S3 and Table S1).
The fibrillation kinetics of Bgl2p was studied at pH 5.0 and 7.6 using ThT. Bgl2p induced specific ThT fluorescence at pH 5.0, but did not induce specific ThT fluorescence at pH 7.6 (Fig. 3). The ability of Bgl2p to form fibrils in water was also demonstrated (pH average 6.4 ± 0.2). Using microscopy it was demonstrated that Bgl2p isolated from the CW without trypsin treatment formed long (up to several tens of µm) fibrils and aggregates resembling "wisps" or "nests" with evident aggregation centers (Fig. Four A-C). When Bgl2p was isolated from the trypsin-treated CW, the fibrils detected were curved, "worm-like" and rather short (up to 10 μm) (Fig. 4D and E). In this case only a few evident aggregation centers were revealed only by TEM (Fig. 4 H–J, arrows). Addition of the major CW component, β-1,3-glucan (laminarin), did not affect the morphology and amount of Bgl2p fibrils (data not shown). However, electron microscopy revealed clear aggregation centers in presence of β-1,3-glucan (Fig. 4I and J). Notably, the common feature of all fibrils was their length of more than 1 μm.
Bgl2p, which was partially purified by ultrafiltration (cut off limit 100 kDa) from the growth medium of mutant strain S. cerevisiae A270 with deletion in SSU/MCD4 gene (Bgl2pGM), at рН 6.7 formed unstructured aggregates in the absence (Fig. 5A) and fibrillar structures in presence of CR (Fig. 5 B–D). CR is known as an agent, which can either inhibit or enhance fibrillation.29,30 Fibrillar structures were also found in growth media subjected to dialysis and partial concentration by ultrafiltration in order to eliminate of components with molecular mass less than 10 kDa (Fig. 5E) without addition of CR.
According to the results presented in the Supplementary Information (Fig. S4), no difference was observed between the mutant growth medium Bgl2p (Bgl2pGM) and wild-type CW Bgl2p in their primary structure and in post-translational modifications. It is important to note as well that Bgl2pGM was able to form denaturation-resistant oligomers (Fig. S5) and CW Bgl2p demonstrated proteinase K resistance (Fig. S6).
Previously, we suggested that the ability to form amyloid-like fibrils allows Bgl2p to be retained firmly in the cell wall.16 In this work we demonstrated that the fibrillation ability of this protein extracted from a cell wall by heating was decreasing with the increase of pH value. We took into consideration such pH dependence tendency of Bgl2p fibrillation and extracted Bgl2p by incubation of cell walls in a solution with alkaline pH (Fig. 6). It was possible to extract Bgl2p at a pH value between 8 and 9, but the extraction was either slow (in this case the cell walls had to be incubated at alkaline pH for more than 24 h) or ineffective (data not shown). At pH 9 efficient extraction conditions were shown, since in a relatively short time period the amount of Bgl2p comparable to the amount of the protein extracted from the cell walls by heating was extracted.
Bgl2p has been studied for a long time but only recently it has been shown that this protein possesses amyloid properties.16 However, conditions influencing on Bgl2p fibrillation have not been studied until now.
According to the literature, one of the important factors that affects the fibrillation is pH value of the medium.18,19 Therefore we focused on studying the pH dependence of Bgl2p fibrillation. The pH-dependence of the ability of Bgl2p to form fibrils was studied using synthetic peptides with potential amyloidogenic determinants (PADs) predicted in the Bgl2p sequence according to a bioinformatic approach.
Structural studies as well as in vitro experiments with proteolytic fragments of amyloidogenic proteins and synthetic peptides revealed that the tendency for a protein to form amyloid is often limited to a short sequence of a full protein, known as a "self-recognition element."31 Self-recognition elements constitute the core of amyloid fibrils,32,33 and "hot spots" for aggregation of the native protein into amyloid fibrils.34 In this article, such self-recognition elements are referred to as "potential amyloidogenic determinants" (PADs).
In this work Bgl2p peptide sequences, determined to be important for the amyloidogenic properties of Bgl2p using various bioinformatics analyses, were validated experimentally mainly using the ThT assay. The selected PAD sequences were predicted to be PADs by at least four out of six prediction algorithms. Furthermore, a sequence only selected by FoldAmyloid and AGGRESCAN was also selected, because these two programs had earlier demonstrated a high level of correlation between the change in aggregation propensity observed in vivo and the change in aggregation propensity predicted in silico.17
Our prediction of amyloidogenic determinants in the Bgl2p sequence correlated well with the data obtained on the fibrillation ability of the peptides (Tables 1 and and22 and Fig. 2). The PAD-containing peptides AEGFTIFVGV (aa 80-89 "B") and VMANAFSYWQ (aa 187-196 "B") formed amyloids at all pH values investigated (Table 1), the peptide NDVRSVVADI (aa 141-150 "B"), which was predicted to be non-amyloidogenic, did not form amyloids (data not shown). The peptides VDSWNVLVAG (aa 166-175 "B" and "NB") also formed amyloids within the pH range 3.2–5.0 (Table 2 and Fig. 2). For comparison some additional experiments revealing the amyloid properties of the peptides investigated (with non-blocked termini) are presented in Supplementary Information (Figs. S1–S3 and Table S1). These experiments supported the presence of amyloid-like aggregates in the samples of peptides that showed a positive ThT signal.
Bgl2p had the same tendency to fibrillate as the peptides VDSWNVLVAG (aa 166-175 "B" and "NB") (Table 2 and Fig. 2): it fibrillated at pH 5.0 and did not fibrillate at mild alkaline pH value 7.6. Thus, apparently the Bgl2p ability to fibrillate at different pH-values was the function of coordinated fibrillation activity of different peptides.
The new insight into the pH-dependence of Bgl2p fibrillation was used to introduce a novel extraction method where Bgl2p was extracted from the cell wall without heating. It is advantageous to avoid heating of Bgl2p although Bgl2p is considered to be a thermally stable protein, since its activity when using the heating method of extraction is rather low,35 which may in some cases lead to misinterpretation of the results.14,15 Furthermore, earlier the isolation of Bgl2p without heating was an extremely complicated and laborious procedure.15 As follows from the results obtained, extraction of Bgl2p at alkaline pH from the cell walls allowed us to purify Bgl2p to near homogeneity in one stage (Fig. 6).
It is important to note that the PAD-containing peptide AEGFTIFVGV (aa 80-89 "B") contains a glutamic acid residue, which possesses a negative charge at mild alkaline pH values. Thereby this peptide at mild alkaline pH may lose its ability to fibrillate due to electrostatic repulsion between negative charges of the glutamic acid residues of the peptides. However, no significant difference was found in fibrillation of the PAD-containing peptides AEGFTIFVGV (aa 80-89 "B") and VMANAFSYWQ (aa 187-196 "B") at mild alkaline pH values (Table 1). Apparently the charges of amino acid residues in peptides with PADs did not impede their fibrillation significantly. On the other hand, PAD-containing peptide VDSWNVLVAG (aa 166-175 "B") contains one residue with a negatively charged side chain (aspartic acid) and it lost its ability to fibrillate at mild alkaline conditions (Table 2). Two peptides AEGFTIFVGV (aa 80-89 "B") and VDSWNVLVAG (aa 166-175 "B") have each one charged residue in the second position from the N-termini of peptide. In the first case it is Glu and in the second one it is Asp. It is interesting that these peptides revealed different amyloidogenic properties in different pH values. This may be explained by the difference in the structure of the Glu residue, which differs from Asp by only a single CH2 group elongation of its side chain. This small change causes big differences in the biological uses of Asp and Glu residues. It was shown for CsgA protein that certain Asp and Gly residues functioned as gatekeeper residues which inhibited the amyloidogenic properties of these repeating units.36 Certain aspartic acid residues were shown to inhibit the intrinsic aggregation tendencies of CsgA – a major subunit of the bacterial cell surface of amyloid curlin.36
Alignment of predicted PADs with Bgl2p homologs (see Supplementary Information section "Search for PAD-containing peptides in Bgl2p fungi and plant homologues") led to the hypothesis that homologs of Bgl2p from S.cerevisiae from different species (including pathogenic yeast species, e.g., C.albicans) also possessed amyloid-like properties. In a previous study we have shown that Bgl2p was not the only protein which could form amyloid in a CW of S.cerevisiae yeasts.37 Our result fit well with the predictions about presence of β-aggregation prone proteins in the cell wall made in the work of Tartaglia and Caflisch.38
The difference in morphology of fibrils formed by Bgl2p obtained from the CW not treated with trypsin, and the fibrils formed by Bgl2p obtained from trypsin-treated CW, was shown (Fig. 4). The first ones looked like "wisp" supramolecular aggregates with evident aggregation centers, and the second ones were much less ordered ("worm-like"). Both morphologies have been observed earlier for well characterized amyloid fibrils, e.g., "wisps" of glucagon fibrils and "worm-like" Aβ fibrils.39,40 The organization of Bgl2p fibrils may be defined by the compound (so called nucleator) susceptible to trypsin hydrolysis. Similar phenomenon is known for E. coli curli extracellular amyloid fibril polymerization which is directed by CsgB nucleator.41 Adding of β-1,3-glucan (major yeast cell wall component) to Bgl2p, isolated from trypsin-treated CWs, did not reveal significant alteration of Bgl2p fibril morphology (data not shown). Electron microscopy revealed slightly more visible aggregation centers (Fig. Four I and J). These results indicated that β-1,3-glucan evidently is not strong nucleator in this case. It should be noted that trypsin did not cleave Bgl2p.16 Apparently the presumptive Bgl2p nucleator (PBN) contained the peptide part with arginine and/or lysine residues which are the prerequisites of trypsin proteolysis sites.
Our studies showed a positive correlation between the predicted degree of amyloidogenicity of various Bgl2p peptide sequences and their experimentally measured ability to fibrillate. We focused on the pH-dependence of Bgl2p fibrillation. Using amyloidogenic peptides as a model for the investigation we demonstrated that pH value was the important factor, which defined the existence of Bgl2p in a soluble or an amyloid form and proved this proposition using Bgl2p isolated from CW by heating. We demonstrated also that at mild acidic and neutral pH values Bgl2p formed fibrillar structures with different morphology (Figs. 4 and and5).5). We suggest that the observed fibrillation of Bgl2p was the result of PADs interaction. On the other hand the morphology of Bgl2p fibrils depended on isolation conditions and apparently on yet unidentified constituents.
Salts and low-molecular weight compounds were of analytical grade or better. Congo Red (3,3′-[(1,1'-biphenyl)-4,4'-diylbis(azo)]bis-(4-amino-1-naphthalene acid) disodium salt; Sigma, product number C6277) and ThT (4-(3,6-dimethyl-1,3-benzothiazol-3-ium-2-yl)-N,N-dimethylaniline chloride; Sigma, product number T3516) were used without further purification. TRIS was purchased from Merck (product number 648309). Qualifications and producers of the other compounds are indicated in the sections devoted to the corresponding methods. Milli-Q water was used throughout.
DBY 746 (genotype MATα ura3-52 leu2-3,112 trp1-289 his3-Δ1) further referred to as "wild type" mating type α kindly provided by M.D. Ter-Avanesyan (Cardiology Research center). This strain is parental with respect to A270 strain.
A270 with the deletion in SSU21/MCD4 gene (genotype MATα ura3-52 leu2 trp1-289 his3-Δ1 ssu21) and its revertant strain bearing pHTSA plasmid with SSU21/MCD4 gene42 were kindly provided by M.D. Ter-Avanesyan and G.V. Fominov (Cardiology Research center). Growing of yeasts in liquid nutritious YPD (1% yeast extract, 2% peptone, 2% glucose) medium was performed in 750 ml flasks with a growth medium volume of 200 ml in an orbital shaker (200 rpm) at 30°C.
Log-phase yeast cells (19 h of growth) were precipitated by centrifugation for 10 min at 1,650 g (OPn-8 with RU 180L rotor, Russia), washed twice with 0.05 М potassium-phosphate buffer рН 8.0 and disrupted in the shaker (Heidolph) using glass beads (0.5 mm; Sigma) on cooling. The extent of cell disruption was estimated using a light microscope (Opton). Only CW preparations containing less than 0.1% of intact cells were used in the further work. CWs were separated from the intracellular contents by centrifugation at 3,000 g for 5 min. CWs and cells formed the double-layer precipitate; CWs forming the upper layer were carefully suspended in water and separated from the cells. Then the CW preparation obtained was washed twice with water, twice with 1% saccharose, twice with 1 М NaCl, twice with 1% NaCl and once with water. According to the data obtained in our laboratory earlier, the treatments allow the complete removal of intracellular contents and cytoplasmic membrane components (Kalebina, unpublished results). The amount of CW was estimated spectrophotometrically [absorbance at 540 nm (A540) of 1 ml of 500 µg ml−1 CW suspension equaled approximately 1.0].
For Bgl2p isolation two different partial deproteinisated CW-preparations were used. One of which was subjected to trypsin and SDS treatment and the other was subjected to SDS treatment only.
Isolated CWs (A540 = 100) were suspended in 3 ml of trypsin solution (10 mg ml−1 in 0.05 М TRIS-HCl buffer, pH 7.5) (Sigma) and incubated for 2 h at 37°C. For trypsin removal, CWs were washed four times with 1 М NaCl and twice with water. To ascertain the absence of proteolytic activity in the preparation, the test with prestained casein (Reakhim) was used. In short, an aliquot of CW suspension was incubated with prestained casein solution (10 mg ml−1) for 30 min at 37°C, and then the protein was precipitated by incubation with 7% trichloroacetic acid (15 min at 4°C) and centrifugation at 11,100 g (Eppendorf Minispin). The supernatant containing proteolytic peptides conjugated with chromophore groups was separated from the precipitate and investigated colorimetrically at 400 nm. The absence of the supernatant color change was evidence of the absence of proteolytic activity in the preparation studied. The solution of prestained casein in the absence of proteases was used as a control. After complete proteinase removal, CW suspension was centrifuged at 3,000 g for 3 min, and the supernatant was discarded.
Treatment with 1% SDS (Amresco, 0227) (1 h at 37°C) was also applied. To remove SDS, the CWs obtained were additionally washed five times with 0.2 M sodium acetate buffer (pH 5.5), three times with n-butanol/water mixture 0.7:1 (vol/vol) and with water, until the smell of n-butanol had disappeared. After each ablution, the suspension was centrifuged at 2,580 g for 3 min, and the supernatant was discarded. CWs were stored at 4°C in 0.05 М TRIS-HCl buffer (pH 7.5), and sodium azide was added to the final concentration of 0.02%, if extended storage was required.
Glucantransferase Bgl2p was isolated from trypsin/SDS- and SDS-treated CWs (A540 = 15) as previously described.16
Purified CWs were incubated in 100 mM TRIS solution, pH 9.2 in ratio 1 optical unit CW (A540) for 2 µl 100 mM TRIS solution during 4.5 h at 30°C. Bgl2p extract was separated from CW by centrifugation at 13,400 rpm (Minispin) during 2 min at room temperature.
The Bgl2p sequence was analyzed using six computational algorithms, and the results obtained from the various algorithms were compared. In this work, the following algorithms were used: FoldAmyloid (http://bioinfo.protres.ru/fold-amyloid/oga.cgi), TANGO (http://tango.crg.es/), AGGRESCAN (http://bioinf.uab.es/aggrescan/), PASTA (http://protein.cribi.unipd.it/pasta/), WALTZ (http://waltz.switchlab.org/), as well as the secondary structure prediction server DHPRED (http://www.fz-juelich.de/nic/cbb/service/dhpred.php). The principles underlying the work of these algorithms can be found in articles devoted to these issues.20-28 Importantly, the average correlation between the change in aggregation propensity observed in vivo and the change in aggregation propensity predicted in silico by the algorithms FoldAmyloid and AGGRESCAN was better than for the algorithms TANGO, PASTA and WALTZ.17
For fibrillation kinetic measurements of peptides with non-blocked termini or Bgl2p protein, a Fluostar Optima platereader (BMG Labtechnologies) was used with 384-well plates with optical bottoms from Nalge Nunc International (Rochester). The samples were assayed in triplicates, and each well contained 50 µl of 25 µg ml−1 peptide or 30 µg ml−1 protein sample in phosphate-citrate buffer. A final ThT concentration of 25 µM ThT (filtered) was introduced to each well. For the ThT concentration determination, a molar extinction coefficient at 412 nm of 36,000 M−1 cm−1 was applied for ThT in water. The wells were covered with Polyolefin non-sterile sealing tape, also from Nalge Nunc International, to avoid evaporation of the samples. ThT fluorescence measurements were performed as bottom/bottom measurements using an excitation wavelength of 450 nm (10 nm bandpass) and an emission wavelength of 480 nm (12 nm bandpass). The fluorescence measurements were performed at 35°C or 45°C every 400 sec. During each cycle of 400 sec, there was 1 mm orbital shaking (600 rpm) for 240 sec.
Fluorescence measurements of fibrillation of peptides with termini blocked were performed with Cary Eclipse fluorescence spectrophotometer with 5 nm excitation and emission bandpass (Varian Inc.). Excitation and emission wavelengths were the same as used for the Fluostar Optima platereader. Peptides were initially dissolved in 100% DMSO (up to concentration of peptides equal 2 mg ml−1). After that they were dissolved in phosphate-citrate buffer and final concentration of DMSO was no more than 1.25%. It should be noted that peptides with blocked termini did not fibrillate without shaking. Fibrillation of peptides with termini blocked was performed using incubator shaker Excella E24 (New Brunswick) at 35°C with 1.9 cm orbital shaking (200 rpm). The samples were assayed in triplicates in 1.5 ml tubes (each tube contained 500 µl of 25 µg ml−1 peptide in phosphate-citrate buffer). A final ThT concentration of 25 µM ThT (filtered) was introduced to each tube.
Higher pH values (more than 7.6) were excluded from the analysis due to the ThT instability under alkaline conditions.43
Synthesis of peptides with non-blocked (not acetylated, not amidated) termini was performed on an n-alcoxybenzyl polymer (containing 0.5 mmol g−1 of hydroxyl groups) using standard procedures of solid phase synthesis for Fmoc\But protective groups of amino acids.44 Removal of a peptide from a polymer and simultaneous unblocking of side groups of amino acid residues was conducted in a mixture of trifluoroacetic acid (TFA): thioanisole: H2O: ethanedithiol (85:5:5:5) for 2 h. The peptides were purified using reversed phase high performance liquid chromatography in a gradient of acetonitryl in 0.1% TFA (10% to 70% in 60 min) at an eluent velocity of 4 ml min−1. The eluent's absorption was registered at a wavelength of 226 nm. The HPLC experiments were performed with a System Gold chromatograph (Beckman), in Jupiter 5μ C18 300A 250 × 4.60 mm (Phenomenex) and Reprosil-Pur C18AQ 5μ 250 × 4.60 mm (Dr. Maisch, Germany) columns in the case of analytical chromatography, and in a Jupiter 10μ C18 300A 250 × 10.00 mm (Phenomenex) in the case of the preparatory one. The molecular weight of the peptides was measured with a VISION 2000 device (Bioanalysis), according to the MALDI method, and was found to correspond with the calculated one.
Synthesis of peptides with termini blocked (acetylated, amidated) was performed on a Rink-polymer using standard procedures of solid phase synthesis for Fmoc\But protective groups of amino acids.44 Removal of a peptide from a polymer and simultaneous unblocking of side groups of amino acid residues was conducted in a mixture of TFA: H2O: ethanedithiol (90:5:5) for 2 h. The peptides were purified using reversed phase high performance liquid chromatography in a gradient of acetonitryl in 0.1% TFA (30 to 70% in 40 min) at an eluent velocity of 4 ml min−1. The eluent's absorption was registered at a wavelength of 226 nm. The HPLC experiments were performed with a System Gold chromatograph (Beckman), in Jupiter 5μ C4 300A 250 × 4.60 mm (Phenomenex) column in the case of analytical chromatography, and in a Jupiter 10μ C4 300A 250 × 10.00 mm (Phenomenex) in the case of the preparatory one. The molecular weight of the peptides was measured with a VISION 2000 device (Bioanalysis), according to the MALDI method, and was found to correspond with the calculated one.
In the majority of the studies the peptides with non-blocked termini were dissolved in deionized water and then buffer was added. Peptides with termini blocked were initially dissolved in dimethyl sulfoxide and then buffer was added (final concentration of dimethyl sulfoxide was no more that 1.2%). Concentrations of peptides were equilibrated to 25 µg ml−1 before experiments.
Protein and peptide concentrations were measured according to the technique of Scopes.45 Sample absorption in the range of 190–350 nm was determined using a Cary 50 Scan UV-Visible Spectrophotometer (Varian Inc.). Bgl2p concentration was 0.03 mg ml−1.
Images of fibrils formed by peptides were obtained using fluorescent confocal scanning microscopes, Carl Zeiss Axiovert 200M LSM 510 META (Zeiss), or Leica TCS SP2 AOBS (Leica).
Bgl2p peptide samples placed on glass slides were incubated in conditions preventing desiccation in case of usage of phosphate-citrate buffer with pH 5.0 or 7.6. Afterwards, samples were stained with 7.5 µM of ThT. ThT was excited using an Argon laser (458 nm), and the emission signal was detected in the range of 475–525 nm.
Images of fibrils formed by glucantransferase Bgl2p were obtained using fluorescent confocal scanning microscopes Carl Zeiss Axiovert 200M LSM 510 META (Zeiss), or Leica TCS SP2 AOBS (Leica).
Bgl2p-containing samples placed at glass slides were incubated in conditions preventing desiccation. After that, samples were stained with mouse primary polyclonal antibody against Bgl2p and secondary polyclonal goat anti-mouse antibody lgG labeled with Alexa-488 fluorophore (Invitrogen).
Negative-staining EM was used. Small volumes (2 µl) of samples containing at least 60 ng of Bgl2p were adsorbed onto glow-discharged carbon-coated, Formvar-filmed 400-mesh copper grids and immediately dried down. 2% uranyl acetate staining solution was then absorbed for 2 min. Grids were allowed to dry in a light-protected environment and were viewed in a JEM-100B (JEOL) electron microscope at the accelerating potential of 80 kV.
Electrophoresis was performed according to Laemmli46 in 10% resolving polyacrylamide gels. Proteins were stained with Coomassie G-250 or immunologically detected by Western Blot analysis. Prestained Protein Molecular Weight Marker (Fermentas, Canada) was used. Bgl2p antiserum was raised in male BALB/c mice (SPF status) using SDS PAGE-purified protein (40 μg per mouse).47
S. cerevisiae ssu21/mcd4 mutant growth medium ultrafiltration was performed using MidGee Cross Flow hollow fiber cartridges (GE Healthcare) with hold-up volume of 0.5 ml and cut-off limit of 100 kDa and using Millipore filters with cut-off limit of 10 kDa.
This work was funded by the Carl Zeiss Program for Support of Research Work of Young Investigators from Higher Education Institutions of Russia (Grant no. MSU 16/11CZ), the Drug Research Academy (Faculty of Pharmaceutical Sciences, University of Copenhagen), the Russian Foundation for Basic Research (Grant no. 09-04-00768-a, 10-04-01821a, 11-04-763a, 12-04-31966-mol_a), the programs "Molecular and Cellular Biology" (01200959110 and 01200957492) and "Fundamental Sciences to Medicine," Howard Hughes Medical Institute (55005607), the Federal Agency for Science and Innovation (grant no. 02.740.11.0295 and 16.740.11.0478) and by the German organization DAAD (German Academic Exchange Service) in the frame of a Scientific Cooperation Agreement between Moscow and Rostock Universities. The authors would like to acknowledge associate professor Osman Mirza (Faculty of Health and Medical Sciences, University of Copenhagen) for providing access to facilities useful for growing yeast, professor Per Amstrup Pedersen (Department of Biology, University of Copenhagen) for the BMA64-1B strain, Stefano Colombo (Faculty of Health and Medical Sciences, University of Copenhagen) for discussion of the results obtained, Dr S.N. Egorova and Dr M.N. Zhmak (Institute of Bioorganic Chemistry, Moscow, Russia) for synthesis of the peptides, and Dr Annette E. Langkilde (Faculty of Health and Medical Sciences, University of Copenhagen) for collecting the X-ray fiber diffraction data. We are grateful to Tatyana A. Sabirzyanova for the discussion of our results and to Aleksandra Zhukova for the assistance in preparing of manuscript.
No potential conflicts of interest were disclosed.
†Current affiliation: Laboratory of Biochemistry and Genetics; National Institute of Diabetes and Digestive and Kidney Disease; National Institutes of Health; Bethesda, MD USA
Previously published online: www.landesbioscience.com/journals/prion/article/22992
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Burkina Faso
Bertrand Traoré will travel with Chelsea's first team to the United States next month after being granted a work permit to play in England
The best goal, the best game and the biggest surprises after an eventful month in Equatorial Guinea was ended with Ivory Coast's shootout success over Ghana
Algeria beat Burkina Faso 1-0 to qualify for the World Cup finals
Burkina Faso are on the brink of reaching the World Cup finals for first time but fear of violence in Algeria sees them return ticket allocation
Jonathan Wilson: Only Algeria, Egypt and Ivory Coast are currently assured of places in the two-leg play-offs for next year's World Cup in Brazil
Nigeria fans in Lagos and South Africa celebrate late into the night after their team beat Burkina Faso 1-0 in the final of the Africa Cup of Nations
Brilliant goal by Sunday Mba ends 13 years of pain for Super Eagles, says Cheta Nwanze
Super Eagles beat Burkina Faso 1-0 to win the Africa Cup of Nations after years of near misses
Sunday Mba's first-half strike settled the final and earned Nigeria a third Africa Cup of Nations victory
Will the fancied Super Eagles outplay the Burkina Faso Stallions? Football experts suggest that it might not be so easy, says Elliot Ross
Paul Put, banned in his native country for fixing matches, has led his team to victory against some of the continent's great names
Jonathan Wilson: Nigeria's opponents in the final prove that the continent's middle-ranking teams can take on and beat Africa's best
Burkina Faso fans celebrate exuberantly as their team pull of an unlikely victory against Ghana to go through to the Africa Cup of Nations final
Guardian Africa network Africa cup of nations: the Super Eagles to take on the Stallions in the final
Nigeria dominated its semi-final against Mali while Burkina Faso had to beat both Ghana and the referee to make it through, says Ant Simms
Burkina Faso reached the final of the Africa Cup of Nations after shocking Ghana in a penalty shootout
Coach Paul But says he aims to get Burkina Faso into the final, further than they have ever been at the tournament before
Burkina Faso coach Paul Put hopes his team's success in reaching the semi-finals of the Africa Cup of Nations can help him restore his reputation
Jonathan Wilson: Nigeria and Ghana are hoping for a return to glory at the Africa Cup of Nations, but both need to beat smaller nations in the semi-finals
Paul Put, the Burkina Faso coach who was banned in Belgium for fixing matches, has claimed the practice has always existed in football
Burkina Faso beat Togo 1-0 in extra-time on Sunday to set up an Africa Cup of Nations semi-final meeting with Ghana
Burkina Faso reached the semi-finals of the Africa Cup of Nations when a header in extra time from Jonathan Pitroipa gave them a 1-0 win over Togo
Will the champions Zambia sneak through to the quarter-finals? Find out with Barry Glendenning
Jonathan Wilson: From the disappointment of Senegal, to Kily Alvarez's goal and Ali Bongo's wild celebrations
Sudan beat Burkina Faso 2-1 to finish second in Group B at the Africa Cup of Nations and reach the last eight for first time in 42 years
Ivory Coast secured a place in the Africa Cup of Nations quarter-finals after Salomon Kalou scored the first goal in a 2-0 defeat of Burkina Faso
Ghana set up a quarter-final encounter with the hosts Angola after an unconvincing display against unfancied Burkina Faso
The tournament continues its recovery in Cabinda after the shock of the gun attack on Togo's squad


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Q & A Library
Flying: Bad Air Up There?
I have been traveling more often lately and the smell of the air on the plane immediately makes me nauseous and "grimy." Is there a clean air device you recommend or something else I can use? I don't have fear of flying.
Answer (Published 3/26/2003)
Aircraft air quality is a real problem for millions of travelers and thousands of airline employees. More than a year ago, a report from the National Research Council found evidence suggesting that a number of problems with the air circulating in passenger cabins may cause health problems. The report listed concerns that ozone levels in the cabin air may exceed regulatory standards, that oxygen pressure may not be adequate to protect passengers with pre-existing heart or respiratory diseases and that the air may be contaminated with traces of engine oil, hydraulic fluid, de-icing solutions, and even pesticides sprayed on international flights.
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Not surprisingly, those most affected are flight attendants and other crew members, some of whom have been complaining for years about headaches, blurred vision, dizziness, nausea and other health problems which they attribute to poor quality cabin air. Two years ago, Alaska Airlines flight attendants won a $725,000 out-of-court settlement based on their contention that design flaws in two types of planes the airline flies had allowed chemical fluids to mix with cabin air and make them sick. However, the flight attendants lost a subsequent suit against the planes'manufacturers.
The aircraft air quality issue may not be satisfactorily resolved without public pressure on the FAA and Congress for better regulation of this important aspect of air travel. In the meantime, purchasing a portable personal air purifier may make plane trips more comfortable for you. You can find a number of products on the Internet and in catalogs, but I'm not familiar enough with any to make a specific recommendation. Be sure to check with the flight crew before using any portable electronic device while in flight.
If you travel by air frequently, another option I can suggest is an expensive one: fly first class if you can upgrade. The first-class cabin provides more outside air per person. You also may feel better during and after air travel if you avoid alcohol during flights, drink plenty of water, and consider taking an immune-boosting herb such as Echinacea or astragalus before you get on the plane.
Andrew Weil, M.D.
Some Rights Reserved Creative Commons Copyright Notice
A portion of the original material created by Weil Lifestyle on DrWeil.com (specifically, all question and answer-type articles in the Dr. Weil Q&A Library) is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 United States License.
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How much pages in a 1500 word essay time or that as- sumes that only the movement of people misreading her. What effect does this say about technical progress, taking in the long debate over documentary was in the.
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This @usabasketball team in 2016 is not going to disappoint / they have all the parts needed to stand tall with the Olympic GOLD MEDAL !
This @usabasketball team in 2016 is not going to disappoint / they have all the parts needed to stand tall with the Olympic GOLD MEDAL !
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Category:1641 in Ireland
From Wikipedia, the free encyclopedia
Articles and events specifically relating to the year 1641 in Ireland
Main article: 1641 in Ireland
This category has the following 2 subcategories, out of 2 total.
- ► 1641 disestablishments in Ireland (1 P)
- ► Irish Rebellion of 1641 (12 P)
Pages in category "1641 in Ireland"
This category contains only the following page. This list may not reflect recent changes (learn more).


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Nimoy to nerds: I'm outta here
Leonard Nimoy has attended his final "Star Trek" convention.
The 80-year-old actor, best-known for playing Mr. Spock in the original TV series that began in September 1966, formed four fingers into a V for Vulcan sign and intoned to fans Spock's most famous phrase: "Live long and prosper."
Nimoy has said the convention in suburban Chicago celebrating the 45th anniversary of "Star Trek" would be his last.
He spoke for an hour about his life and career, and thanked fans for their support over the years. Some held signs saying: "We love you Leonard! Live long & prosper."
A bad judge of ratings
Simon Cowell acknowledges that he regrets saying ratings less than 20 million for the U.S. version of "The X Factor" would be a failure.
The show's September debut earned 12.5 million viewers. In week two, ratings hovered around 12 million viewers for each episode.
"I'm not going to lie. I wanted 20 million when we launched," says Cowell, "but now I'm kind of back in the real world and I'm seeing this grow naturally. I'm as happy as I've ever been."
Hurley and cricketer
Former Australia cricketer Shane Warne says he and British actress Elizabeth Hurley (the "Austin Powers" movies) are engaged.
The couple have been together for 10 months.
Hurley dated actor Hugh Grant for more than a decade and was previously married to Indian businessman Arun Nayar.
The birthday bunch
"The View" co-host Joy Behar is 69. Christopher Norris ("Trapper John, M.D.") is 58. Drummer Tico Torres of Bon Jovi is 58. Gospel singer Michael W. Smith is 54. Simon Cowell is 52. Singer Toni Braxton is 44. Singer Thom Yorke of Radiohead is 43. Allison Munn ("One Tree Hill") is 37. Singer Taylor Hicks ("American Idol") is 35.
Saving for the future
sponsored According to two 2015 surveys, 62 percent of Americans do not have enough savings to handle an unexpected emergency, much less any long-term plans.


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Nick Heyward and marriage advice
Join Tim for your guide to Oxfordshire's weekend. It's a "fantastic day" as Tim Smith welcomes Haircut 100's Nick Heyward to Oxfordshire, Joe McGann explains how a trip to Paris can save your marriage, and hits n headlines from a mystery year when a new Cavalier 1.6 4dr saloon cost £7800.
Last on
Sat 2 Aug 2014 09:00


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Australian Prime Minister Julia Gillard called a special ballot on the leadership of the ruling Labor Party for later Wednesday amid speculation of a leadership challenge, according to local reports.
Australian Associated Press said the "special caucus meeting" of party lawmakers will be held at Parliament House in the capital Canberra at 7 p.m.
According to AAP, Gillard expressed confidence of winning the vote, and said she will leave federal politics if she loses.
Former Prime Minister Kevin Rudd said he will challenge Gillard for the leadership, according to AAP.
"I will be a candidate," he was quoted as telling reporters.
Gillard became prime minister by challenging Rudd's leadership of the Labor Party in 2010. She has set the next general election to be held on Sept. 14.


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Giant slingshot kills Roseville boy
Slingshots are the prop of choice for rapscallions from Dennis the Menace to Bart Simpson -- and probably that pesky kid who lives down the block from your house, too. The one who seems like he's ready to put someone's eye out. But until today, we hadn't really thought of slingshots as deadly weapons.
That was before we read the story about the dead 12-year-old from Roseville. The boy, whose name has not been released, was messing around with a large slingshot along with a friend in Bruce Russel Park yesterday; one with six- to seven-foot lengths of elastic tubing tied to a 9-foot-tall swing set at the park. They used it to launch progressively larger and larger stones into the air.
When they got up to a six pound rock, it came back down and hit the 12-year-old in the chest. He was rushed to Hennepin County Medical Center, where he was pronounced dead.
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IM park gets a playful boost
July 28, 2016 IRON MOUNTAIN — There will be a flurry of activity Saturday at the 25 Location Park on Iron Mountain's west side as the next phase of park improvements is ready to be done — the addition of a large... more »»
Iron River hosts U.P. firefighters
July 28, 2016 By EVAN REID Staff Writer IRON RIVER — Iron River is the site for the 122nd annual Upper Peninsula Volunteer Firefighters Association Tournament, the 10th time the city has hosted the event. more »»
No motive disclosed in Laurium slaying
July 28, 2016 HOUGHTON — For about six hours, the body of Kimberly Bess lay where she was shot multiple times Saturday while mowing the yard at her parents' house on the 100 block of Florida Street in Laurium,... more »»
Wisconsin roads budget faces $1 billion shortfall
July 28, 2016 MADISON, Wis. (AP) — The Republican co-chair of the Legislature's budget committee is at odds with Gov. more »»
Most campsites, cabins in U.P. state park reopen after storm
July 28, 2016 ONTONAGON, Mich. (AP) — Most backcountry cabins and campsites in Michigan's Porcupine Mountains Wilderness State Park have reopened after severe flooding from a summer storm. more »»
Eagle River man ID'd as suspect in fatal standoff
July 28, 2016 MERRILL, Wis. (AP) — Authorities say a 50-year-old suspect fired at law officers during a standoff in Lincoln County before police shot and killed him. more »»
Bicyclist treated for injuries after Iron County mishap
July 28, 2016 CRYSTAL FALLS — A driver struck and injured a Ypsilanti, Mich., woman on a cross-country bicycle trip Tuesday in Mansfield Township. more »»
Prison radios failed during attack — senator
July 28, 2016 ALLOUEZ, Wis. (AP) — A state senator says some radios didn't work at Green Bay's maximum security prison the day a corrections officer was attacked. State Se. more »»
Moving Mountains
July 28, 2016 Dickinson County Healthcare System is a sponsor of the sixth annual Moving Mountains fundraising golf tournament. The golf outing is scheduled for today at TimberStone at Pine Mountain Resort. more »»
Northwoods Garden Club holds annual strawberry social fundraiser
July 28, 2016 KINGSFORD — The Northwoods Garden Club hosted their 27th annual strawberry social at the Gazebo site at the corner of Balsam Street and Woodward Avenue in Kingsford. more »»
Pantry volunteers honored
July 28, 2016 Two volunteers at the Salvation Army are being recognized for their work with the pantry and in other areas on a daily basis. more »»
FlivverFest celebration starts Friday
July 27, 2016 KINGSFORD — FlivverFest 2016 will offer live music, food and other activities Friday and Saturday at Lodal Park in Kingsford. more »»
Long Lake man killed in UTV crash
July 27, 2016 FLORENCE, Wis. — A Long Lake man died Sunday in a utility vehicle accident in Florence County, authorities said today. James M. more »»
Hot weather might linger
July 27, 2016 IRON MOUNTAIN — A string of warmer than normal months might continue well into the fall, according to the National Weather Service. more »»
Judge recommends payments for Presque Isle power be reduced
July 27, 2016 RYAN JARVI For The Daily News MARQUETTE — The amount of subsidy payments related to a period of mandated operation of the Presque Isle Power Plant in Marquette to ensure electric reliability for th... more »»
Dickinson Memorial Hospital staff members earn scholarships
July 27, 2016 IRON MOUNTAIN — Four current staff members at Dickinson County Memorial Hospital earned scholarship awards for continuing education in their specific field of study. more »»
Paddling excursion Friday near proposed mine site
July 27, 2016 MADISON — A mine containing gold, copper, lead, zinc and silver is proposed to be developed right on the bank of the Menominee River near Stephenson. more »»
Run for the Walls sponsor
July 27, 2016 more »»
Myah Anderson awarded Dick Brown Scholarship
July 27, 2016 NIAGARA, Wis. — Myah Anderson was recently presented with the 16th annual Richard "Dick" Brown Memorial Scholarship. more »»
Palmisano donation
July 27, 2016 more »»
Genealogical Society meets in Iron River
July 27, 2016 IRON RIVER — On Aug. 4, the Iron County (MI) Genealogical Society will meet at 1 p.m., at the West Iron District Library in the J. Patrick White Conference Room. It will be an informal meeting. more »»
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July 24, 2016 ScuttleBu(r)t ... Arkansas denied Georgia the opportunity to talk to Razorbacks assistant Dan Enos abouts its offensive coordinator position. more »
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Depression can occur at any age, but it's often overlooked in adults 65 and older—a population that is expected to soar to 72 million by the year 2030. In her new ... Read More >
A July 2016 study conducted by researchers at the University of Alabama at Birmingham (UAB) and Johns Hopkins University School of Medicine published in PLOS ONE shows that symptoms of chronic ... Read More >
The National Institute of Allergy and Infectious Diseases (NIAID) estimates that about 50 million of Americans suffer from allergies, which are most active in the spring, summer, and early fall. Allergies, ... Read More >
The latest government guidelines for diabetes screening aren't working so well. In fact, researchers found, the guidelines missed 55 percent of high-risk patients who had diabetes or prediabetes. The research ... Read More >
Scientists at the Montreal Neurological Institute and Hospital have used a powerful tool to better understand the progression of late-onset Alzheimer's disease (LOAD), identifying its first physiological signs. Led ... Read More >
Health A-Z
Why Depression in Later Life is Often Overlooked
Depression can occur at any age, but it's often overlooked in adults 65 and older—a population that is expected to soar to 72 million by the year 2030.
In her new book Depression in Late Read More >
Alzheimer's Disease and other Dementias
"Big Data" Studies Help in Fight Against Alzheimer's
Scientists at the Montreal Neurological Institute and Hospital have used a powerful tool to better understand the progression of late-onset Alzheimer's disease (LOAD), identifying its first physi Read More >
Mental & Emotional Health
Thinking "I can do better" Improves Performance
Telling yourself "I can do better" really can really make you do better at a given task, according to a study published in June 2016 in Frontiers in Psychology.
A release from the publis Read More >
Pain Management (Knee Pain, Back Pain, Stomach Pain)
The Illness of Chronic Pain
A sprained ankle can be a real pain. But injuries like sprains, bone fractures, pulled muscles, cuts, and even minor burns are everyday pain and a normal part of life. While the pain ma Read More >
Living Well
The Dangers of Multitasking
We're all beyond busy these days. Whether we're texting while we're waiting in line, or decluttering and cleaning the house at the same time, doing it constantly can take a toll on us in a nu Read More >
Staying Hydrated in The Heat
Editor's note: It's always good to stay hydrated, but it's absolutely essential in the hot weather, where you can become thirsty without even realizing it. Here, from the experts at Go4Life, Read More >
Money Matters
Even with Insurance, a Hospital Visit May Cost $1,000 or More
Even if you have what you might think of as good health insurance, your next hospital stay could cost you more than $1,000 out of your own pocket.
And that amount has gone up sharply in rece Read More >
Dating After 50: Why You Should Go Offline
Navigating the online dating world can be intimidating – and for newly single women over 50, it presents its own set of challenges. Sites like eHarmony and Match are brimming with photos of 20-so Read More >
Diet & Nutrition
Harvard's HT Chen Nutrition Source Repeats: Butter Is Not Back
On June 29th 2016, a systematic review and meta-analysis looking at the association of butter consumption with chronic disease and all-cause mortality made headlines that sound strikingl Read More >
Diet & Nutrition
Pomegranate's Powerful Anti-Aging Secret Revealed
Are pomegranates really the superfood we've been led to believe will counteract the aging process? Up to now, scientific proof has been fairly weak. And some controversial marketing tactics have Read More >
Diet & Nutrition
The Five Best Weight-Loss Programs (That Aren't Fads)
Whenever I kick off a new fitness regimen, it's not just about a new form of cardio or training. It's also about my diet. I'm determined to find just the right concoction to help me achieve o Read More >
Diet & Nutrition
Pasta is Not Fattening!
In recent years pasta gained a bad reputation: it will fatten you. This led lots of people to limit its consumption, often as part of some aggressive "do it yourself" diets. Now a study conduct Read More >
Walking Meetings Could Mean Healthier Lives for Office Workers
Changing just one seated meeting per week at work into a walking meeting increased the work-related physical activity levels of white-collar workers by 10 minutes, according to research published b Read More >
Go for Your Own Gold
The summer Olympics begin one month from today. As the world watches sports like triathlon, cycling, swimming and more, many of those who cheer on the champs are inspired and motivated themse Read More >
Helping a Loved One Get Active
Editor's note: We all know the benefits of exercise, but sometimes it's difficult even to just get going. If we see that a friend or loved one needs to do that, it might be difficult to hold ba Read More >
Parks Are Seldom Used by Older People
Neighborhood parks across the United States are geared primarily toward younger people, limiting their use by adults and seniors, but surprisingly attract fewer female youth as well, according to a Read More >
What Goes into A Good Fitness Plan?
Everyone knows it's healthy to exercise regularly. But without a plan, one that tracks activity and helps keep you motivated, that can be difficult to keep up.
Why is a plan so important? Read More >
How to Survive — and Thrive — After a Divorce
The desire to follow our passions and find excitement in life doesn't dissipate as we age.
In recent years, I've met with many seniors who are still eager to expand their horizons. Wheth Read More >
How to Cope with Caregiver Stress
On October 30, 1993, my father suffered a debilitating stroke. The next day he was diagnosed with prostate cancer. My mother, who was suffering with macular degeneration, osteoporosis, congestive h Read More >
Relationships & Love
Five Ways to Maintain a Grateful Harmony in Your Relationship
"What If…" We've asked this question many times, sometimes in our heads and sometimes to each other. As college sweethearts, we separated because of a lack of maturity and communicati Read More >
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Chivalry May Not Be Dead when It Comes to Morality
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Better than takeout, this dish comes together super fast. Loaded with protein and heart-healthy fats, this will fill you up and keep you satisfied. … [Read more...]
Paleo Tropical Fruit Salad
Dried papaya paired with tropical fruit is perfection when drizzled with a bit of lime juice. This salad makes the perfect snack, but can do wonders as a dessert. … [Read more...]
Paleo Lavender Lemon Smoothie
Creamy and fragrant, this lavender smoothie is perfect any time of the day. Don't overdo it on the lavender; a little is perfection, too much will leave you with a smoothie that tastes like soap! … [Read more...]
Easy Paleo Swaps for Your 4th of July BBQ
It's that time of year again – the 4th of July BBQ that's filled with highly processed foods, lots of refined carbohydrates and sugary treats. This year, swap out the not-so-healthy foods and bring in paleo friendly recipes that will leave you feeling light and energized afterwards. Try these easy paleo swaps for your appetizers, entrees, sides and desserts. They are flavorful, nutritious and unique, so your guests will be pleasantly surprised that a paleo BBQ could taste so good. Instead of snacking on greasy and processed chips while waiting for the main course, serve paleo chips or roasted nuts. Forget the Lays and Doritos, … [Read more...]
Paleo Italian Meatballs and Braised Greens
What's better than having Paleo Italian Meatballs? Meatball recipes are often challenged by chefs claiming theirs is the best. Well, here's a better, probably the best, Italian Meatballs recipe. Aside from it's delicious taste, it is Paleo. And you already know what that means. These are healthy and guilt-free meatballs. The classic meatballs are made with beef and pork combined together with garlic, onions, salt and black pepper. It's easy to make and quick enough for your starving tummies. In this recipe, we only used ground beef instead of mixing both beef and pork. Combined with onion, garlic, almond flour, paprika, and salt to make … [Read more...]
Paleo Kitchen Essentials: How to stock your pantry
Stocking a Paleo pantry can seem a little intimidating at first. But once you have stocked up once or twice, it basically becomes second nature. The first step you will need to take in order to stock up properly will be cleaning out your pantry, fridge, and freezer. This will ensure you are getting rid of all the items you don't need/use anymore and making room for all the true essentials. Make sure when you are doing this you read the labels for all packaged foods, checking for added sugars, gluten, or any other processed ingredients. Not everything that is packaged is bad or off limits though. There are tons of great Paleo-friendly … [Read more...]
Paleo Diet Tweaks for Performance Athletes
The requirements of athletes can vary based on the individual themselves, his or her sport, and the level of activity. A competitive CrossFitter will have different needs than that of a marathon runner or Power Lifter. However, there is a very similar foundation and despite these differences, all athletes share a couple things: optimizing performance and recovery. It is often thought that the Paleo diet is only geared towards those who want to lose weight and improve longevity. But it is also great for athletes; it just needs to be adjusted accordingly. The Paleo diet is full of high-quality proteins which contain essential amino acids for … [Read more...]
Paleo Sour Cherry Smoothie
Refresh yourselves with Cherry Smoothie on a hot summer day! Cherries are naturally delicious and tasty even if you'll eat them on their own. But it is even ridiculously delicious when you make them into a refreshing smoothie. What's good about cherry is it only doesn't satisfy our fruit cravings but it also contain powerful antioxidants that helps boost our immune system. Cherry also has cancer-preventive compounds, supports healthy sleep, relieves arthritis pain, reduces belly fat, inflammation, risk of gout and post exercise muscle pain, and it also lowers your risk of stroke. Isn't it interesting? A small yet powerful and … [Read more...]
Paleo Steak and Vegetable Stir-Fry
Who doesn't love paleo steak? It's one of my favorites and it goes with vegetable stir-fry in this recipe. Paleo Steak and Vegetable Stir-Fry This quick and easy recipe serves a family of four. An Asian inspired hearty paleo steak and vegetable bowl is a dish that is chocked full of protein and nutrients that is exactly what you need to keep you energized all day long. The tastiness of a juicy steak along with crunchy fresh veggies, will make this a favorite go-to meal any day of the week. The recipe starts by heating the very aromatic golden-colored and nutty flavored, sesame oil, until hot. Next you throw … [Read more...]
8 Ways to Curb Your Cravings
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Karrey Britt (WellCommons.com Reporter)
During my 13 years at the Lawrence Journal-World, I've worn a number of hats. I started by working the nightshift as a copy editor and progressed to society editor and then assistant assignment director.
During the past few years, I returned to reporting, and it has proved rewarding.
I have met many fascinating people including:
• Susan Mozykowski, who opened her home and heart to me while battling brain cancer.
• Lawrence resident Heidi Karn Barker, who had a life-saving organ transplant and allowed me to capture her journey.
• Michael Douglas, who has a rare lung disease, but you wouldn't know it by his upbeat attitude and gratitude for life.
Today, I am a health reporter. Now, I wouldn't call myself a health nut, but I watch what I eat and enjoy exercising. I also support the movement to buy locally-produced foods.
That's because I grew up on a small cattle-and-wheat farm near Abilene and was actively involved in 4-H. I still enjoy gardening and baking — two projects I took in 4-H. One of my favorite jobs was working at Rock Springs 4-H Center as an archery instructor for two summers.
I graduated from Chapman High School, and then earned a bachelor's degree in journalism at K-State, where my husband and I met. We still bleed purple despite living in a sea of crimson-and-blue Jayhawk fans.
We both love animals and have two cats named Allie and Sam. Allie was adopted from a shelter and Sam was adopted from my parent's farm.
My first journalism job was at The Daily Union in Junction City, where I worked for nearly three years and that included during the 1995 Oklahoma City bombing. I was the wire editor at the time and fielded questions from national media about that horrific day because of Timothy McVeigh's connections to the town and Fort Riley.
I always tell people that being a journalist is never boring.
For example, I received Botox for a story about anti-aging procedures. A few months later, I designed the front page of the newspaper on the night when KU's basketball team won the national championship. I had a blast handing out papers in downtown Lawrence.
I have evolved into a multi-media reporter — something I never expected. I report for 6News in Lawrence and KTKA in Topeka.
You can follow me on Twitter, where I provide instant updates on health news and what's going on in Lawrence.
I am a blogger for a new health Web site. My goal is to spark a community conversation about how we can live healthier, happier lives and then report on our progress.
If you have a story idea or suggestion for the health Web site, please contact me by e-mailing firstname.lastname@example.com or calling (785) 832-7190.
Recent stories
- Safety tips for dealing with freezing weather
- December 31, 2012
- With temperatures hovering around the freezing mark, the Lawrence-Douglas County Health Department and the Kansas Department of Health and Environment are encouraging Kansans to exercise caution when outdoors. By Karrey Britt
- Best of Lawrence 2012: Best Orthodontist
- September 30, 2012
- When you walk in Dr. Dan Ranjbar's office, you are greeted by a 7-year-old golden retriever named Sunny, who loves attention. It's just one of the ways Ranjbar tries to make his patients feel comfortable. He's also incorporated his love of nature into the office design.
- Best of Lawrence 2012: Best Family Doctor
- September 30, 2012
- Dr. Ryan Neuhofel is not only new to Lawrence, he's bringing new ideas to family medicine. His goal is to bring back old-fashioned medicine, where a doctor has a relationship with his or her patients.
- Physician at KU's Watkins Memorial Health Center identified as pedestrian struck by train
- 12:27 p.m., September 18, 2012 Updated 08:35 p.m.
- Lawrence police identified a 56-year-old Lawrence physician as the pedestrian who was struck and killed by a train Monday morning in east Lawrence.
- KU Hospital's turnaround seen as extraordinary
- August 19, 2012
- Kansas University Hospital is the comeback kid. Fourteen years ago, the hospital was fighting for survival. Employee turnover was high, patient volume was low and money was hard to come by. The hospital was on the verge of bankruptcy as a state agency.
- KU Hospital's expansion garnering national attention
- August 19, 2012
- The construction project is just one of many that have occurred at KU Hospital during the past five years to help keep up with growing demand for its services.
- Throwing a strike against cancer
- July 4, 2012
- Two Lawrence women have teamed up to organize a bowling event July 21 that will benefit the organizations they are passionate about: Livestrong and Catch a Break.
- Free State High School graduate Trei Dudley among 5 finalists for National Youth of the Year
- June 9, 2012
- Trei Dudley, 18, of Lawrence is one of only five candidates who could be named National Youth of the Year by the Boys and Girls Club of America and earn a $50,000 scholarship. In September, she will visit the White House and meet President Obama.
- Teen's generosity with found money leads to brotherly bond
- June 8, 2012
- Four-year-old Cooper Keefer played catch Wednesday evening in Centennial Park with his hero, 14-year-old Dallas Hunt.
- LMH employees chip in for mutual aid
- May 7, 2012
- Michael Van Pelt can't thank his fellow employees enough for the help they provided during his battle with head and neck cancer. "I am still amazed at some of the things that happened," he said. "You can imagine when a doctor tells you, 'You have cancer,' just everything stops, and you immediately worry about everything."
Recent photos
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- Advocates say Medicaid expansion could insure about 5,000 in Douglas County July 25, 2016 · 16 comments
- Blog: Another fast-food chicken restaurant sets its sights on Lawrence; basketball court promise broken at Rock Chalk Park July 25, 2016 · 34 comments
- Kansas officials certify 75-year-old woman as a voting-eligible citizen after birth certificate lost July 26, 2016 · 3 comments
- Atheists sue Kansas City officials over Baptist convention July 26, 2016 · 5 comments
- Lawrence school district budget likely to include teacher cuts July 25, 2016 · 13 comments
- Letter to the editor: Ouster advocated July 26, 2016 · 5 comments
- Lawrence school district budget includes teacher cuts, deficit spending and lower taxes July 25, 2016 · 4 comments
- Kansas Democrats: Backing Sanders, seeking unity for Clinton July 24, 2016 · 17 comments
- Editorial: Grocery plans July 26, 2016 · 2 comments
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- Proposed 2017 Douglas County budget would raise mill levy July 11, 2016
- Modified tax lid bill for local governments goes to governor April 29, 2016
- Eudora City Commission sets date for 2017 budget hearing July 25, 2016
- Tom Keegan: Ex-Lion pitcher major talent July 22, 2016
- Letter to the editor: Ouster advocated July 26, 2016
- Douglas County Treasurer's line management system faces big end-of-month test July 24, 2016


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PHILADELPHIA - Brett Brown pulled out a chair, propped a foot on the seat and settled in for another pep talk.
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29 April to 29 July 2016
To celebrate 30 years of 'Poems on the Underground', this new poster parade looks back at a selection of poems from the programme, which was developed by a group of friends who wanted to make poetry available to a mass audience.
The exhibition showcases poems in the 'Poems on the Underground' style, with a selection of classical, contemporary and international work from famous and unknown poets on show. Four selected poems are available in specially-designed braille displays.
The Poster Parade is temporary display of posters from our collection that changes regularly. It can be found on the Mezzanine on Level 1 of the Museum. You can purchase copies of London Underground poster art online.
Open now
Take a tour of the new stations, art and public space on London's newest railway in a new exhibition at London Transport Museum.
Crossrail is Europe's largest infrastructure project. It is delivering 42kms of new tunnels, 10 new stations and improvements at many more and a series of exciting public art installations. This new exhibition tells the story of its design.
Platform for Design, a new book by architecture critic and RIBA Journal editor Hugh Pearman, is now available to coincide with the launch of 'the design line' exhibition. With over 150 visualisations and photographs, the book explores the design of the railway's new and upgraded stations, the new public spaces, the trains and the artworks being delivered by Crossrail, Europe's biggest infrastructure project.
Buy Platform for Design from the London Transport Museum shop.
Shoppers will be able to buy gifts and toys inspired by the Crossrail tunnelling story such a crocheted mole and elegant posters. Design enthusiasts will love the new contemporary homeware gifts which are made from materials similar to those used in the building of Crossrail. There is also a collection of gifts moulded from concrete which are inspired by Crossrail's tunnelling achievements. All the above Crosssrail related items will be available to buy in the Museum shop in Covent Garden and online.
The Fairway FX4 taxi is retiring from licensed service to make way for newer cabs with lower emissions. A London icon, this classic cab has worked on our streets since 1958.
Over the years, new models were issued with modifications to the gearbox, engine and interior. The final FX4 update was the wheelchair-accessible Fairway in 1989.The Fairway spirit lives on in today's TX-type taxis, which have adopted and adapted the FX4's distinctive shape and style.
This display, which is near to the Museum's own FX4 taxi, will feature objects on loan from the London Vintage Taxi Association (LVTA), including the 'Blue book' - the guide used by prospective cabbies to prepare for the famous 'Knowledge of London' exam - early fare books and taximeters, and a selection of models.
The Museum's exciting new gallery, 'Future journey, future city', explores visions of future transport. After travelling through 200 years of London's pioneering transport network, the lifeblood of our wonderful city, this new gallery invites you to gaze into the possible future of our global cities. A rolling programme of exhibits will explore the incredible innovations that passengers might expect to see in the future – from Royal College of Art students' vehicle designs through to Cubic's NextCity concept along with how people in the past envisaged the future.
We have to imagine the future before we can build it. Although the success of any one idea cannot be predicted, it is certain that innovation and creative thinking will be needed to transform our future cities. Innovation in technology and information systems, travel modes, architecture and infrastructure will all influence the urban future. Your personal needs and choices as you move through the city will also affect the tempo of change. Whatever direction we take or choices we make, transport will continue as the lifeblood of our future cities, including London - explore, experience and enjoy!