text:
Flu Still Hitting Hard Locally, Nationally
More than 60,000 people nationwide have contracted the flu during the 2017-2018 influenza season as of Jan. 6, according to the Centers for Disease Control and Prevention (CDC).
Emergency rooms across the country, including those at all Lehigh Valley Health Network (LVHN) hospitals, have been extremely busy with flu patients. The CDC reports flu activity as widespread in 49 of the 50 states, and 20 pediatric deaths reportedly have been flu-related.
"The good news is the flu season has likely peaked, although it will be a few weeks before we see it declining," says Luther Rhodes, MD, Chief of Hospital Epidemiology at LVHN. "This is far from the worst flu season we've had. I'd call this an average year."
As of Jan. 6, 11,530 flu cases had been reported across Pennsylvania, including 537 cases in Northampton County and 506 in Lehigh County, according to state Department of Health statistics. The flu has hit especially hard in western Pennsylvania, with 1,842 cases reported in Allegheny County, 658 in Westmoreland County and 588 in Washington County. There have been 560 cases reported in Berks County.
Flu Symptoms
For most healthy people, the symptoms of the flu are like a bad cold. Usually, symptoms come on more abruptly and are more severe. Common symptoms include:
- Cough
- Sore throat
- Runny nose
- Fever
- Muscle pain
- Headache
- Fatigue
- Sometimes vomiting and diarrhea
Avoiding the Flu
Even though flu season appears to have peaked, getting a flu shot remains your best deterrent in case you come in contact with the influenza virus. Check with your primary care provider about a flu shot.
Other things to keep in mind when trying to avoid the flu:
- Wash your hands often with soap and water
- Avoid touching your eyes, nose or mouth. Germs on your hands can easily spread this way.
- Avoid close contact with sick people.
If You Have the Flu
If you need medical attention, chances are you will find local emergency rooms very crowded. We recommend you visit one of Lehigh Valley Physician Group's 19 ExpressCARE locations, including the region's only Children's ExpressCARE specifically for kids at the Health Center at Palmer Township. Walk in to any ExpressCARE for care without an appointment. Visit our ExpressCARE page on LVHN.org to see the latest average wait time at most locations.


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Mata Ortiz Collection
About Mata Ortiz Pottery and the CCA Collection
Mata Ortiz is a small village in the state of Chihuahua in northern Mexico. It is very near the archaeological site of Paquime (Casas Grandes) which dates from the 1100's. In the 1970's, a young man named Juan Quezada became interested in the pottery shards he found in the hills around his home. He began reproducing pottery in the style of the archeological site of Paquime (Casas Grandes) with materials he found locally. Soon others from the village joined him and today there are over 450 potters in the village and surrounding area.
Many potters still use the traditional designs but the movement continues to evolve as many potters today are third generation. They are very innovative and continue to explore new ideas.
The potters do not use a wheel but form the pots with their hands. The clay is obtained from around the village using pick-axes and shovels. The paints are natural. The pottery is dung-fired on the ground, not in a kiln. Each piece is signed by the artist.
You will find Mata Ortiz pottery in museums around the world. The collection of Mata Ortiz pottery currently on display at CCA is on permanent loan by a group of Chatfield High School graduates that were introduced to Mata Ortiz pottery by Mary (Chase) Borgen in spring of 2014. While visiting LaDonna (Jackson) Boyd in Arizona, Darold and June (Borgen) Olson were immediately smitten and began looking for pieces in galleries in the Phoenix area, Tucson and Silver City, New Mexico. They were told by many gallery owners that it was very dangerous to visit Mata Ortiz and that the gallery owners bought their pottery from potters that came north to sell their wares.
Darold and June soon became aware of several websites that sold Mata Ortiz pottery. Darold ordered a few pieces online and discovered that one of the websites was in Hastings, Minnesota, just a few miles from where they live. Darold went to visit the shop and met Ron Martino who has made several trips to the village to buy pottery to sell in his shop. Ron encouraged Darold and June to make a trip to Mata Ortiz ourselves — telling them that they would absolutely fall in love with the people and that it was quite safe to travel into Mexico.
They searched online and found a guide, John Hatch, who is a dual citizen of Mexico and the U.S. John was born in Mexico in a Mormon settlement and has lived in the state of Chihuahua all his life. He speaks both Spanish and English like a native and is very knowledgeable about the history of the area.
Darold, June, and Ladonna decided they would make the trip and began to make plans — which included getting two more friends from Chatfield interested in going to Mata Ortiz — Tom Touhy and Judy Stites.
They first arranged to meet John Hatch in Douglas, Arizona on February 16, 2016 and made several more trips on this new adventure.

subset: commoncrawl


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Frataxin is a highly conserved mitochondrial protein whose deficiency in humans results in Friedreich's ataxia (FRDA), an autosomal recessive disorder characterized by progressive ataxia and cardiomyopathy. Although its cellular function is still not fully clear, the fact that frataxin plays a crucial role in Fe–S assembly on the scaffold protein Isu is well accepted. In the present paper, we report the characterization of eight frataxin variants having alterations on two putative functional regions: the α1/β1 acidic ridge and the conserved β-sheet surface. We report that frataxin iron-binding capacity is quite robust: even when five of the most conserved residues from the putative iron-binding region are altered, at least two iron atoms per monomer can be bound, although with decreased affinity. Furthermore, we conclude that the acidic ridge is designed to favour function over stability. The negative charges have a functional role, but at the same time significantly impair frataxin's stability. Removing five of those charges results in a thermal stabilization of ~24 °C and reduces the inherent conformational plasticity. Alterations on the conserved β-sheet residues have only a modest impact on the protein stability, highlighting the functional importance of residues 122–124.
Reduced expression of frataxin, or in some cases, expression of frataxin variants, leads to the development of Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative and cardiodegenerative disease characterized by progressive ataxia and cardiomyopathy [1–3]. In spite of the availability of multicellular models for the disease [4,5], insights into possible therapeutic strategies [6,7] and broader systematic clinical studies [8], the pathogenesis of FRDA and its relationship with frataxin remains to be fully understood. Frataxin is a highly conserved protein localized in the mitochondria whose function is associated with iron homoeostasis. Frataxin deficiency, as shown recently using conditional knockout mice, down-regulates molecules involved in mitochondrial iron-utilization pathways (Fe–S cluster and haem biosynthesis, and iron storage), while cellular iron uptake and its availability for mitochondrial uptake are increased [5]. This illustrates the complex interplay between frataxin deficiency and iron homoeostasis in the context of FRDA [5].
The human and yeast frataxin (Yfh1) orthologues have a high degree of amino acid (65%) and structural identity. The phenotype observed as a consequence of frataxin's deficiency is very similar in human and yeast cells [9,10]. Hence, the yeast model system has been used frequently to study frataxin's function, as well as the pathological events associated with FRDA. Although a detailed understanding of frataxin function is still lacking, data suggest a role as an iron metallochaperone [11–14], delivering iron to its binding partners, promoting haem and Fe–S clusters biosynthesis, as well as Fe–S cluster repair [15–17]. Although, initially, oligomerization was believed to be dispensable in vivo, it has been shown subsequently that oligomerization impairment is harmful under oxidative stress conditions and it leads to a decrease in yeast chronological lifespan [11,18,19]. Yfh1 is not essential, but its absence causes severe growth defects and a reduction in the activity of Fe–S-cluster-containing enzymes, such as aconitase [9,10,19,20]. Iron binding by frataxins, a requirement for a role as a metallochaperone, has been well documented by several laboratories [16,21,22]. Frataxins bind iron with micromolar binding affinities, but different stoichiometries: while monomeric human frataxin binds six or seven iron ions (Kd ~12–55 μM [16]), monomeric frataxin from Drosphila binds one ferrous iron (Kd ~6 μM [22]) and yeast frataxin binds two ferrous irons (Kd ~2.5 μM [21]). This variability suggests a certain plasticity of the frataxin fold in respect to binding iron. In fact, an NMR-based investigation of the iron binding to Yfh1 at low stoichiometry (up to 2:1 Fe/Yfh1) denoted changes in ten distinct residues [23], which could indicate long-range effects upon iron binding, or lower-affinity sites. More complex effects arise at higher Fe/Yfh1 stoichiometries. Like human frataxin, Yfh1 undergoes iron-dependent oligomerization, above a threshold of two irons per monomer, according to the progression α→α3→α6→α12→α24→α48 [11,18]. This property has been established as important for iron-induced oxidative stress protection, but not for Fe–S cluster biogenesis [19,24].
The conserved frataxin fold consists of an α/β sandwich motif, with two α-helices (N- and C-terminal) that construct the helical plane and five antiparallel β-strands that form the β-sheet plane. A sixth (seventh in the human protein) β-strand intersects the helical and β-sheet planes [23,25–28]. The first helix (α1) and the edge of the first β-strand (β1) form a semi-conserved acidic ridge that constitutes the putative iron-binding region of the protein [21,23,28]. A set of conserved residues in the β-sheet surface constitutes the other putative functional region of the protein, believed to be involved in the binding of frataxin to its protein partners [23,29]. Recently, we have shown that mutations at the conserved β-sheet residues 122–124 affect neither iron-binding capacity nor the oligomerization properties of frataxin, although these mutations do compromise the interaction with Isu [30].
To complement previous functional characterization of YFH1 mutants, in the present study we address the structural and conformational effects of eight functional mutants in yeast frataxin that alter either the acid ridge or conserved residues on the β-sheet surface. We found that the negatively charged residues in the acid ridge that are important for iron binding reduce Yfh1's stability, indicating a trade off between functionality and stability. In addition, alterations in the conserved residues of the portion of the acid ridge found in the β-sheet affected interaction with the scaffold protein Isu, but did not compromise overall protein stability.
Yeast strains, plasmids and media
Saccharomyces cerevisiae strains carrying YFH1 mutant or wild-type genes on plasmids were created by sporulation and dissection of YFH1/yfh1Δ (W303 background) transformed with the plasmids. YFH1 mutants were generated by site-directed mutagenesis using pCM189-YFH1 as a template, having YFH1 under the control of the tetO promoter [31]. Constructs containing the following mutations were prepared: yfh1-D86A/E90A/E93A, yfh1-D101A/E103A, yfh1-D86A/E90A/E93A/D101A/E103A, yfh1-N122A, yfh1-N122K (corresponding to the human clinical mutation N146K), yfh1-K123T, yfh1-Q124A and yfh1-N122A/K123T/Q124A. Doxycycline was used at 1 μg/ml to reduce expression. Cells were grown in minimal synthetic medium with −ura DO (uracil drop-out) at 30 °C for 2 days.
Aconitase activity in isolated mitochondria
Mitochondria were isolated, according to the method described in [32], from cells grown in synthetic minimal medium (−ura DO), in the absence of doxycycline. Aconitase activity was measured in isolated mitochondria by monitoring the decrease in the absorbance of the substrate isocitrate at 240 nm as described in [33].
Gene expression and protein purification
The mature form of Yfh1 (amino acids 52–174) with an N-terminal His6 tag and a thrombin cleavage site in between was cloned into pET-3a vector (Novagen) [19]. Protein expression was induced over 3 h at 37 °C in BL21(DE3) Escherichia coli cells by adding IPTG (isopropyl β-D-thiogalactoside) at a final concentration of 0.5 mM. With the exception of Yfh1-K123T, which was expressed at much lower levels (approx. 30%), all other mutant variants had expression levels identical with those of the wild-type Yfh1. The reduced expression levels of Yfh1-K123T in yeast have been described previously [30]. Under the conditions used, wild-type Yfh1 was exclusively expressed as a soluble protein, as were most of the mutants. The only exceptions were Yfh1-D86A/E90A/E93A, Yfh1-N122A and Yfh1-N122A/K123/Q124A, in which ~15% of the expressed protein was found in the insoluble fraction. After harvesting the cells, they were lysed on a French press. Cell lysate was subjected to His-binding resin (GE Healthcare) chromatography and the protein was eluted with 500 mM imidazole in binding buffer (50 mM Tris/HCl, pH 8.0, 200 mM NaCl and 10 mM imidazole). The His6 tag was cleaved using biotinylated thrombin, and thrombin was removed by streptavidin–agarose (GE Healthcare). When necessary (purity less then 90%), the protein was purified further by applying the sample on a Superdex 75. At the end, the buffer was changed to 10 mM Hepes and 50 mM NaCl (pH 7.0) using Centricons (Millipore). Protein concentration was determined using the molar absorption coefficient ε280=15470 M−1·cm−1. The mature form of Isu1 (amino acids 35–165) with a His6 tag and a TEV (tobacco etch virus) protease cleavage site in between was cloned into the pKLD37 vector. Protein expression was induced over 4 h at 30 °C in C41 E. coli by adding IPTG at a final concentration of 1 mM. The purification was performed as above described for the Yfh1 variants, but using TEV to cleave the His6 tag and passing through a His-binding resin to remove it. Protein concentration was determined using the molar absorption coefficient ε280=14161.4 M−1·cm−1.
Spectroscopic methods
UV–visible spectra were recorded at room temperature (20 °C) in a Shimadzu UVPC-1601 spectrometer. Fluorescence spectroscopy was performed on a Cary Varian Eclipse instrument (λex=280 nm, λem=340 nm, slitex=5 nm, slitem=10 nm, unless noted otherwise) equipped with cell stirring and Peltier temperature control. Far-UV CD spectra were recorded typically at 0.2 nm resolution on a Jasco J-715 spectropolarimeter fitted with a thermostatically controlled cell holder.
Thermal denaturation
Thermal unfolding was followed by ellipticity (Δεrmw at 222 nm) variations. In all experiments, a heating rate of 1 °C/min was used, and the temperature was changed from 10 to 90 °C. All reactions were found to be reversible under the conditions used, as inferred from obtaining identical far-UV CD spectra of the proteins before the thermal ramping and after cooling the heated sample; a second temperature ramp of the same sample also yielded identical melting transitions. Protein aggregation was not observed during thermal unfolding. Data were analysed according to a two-state model, and the thermodynamic data parameters were determined [34].
Limited trypsin proteolysis
Frataxins were incubated with trypsin (bovine pancreas trypsin, sequencing grade, Sigma) at 37 °C in 0.1 M Tris/HCl (pH 8.5), in a 150-fold excess over the protease. Aliquots (300 μmol of protein) were sampled at different incubation periods and the reaction was stopped by adding loading buffer with 5 μM BSA, followed by a 10 min incubation at 100 °C. The products of the proteolysis reaction were analysed by SDS/PAGE [35].
Iron-binding assays
Yeast frataxin oligomerizes in an iron-concentration-dependent fashion, but at up to a stoichiometry of two irons per protein, the protein remains in the monomeric form [11,18,23,36]. To avoid side effects from different oligomerization behaviours, we have measured iron binding before oligomerization takes place, i.e. up to an iron/protein ratio of 2. Tryptophan fluorescence emission spectroscopy was used to monitor iron binding, which quenches tryptophan emission as the iron–frataxin complex is being formed. As shown previously, this methods yield results identical with those obtained by isothermal titration calorimetry [16,37]. For the purpose, the quenching of the tryptophan fluorescence (λex=290 nm; λem=340 nm) of a 50 μM solution of apo-frataxin in 100 mM Hepes (pH 7.0) and 50 mM NaCl was measured upon stepwise addition of an iron citrate solution, in 1 mm-pathlength quartz cuvettes under continuous stirring [35,37]. These data were used to calculate the fraction of binding sites occupied. The stoichiometry, p, and apparent dissociation constant, Kd, were then determined as described by Winzor and Sawyer [38].
Isu–Yfh1 interaction assay
Holo-Yfh1 was prepared using a stoichiometry of two irons per frataxin to avoid interference from oligomer formation. The formation of a Isu–holo-frataxin complex was followed monitoring the tryptophan fluorescence, taking advantage of the fact that yeast Isu does not contain any tryptophan residues. Thus, when Isu binds to holo-Yfh1, the tryptophan fluorescence emission (λex=290 nm; λem=340 nm) of the latter is quenched and this can be used to monitor binding. Holo-Yfh1 was prepared in 100 mM Hepes (pH 7.0) and 50 mM NaCl. Aliquots of Isu were added to 2.5 μM holo-frataxins with constant stirring. The stoichiometry, p, and apparent dissociation constant, Kd, were then obtained as described by Winzor and Sawyer [38].
Alterations in the conserved β-sheet surface result in functional effects
To gain a better understanding of the relationship between the structure and function of frataxin, we compared four Yfh1 variants. Three (Yfh1-D101A/E103A, Yfh1-D86A/E90A/E93A and a combination of these two, Yfh1-D86A/E90A/E93A/D101A/E103A) alter the putative iron-binding region at the α1/β1 acidic ridge and oligomerization region (Figure 1). The fourth, Yfh1-N122A/K123T/Q124A (hereafter referred to as Yfh1-122-4), alters the conserved β-sheet, the putative binding surface for interaction with Isu, the scaffold on which Fe–S clusters are built [30]. Since Yfh1 cellular levels can be reduced significantly before any growth defects are detected [39], wild-type and mutant YFH1 genes were placed under the control of the tetO promoter allowing the modulation of protein expression using doxycycline. In this way, in vivo function could be tested under both normal and reduced levels of expression. At normal Yfh1 expression levels (without doxycycline), all mutant gene were able to support growth as well as the wild-type gene (Figure 2). In addition, as we reported previously [19,30], Yfh1-122-4 cells grew poorly when the variant was expressed at reduced levels (with doxycycline), whereas cells expressing low levels of Yfh1-D86A/E90A/E93A grew as well as wild-type cells. However, Yfh1-D101A/E103A was slightly compromised, and when these mutations were combined with those of Yfh1-D86A/E90A/E93A, generating alterations in five of the charged residues in the acidic ridge, growth was severely compromised. Consistent with this enhanced growth phenotype, mitochondria isolated from Yfh1-D101A/E103A had lower levels of activity of the Fe–S enzyme aconitase, even when the mutant protein was expressed at normal levels (Figure 3). This effect was more extreme in Yfh1-D86A/E90A/E93A/D101A/E103A mitochondria, which had only 40% the aconitase activity of wild-type mitochondria. Consistent with the affects on growth, Yfh1-122-4 mitochondria had only 20% of the aconitase activity quantified for wild-type mitochondria.
Yfh1 ribbon structure generated using PyMOL (PDB code 2GA5)
Yeast growth rescue of (Δyfh1) cells by Yfh1 variants
Aconitase activity
None of the mutations abolishes iron binding
Since iron binding is an essential functional feature of frataxin, we asked whether iron-binding properties of the Yfh1 variants were affected. In order to exclude iron-induced frataxin oligomerization effects, we have investigated iron binding at low stoichiometry [up to two Fe(II)/Yfh1], i.e. under conditions in which oligomerization does not take place. We have used tryptophan fluorescence to monitor iron binding to Yfh1 variants, as tryptophan emission is a specific reporter for iron binding to frataxin. Our measurements indicated that mutations altering the iron-binding region, D86A, E93A, D101A and E103A, had no effect on the iron-binding capacity at low stoichiometry, but did decrease binding affinity somewhat (Table 1). Conversely, iron must be able to bind to frataxin through other residues, as even the quintuple mutant Yfh1-D86A/E90A/E93A/D101A/E103A retained the ability to bind ~2 Fe(II)/Yfh1. We hypothesize that iron is binding to secondary sites with lower affinity, as evident by the higher dissociation constants (~20 μM). In fact, a previous NMR study has shown that, under identical conditions at low iron stoichiometry, iron binding to Yfh1 affected multiple sites: mainly interactions with carboxy groups and nitrogen from acidic residues within the α1/β1 ridge (His83, Asp86, Glu93, His95, Asp101 and Glu103), but other residues (Ala94, Leu104, Ser105 and Asn140) were also found to change their resonance positions upon binding of up to two irons per frataxin [23]. The lower binding affinity that we have determined in the Yfh1 variants (nevertheless still in the micromolar range comparable with that of human frataxins) could possibly indicate the recruitment of secondary positions, rather than unspecific binding. However, this is not the case, as the observed iron binding is functional, as shown by the fact that yeast expressing these variants still has some aconitase activity (Figure 3), which depends on frataxin-mediated iron transfer [15]. In addition, a previous study confirms our observations: it has been shown that single point mutations to alanine on residues 86, 90, 93, 101 and 103 reduce Yfh1 affinity for iron, but do not abolish iron binding [24]. Single point mutations in the Asn122–Gln124 segment seem to have an intermediate effect in respect to the binding affinity (~14–18 μM). Presumably, alterations in the protein–protein interaction region of Yfh1 result in long-range effects on the iron-binding acid ridge leading to a decrease in the iron-binding affinity.
|ΔH (kJ·mol−1)
|ΔG* (J·mol−1)
|Tm (°C)
|ΔTm (°C)
|Δ(ΔG)† (J·mol−1)
|Kd (μM)‡
|ΔH (kJ·mol−1)
|ΔG* (J·mol−1)
|Tm (°C)
|ΔTm (°C)
|Δ(ΔG)† (J·mol−1)
|Kd (μM)‡
ΔG at 25 °C, considering an estimate for Δ Cp of 50.2 J/mol per K.
Δ (ΔG)=[Δ(Tm)]×ΔSm=[Δ(Tm)]×(ΔHm/Tm), where ΔSm and ΔHm are values for the wild-type.
Isu binding may also involve residues from the acidic ridge
Since low-stoichiometric iron binding was not impaired in the mutants, we next evaluated whether the interaction between Yfh1 and Isu was compromised. This interaction is mediated by iron, as only holo-Yfh1 interacts with Isu [16]. Yfh1-D86A/E90A/E93A was found to bind to Isu with a wild-type-like affinity (Kd ~5 μM), whereas no interaction with Yfh1-122-4 was detected, consistent with previously published results ([19] and [30] respectively). The alteration of residues 122, 123 and 124 individually (N122K, N122A, K123T or Q124A) severely affected the Isu interaction, supporting the hypothesis that all three residues are important for this interaction.
No interaction between Isu and Yfh1-D101A/E103A (or Yfh1-D86A/E90A/E93A/D101A/E103A) was detected in our in vitro assay. This reduced interaction is somewhat surprising considering that Yfh1-D101A/E103A could significantly rescue the growth defect of Δyfh1 cells, even when expressed at low levels (Figure 2). Mutating these two residues has only been found to cause a growth defect when the alterations are to lysine residues and the medium is supplemented with high levels of iron [20]. In vivo, other cellular factors may promote the interaction between Yfh1-D101A/E103A and Isu, explaining the difference between the in vivo and in vitro results. Indeed, frataxin was found to interact with Isd11 of the Nfs1–Isu complex and multiple mitochondrial chaperones [40]. Alternatively, significantly reduced affinity may be tolerated in vivo.
Charge-to-neutral alterations in the α1/β1 acidic ridge increase stability
In order to evaluate whether the functional impairment may result from decreased protein stability, the effect of the alterations on Yfh1 folding thermodynamics was analysed by comparing the thermal stability of mutant variants with that of wild-type. According to the analysis of the far-UV CD spectra at 20 °C, before and after thermal denaturation, thermal unfolding was reversible for all protein variants studied and no aggregation was observed after thermal unfolding. The results showed that charge-to-neutral alterations in the acidic ridge result in an impressive stabilization of the protein fold: an increase of up to ~24 °C was noted for the Yfh1-D86A/E90A/E93A/D101A/E103A variant, whereas alterations in the β-sheet surface had almost no effect on protein thermal stability (Figure 4 and Table 1).
Thermal denaturation curves at pH 7.0 following tryptophan emission: impact of mutations on Yfh1 thermal stability
The protein stability decreased in the order: Yfh1-D86A/E90A/E93A/D101A/E103A>Yfh1-D86A/E90A/E93A>Yfh1-D101A/E103A>Yfh1-N122K>Yfh1-K123T>wild-type>Yfh1-N122A/K123T/Q124A>Yfh1-Q124A>Yfh1-N122A. The exceptions to the effect on the β-sheet surface are the mutations in Asn122: changing to an alanine (ΔTm=−4.5 °C) or to a lysine (ΔTm=+4.3 °C) residue had opposite effects, probably due to the effect these alterations would be expected to have on the β-hairpin between strands β3 and β4, which involves two hydrogen bonds (Asn122–Trp131 and Val120–Ala133). Whereas the insertion of an alanine residue probably disrupts the hydrogen bond with Trp131, the positively charged lysine residue might strengthen it, stabilizing the protein. The D101A/E103A alterations stabilize the protein in spite of compromising two of the three hydrogen bonds involved in the β-hairpin connecting strands β1 and β2. This suggests that minimizing repulsive interactions overcomes the stabilization obtained by the two hydrogen bonds.
Since protein conformational plasticity affects both protein function and degradation rates, we next analysed whether the functional mutations were also affecting frataxin flexibility, by performing limited proteolysis experiments using trypsin. Our underlying rationale was that mutations resulting in an increased structural flexibility would increase trypsin access to cleavage sites and consequently increase the degradation rate. The results show two distinct patterns, depending on the region in which the alteration is located (Figure 5). Alterations on the acidic ridge had a pronounced effect on frataxin dynamics, making the protein more rigid and substantially less susceptible to proteolysis. In fact, the Yfh1-D86A/E90A/E93A/D101A/E103A variant remains essentially intact under conditions in which wild-type frataxin is essentially completely digested (80% compared with 10% integrity after 100 min of digestion). On the other hand, alterations on the β-sheet surface (Yfh1-N122A/K123T/Q124A) behave almost identically with the wild-type, suggesting that modifications in this region have either a very small or no effect on the protein conformational plasticity.
Time course of limited trypsin proteolysis
Overall, alterations in the acidic ridge that prevent iron binding at the primary sites increased substantially the protein stability, and decreased its flexibility. Decreases in the structural flexibility may prevent conformational changes necessary to allow the interaction with protein partners. This increase in both thermal stability and resistance to proteolytic degradation suggests that the iron-binding region is particularly susceptible to an activity–stability trade off.
In the present paper, we describe a detailed characterization of eight yeast frataxin functional variants that either alter the acidic ridge between α-helix 1 and β-sheet 1, or the conserved β-sheet surface between strands 3 and 4. Changing the conserved β-sheet residues Asn122–Lys123–Gln124 had almost no effect on Yfh1 stability and plasticity, indicating that changes in this region did not disrupt overall conformation, but are relevant for the Yfh1–Isu interaction. Alteration of up to five residues in the acidic ridge region, four of which had been identified as iron-binding sites [23], significantly increased frataxin stability. Thus illustrates a rather interesting trade off between activity and stability in this region. In addition, the present study suggests that residues Asp101 and Glu103 are involved in the iron-mediated interaction between Isu and Yfh1, but their alteration does not abrogate the interaction, as is evident by the rescue of the Δyfh1 phenotype.
Ana Correia and Tao Wang designed and performed experiments, analysed data and wrote the paper. Elizabeth Craig and Cláudio Gomes conceived the study, designed experiments, analysed data and wrote the paper.
This work was partly financed by the Fundação para a Ciência e Tecnologia [grant number PTDC/QUI/70101], National Ataxia Foundation (to C. M. G.), the National Institutes of Health [grant number GM27870] and Muscular Dystrophy Association (to E. A. C.). A. R. C. and T. W. were recipients of a Fundação para a Ciência e Tecnologia Ph.D. Fellowship [grant number SFRHBD/24949/2005] and an American Heart Association Postdoctoral Fellowship [grant number 0525728Z] respectively.
Author notes
Present address: Hillman Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15213, U.S.A.


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The David of America's restored beaches took down one of the country's Goliaths this week to win the American Shore and Beach Preservation Association's coveted "Best of the Best Restored Beaches" contest.
The Village of West Hampton Dunes, which boasts 4.5 miles of restored beach, received 8,744 votes during the online contest, nipping Panama City Beach, Florida, which boasts 26 miles of beach, by 704 votes, according to results announced on Tuesday. Isles of Palms, South Carolina, finished a distant third with little more than 3,000 votes.
Online voting began on March 30 and ended on Friday, April 27. People were allowed to vote one time per day.
"As in many of these instances, there were two communities that really got motivated for this contest," said Ken Gooderham, co-executive director of the American Shore and Beach Preservation Association.
"Basically, it's for bragging rights," he added, referring to the contest. "We're a small nonprofit; it's not like we can give them a big check. We'll single them out for some sort of honor."
As of last week, West Hampton Dunes stood in second place in the contest's urban beach category, the most competitive of the three categories.
Gary Vegliante, the mayor of West Hampton Dunes and a member of the American Shore and Beach Preservation Association's Board of Directors, on Tuesday thanked all of the "friends of West Hampton Dunes" for putting his village's beach over the top.
"This was a lot of fun but, seriously, it only goes to show just how passionate people on Long Island are about our beaches," he wrote in an email. "It was truly an honor to be a part of something so important, passionate and fun."
Mr. Gooderham added that his group's goal for the contest was to show that beaches are popular and to "remind people that many of these beaches have been restored."


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5 Key Takeaways on the Road to Dominating
June 15, 2019
Some of the Areas that You Can Go to When You Need the Best Interior Design Ideas
When you build a home, you will need to improve its aesthetic factor. The interior design companies are in place for this reason. It will be vital to think of these since they will get to improve the appearance of your room. It is vital for the interior design companies to come up with different ways in which they can improve the home. You will have easy interior design ideas that you can work on by yourself. You will thus need to find the best interior design to implement for your home. You will have different places where you can get more info on interior design. It will thus be vital to read more from the section that follows when you need to get interior design ideas.
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You Say You Want a Revolution
How many times a day are we confronted with some catchy slogan that says, in one sentence or less, "Changing your life is as easy as X." The X is often some pithy philosophical idea that questions our perception of reality. Often these messages are accompanied by images from a movie or a link to a TED Talk.
These philosophy-packed one-liners show up in our inboxes and Facebook pages because our friends agree that "thinking differently is good." What is interesting to me is that, even though we all see these, we end up not living up to their injunctions. Our busy adult reality is too often dominated by a day-to-day routine that is monotonous and overwhelming. If we were to think differently, life might in fact be different. Is this too scary an idea?
Here are some examples:
- "This is Water". "An old fish swims by two younger fish and says, 'How's the water, boys?' to which the younger fish say, 'What's water?'" From the video: "The most obvious important realities are the ones that are the hardest to see and talk about."
- Morpheus offering Neo the red pill or the blue pill in The Matrix. How many of us are aware of the "constructs" around us? How many of us choose to go back to sleep when confronted with uncomfortable realities by taking the blue pill instead of the red pill?
- Various images about how we grownups once played dangerously (e.g., big slides, running around at recess, holding toy guns or sticks that look like them), and how those opportunities are gone (though no one seems to do anything about it, apart from post a picture).
- Simple quotes that leave you wondering. "We ask 18-year-olds to make huge decisions about their career and financial future, when a month ago they had to ask to go to the bathroom." (Adam Kotsko, via Facebook)
"We all want to change your head." (Lennon/McCartney)
Most of us want to see the school system changed (some of us in a revolutionary way), but the fact of the matter is that nothing much has changed since this song was written (and Sudbury Valley School opened) in 1968 — even with the recent proliferation of these catchy phrases and pictures. Why? I ask myself that all the time.
I assert that the reason for the constant reminders in these slogans and images is that too many of us grew up in a conventional schooling system explicitly designed to make us fit into a monotonous reality and feel good about it, because everyone else did it too. Now, however, with the Internet constantly screaming a different message, many suffer from cognitive and psychological dissonance. We live one way and see/hear another. Confronted with this dissonance, we tend to default and stay with the status quo–because that's what we were trained to do in school.
When my friends John and Paul sang "Revolution" , one thing they came right out and said was that "you better free your mind…" If we want a better world, we all have to be able to see reality, and that means liberating your mind from the constructs around you (as Morpheus might say). You may end up choosing to adopt the dominant paradigm, but for the sake of the children, at least understand why conventional schools exist today. For children, the biggest construct is the idea that they cannot be free until they have submitted to an arbitrary master for 12 years of their lives. Most of us grew up this way too — okay, so how did it affect you? Are you more likely to act on your dreams or play it safe?
Children who grow up in Sudbury schools learn to see reality very early in their lives. They learn that their choices are theirs and that their actions have consequences. When our Sudbury alumni go out into the world, they may find monotony and boredom, but I think they are uniquely prepared to listen to all the platitudes we get in our inbox and on social media. Chances are they will not even need to see them: they will have the ability and desire to actually make the change that some of us from a previous generation only wish that we could.
Written by Larry Welshon – staff at Alpine Valley School
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I'm not sure how to address this, but I think it is BS I am running this job, it's not in my job description, and then I'm not getting paid the same as the person who is supposed to do it.
Or am I out of line?
Or am I out of line?
Why did you do all the leg work if you're going to turn around and say not my job? Did you negotiate anything before you did all the extra work? I don't really know what to tell you but you should come up with solutions and ideas instead of digging in your heels if you don't want to work on the project.
I didn't say I am not doing it. I asked how to address the pay inequity. I did it because my customer needed something, no one else was addressing it, so I did. And like I said, why would I expect them to give me the job when the normal process is for them to give it to project management.
So I went above and beyond, and continue to go above and beyond, but I'm not supposed to try to address this? I feel like it sets a precedent.
ETA- I'm also not sure why you think I'm not trying to come up with solutions, that's the entire reason I posted this.
Can you address it with your supervisor? At the places I've worked at, a one-time major contribution like that is generally addressed with a bonus.
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I think the problem is that you don't know what the project manager's comp structure is. So, yes they're getting a percent of commission on the projects they complete, but you don't know how their base is structured. Maybe they make a lower base and make up for it in commission on projects.
If that's the case, basically your argument wouldn't be that you should get commission. It would be that you should be paid more for taking on more work.
Which may 100% be true, but is probably hard to say after just one such project.
(Also, if you're good at this and like the project management part of it -- maybe you can consider switching roles after you have some of these projects under your belt.)
DS1 9/29/14 (38.5w)
I would just go ask nicely. Remind your boss that you are non bonus eligible where as the sales guys and the project managers are all bonus eligible. So you don't mind doing the job but you want the bonus that goes along with this work.
I would assume they aren't trying to screw you, it just isn't a normal part of your job and they aren't going to be immediately thinking...."hey we don't have to pay her a bonus"
You need to ask to work it out. Sales guys don't hesitate to just ask for anything. Be that.
On managing the project I would need more info and I think you might, too. Be nice, be respectful. Maybe this is a career step up for you instead of just extra work.


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Is a HELOC From a Rental Home Deductible? | Pocketsense – The tax-deduction rules for rental homes are completely different than the rules for your own home. You can usually deduct the interest on a home equity line of credit taken against a rental home, relative to that rental home's income. However, calculating how that deduction affects your overall taxes can be more.
Home equity is a valued resource, and if you have it, you might be tempted to tap that wealth for other purposes. A home equity loan, which allows you to use your home's equity as collateral, is a great way to do this. But depending on your personal situation, it may not be the right thing to do.
You can use your HELOC for the down payment on the purchase of a single family home that you will rent out. Rental property loans typically require a 25 percent down payment.
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|Classic Airframes
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Fiji needs to improve some areas of their game: Forbes
May 7, 2019 5:10 am
Former New Zealand 7s Captain DJ Forbes says Fiji need to improve some area of their game plan if they are to beat table leader USA.
With only two tournaments left in the World Sevens Series, Forbes says the rivalry for the top four spots will get even harder as all teams wants to qualify for next year's Tokyo Olympic.
"Base on what I saw last time, Fiji will need a few things to go their way in order for America to trip up but yeah, it's going to make for an exciting finish to the World Series and whoever ends up in the top four is probably going to off-season a little bit more easier knowing that they have qualified."
Fiji Airways 7s Coach Gareth Baber says they aim to finish at the top of the table standing at the end of the London and Paris 7s.
Baber is also expected to name his squad for the London 7s next week.
Meanwhile, Fiji is pooled with Samoa, France, and Kenya.
Fiji plays Kenya in its first pool match, they will then take on France, before meeting Samoa in its last pool campaign.


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Posts Tagged: Xerces Society
Saving the Native Bees in Yolo County
Xerces Society scientists just developed a first-of-its-kind conservation strategy summarizing the threats facing native bees in the diverse...
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Declining Bumble Bees
The bumble bee population is declining and some species are teetering on the brink of extinction. That's the gist behind why three conservation...
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Harken Health Wellness Curriculum
How might we utilize our health coaches to deliver meaningful information based on a member's needs and goals?
Workshop facilitation
Synthesis presentation
Prototype development
All 10 Health Centers had representation and collaborated for the first time
Developed personalization tool and content template prototypes
Harken Health
February - May 2017
Lead Service Designer
Eliminate wellness programs and create a Health Coaching curriculum. 4 or 6-week programs inherently have scheduling and timing conflicts. While some members enjoyed a group setting most really needed personalized content on their time. We needed to create a curriculum that Coaches could implement on a member's very first visit or 10th visit.
Repeatable tools with individualized outcomes. Our Health Coaches all had various backgrounds and did not follow a standard protocol. This led to inconsistency in Member's experiences and lack of confidence in Coaching skills. We needed a set of tools as apart of the curriculum that would create a standard practice, while preserve the personalization to an individual Member.
Make the Coach relatable. Coaches needed to share personal stories with Members to help establish a connection. Many Members didn't bother opening up to their Health Coach (who were usually young and fit) because Member's didn't feel like their health coach could relate to their experiences.
Meet members where they are, not where you think they should be. Members shared horror stories of other places telling them to drink only protein shakes, or go to the gym every day, only to be set up for failure. Members appreciated when Health Coaches approached recommendations as a lifestyle change and gave incremental changes that fit into their life.
Make information actionable. Members didn't just want the theory on a topic. They wanted tactics, tools and clear takeaways of what to do with the information they were presented.
Our workshop session with Health Coaches and Providers from multiple Centers lead to the creation of a personalized wellness curriculum that took into account a Member's learning style, current ability to make changes, topics of interest and level of knowledge desired.
The Wellness Curriculum walks a Member through a series of tools to identify: their top topics of interest, areas where they need support, their current knowledge level and their willingness to make a change. The output indicates what content and techniques the Health Coach can leverage to deliver a personalized plan to the member. This database houses content, techniques and examples for Care Team members to use in order to deliver a consistent experience across all Centers and reduce the amount of time spent on making content.
After the workshop, Health Coaches in both Chicago and Atlanta split into two teams, one focused on the tools and the other on content. The content team created a template to structure the various topics so that any health coach could deliver the information regardless of their background and training.
I guided the Care Teams through how to prototype, provided resources and reassured them when they became uncomfortable with the ambiguity of not knowing what the final product would be. I instructed the Care Teams to develop prototypes ranging from very literal (i.e. survey questions) to abstract (i.e. metaphors / images) to stretch their creativity.


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. ABOUT MAMBOS · KIDS EAT FREE! FIND US · XMAS MENU. Our Main Menu. Mambo High Rise £14. 95 double stacked burger, cheese, BBQ sauce, onion. Our 2018 Christmas Menu, bookings. Announcing a Special Lunchtime menu offer at Mambo Jambo. 2 or 3 course set price menu. Monday to Friday. FIND US · XMAS MENU. In the heart of historic Norwich sits a hot 'n' hopping place to eat called Mambo Jambo's Restaurant. Take a look at our menu, see our special offers and enjoy browsing through our great new website packed with.
Cuban Restaurant in Alpharetta, GA. Foursquare. They opened up early to get the food ready for our Christmas. mambo jambo alpharetta • mambo jambo cafe. Menu. News. chacha vhashti choti January 17, 2018 No Comments. Amar nam reazul islam, 18 bochor. Sobai amke reaz bole dake. chacha vhashti choti. XMAS MENU; Our Main Menu vegetarian. Appetizers. Gulf Coast Prawn Cocktail £6. 50. Mambo´s Combo £18. 95 half rack of ribs, goujons of chicken and either deep.
See 69 photos and 32 tips from 627 visitors to Mambo Jambo Cafe. They opened up early to get the food ready for our Christmas luncheon. Owner is very nice. FIND US · XMAS MENU. Saturdays and Sundays, we have to ask that each diner has one main meal per person. Mambo Jambo. Add to itinerary. A hot 'n' hopping place to eat!. the mambos Xmas 2013 menu is now available for download off the site. view it and make your. Our fixed price menu is perfect for a working lunch. Please only use this pre-order option if you have a confirmed table reservation with Middletons.
Tex-Mex Restaurant in Norwich, Norfolk. People talk about mexican food, fabulous steak meal and enchiladas. See reviews and recommendations. At Middletons Steakhouse& Grill Restaurants we pride ourselves on serving delicious meats carefully prepared at our very own butchery Get menu, photos and location information for Mambo Jambo in Milton, GA.
Or book now at one of our other 6153 great restaurants in Milton. Mambo Jambo @ 'Nottingham Contemporary' Christmas Party - Duration: 3 minutes, 3 seconds. Play now; Mambo Jambo Play Bergmans Polska (Hambo)& E Bro Dreger -Traditional tunes - Duration: 2.
FIND US · XMAS MENU. PLEASE. Mambos Mint Mallow. On Fridays, Saturdays and Sundays, we have to ask that each diner has one main meal per person. xmas menu In the heart of historic Norwich sits a hot 'n' hopping place to eat called Mambo Jambo's Restaurant. Mexican and South American flavours, combined with a themed atmosphere to make our venue the perfect place to have your birthday party, corporate function, leaving do or a great night out with friends and loved ones.
XMAS MENU; In the Mambo jambos xmas menu of historic Norwich sits a hot 'n' hopping place to eat called Mambo Jambo's Restaurant.
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text: Perceptions of the CSI Effect Has the CSI Effect altered your perception of the crime scene investigative process? This short survey will be used as part of my research into the CSI Effect on the public and trained investigators. This survey consists of ten questions. The results of this will be used to develop a movement into re-educating people of the reality of crime scene investigating verses that of what tv portrays. No personal information will be needed in this survey. All responses are entirely anonymous. If you are unsure of the answer please state this. Thank you for taking your time to participate in this survey. Teesside University Student - Courtney. OK Question Title * 1. What do you think the CSI effect is and how does it alter the public view of CSI services? OK Question Title * 2. How often do you watch crime investigation or police based televised dramas? Never Always Clear i We adjusted the number you entered based on the slider's scale. OK Question Title * 3. What are your opinions on the accuracy of the portrayal of these services? Are these tv programmes realistic? OK Question Title * 4. How much of a role do you believe police officers play in crime scene investigations, what do you think they do at the scenes? OK Question Title * 5. What do you think is the role of a crime scene investigator, and what do they do at scenes? OK Question Title * 6. In terms of time, how long does it take for evidence to be found and collected at a crime scene? Less than one day One to three days One week to two weeks One month More than one month OK Question Title * 7. How much of a role do crime scene examination officers play in the forensic examination of evidence? OK Question Title * 8. Once evidence has been collected, what is the time period between evidence being sent off, forensic analysis and the return of results? Less than 24 hours 2-14 days 14-28 days 1 month - 6 months 6 months plus OK Question Title * 9. Can you name any evidence types? If so, which one is the most useful and why? OK Question Title * 10. What do you think can be done to improve the public's views on Crime Scene Investigatory Services? OK DONE


text:
The Klaxxi Council
|The Klaxxi Council
90 (Requires 89)
+250 The Klaxxi
[90] Wakening Sickness, [90] Ancient Vengeance
[90] The Dread Clutches, [90] Not Fit to Swarm, [90] Dead Zone, [90] Amber Arms
Travel to Klaxxi'vess at the center of the Dread Wastes. You may speak with Kil'ruk the Wind-Reaver in the Terrace of Gurthan for a ride, or travel on your own.
- Travel to Klaxxi Hub
I must report to the Klaxxi. They would not have woken me unless the situation were dire.
You have not been entirely useless. For that, I will spare your life.
The council may even find a use for you.
|[Gurthan Locket]
|[Council Choker]
|[Swarmborn Necklace]
|[Kil'ruk's Pendant]
|[Sacred Stone Amulet]
This is a sacred meeting place for the Klaxxi.
It would be wise of you to let me do the talking.
- +250 reputation with The Klaxxi
Speak with Kil'ruk again:
- If you are so curious about the mantid, I will tell you more on the way to Klaxxi'vess.
- Otherwise, leave me to my work.
- Take me to Klaxxi'vess.
- Kil'ruk picks up the adventurer and starts flying westward.
- Kil'ruk the Windreaver says: I am Kil'ruk, a warrior from an era long passed.
- Kil'ruk the Windreaver says: In my time, this was a mogu stronghold. It was my honor to paint the walls with their blood.
- Kil'ruk the Windreaver says: For my deeds, I was named a Paragon. A hero to be preserved in amber for a time when the empire would need... correcting.
- Flying by Kypari Ik.
- Kil'ruk the Windreaver says: Wakener, what power is this that burns our land and corrupts our trees?
- Getting close to the Heart of Fear.
- Kil'ruk the Windreaver says: The palace! This is... not our way. I see why the Klaxxi have been forced to act.
- Kil'ruk the Windreaver says: They are a council of our wisest cultural leaders. They serve to check the power of the Empress.
- Kil'ruk the Windreaver says: Should the Emperess ever fall to corruption or endanger our civilization, the Klaxxi are sworn to unseat her.
- Kil'ruk the Windreaver says: There are others like me. Paragons of each era, frozen in time and buried safely beyond the whispers of the Empress.
- Kil'ruk the Windreaver says: We answer only to the council. And we rest only when their will is done.
This quest completes the "Kil'ruk the Windreaver" criterion of [Amber is the Color of My Energy].
On complete:
- Klaxxi'va Vor says: Wind-Reaver. You have awakened. What of Klaxxi'va Tik?
- Klaxxi'va Set says: And what is this filth you've brought to our sanctuary?
- Kil'ruk the Windreaver says: The Klaxxi'va you sent has fallen to an adjunct of Her majesty.
- Kil'ruk the Windreaver says: This... "filth" defeated the adjunct and saw to my Wakening.
- Klaxxi'va Set says: We do not need help from the lesser races! Dispose of it!
- Kil'ruk the Windreaver says: Right away, Klaxxi'va.
- Klaxxi'va Vor says: No.
- Klaxxi'va Vor says: The Wakener may prove useful. One way or another.
- Klaxxi'va Set says: As fodder for the swarm, perhaps!
Up next: [90] The Dread Clutches and [90] Not Fit to Swarm from Kil'ruk, [90] Dead Zone from Kor'ik, and [90] Amber Arms from Ambersmith Zikk
Optional breadcrumbs: [90] The Klaxxi, [90] Dread Space, [90] The Threat in the South, [90] Better Dead than Dread
- [90] Falling Down & [90] Nope
- [90] Psycho Mantid
- [90] Preserved in Amber
- [90] Wakening Sickness & [90] Ancient Vengeance
- [90] The Klaxxi Council
Patch changes
- Patch 5.0.4 (2012-08-28): Added.


text:
Brendan Fanning: 'Could Joe Schmidt be enticed into leading the Lions in 2021?'
The only surprise in the IRFU statement this morning about Joe Schmidt's exit plan is that seemingly he is folding his tent altogether and not pitching it somewhere else.
He turned 53 in August and appears fit as a fiddle. If you watch Ireland warm-up on matchdays you will get a clear picture of what coaching category he slots into: if Warren Gatland for example is hands-off, a peripheral figure in a suit and tie as the players run the show, Schmidt is the polar opposite.
He is all over every aspect of the operation like a rash. By the time the players are leaving for the final pit-stop before kick-off he is heading towards the coaches' box in a sweat, with his heart pounding. And he's going to stop coaching altogether a few months after his 54th birthday?
Clearly family priorities are paramount but the idea of having this man around the house all day may not he every partner's dream.
So how will be occupy his time? With great difficulty you'd imagine if it doesn't involve regular involvement in a sport that has dominated his life. Joe Schmidt is clearly not a one-dimensional character but rugby has defined largely his identity.
By mid 2020 he will have had a good few months off, caught up with extended family and friends in New Zealand, and applied himself to creating the best environment possible for his family who have done most of their growing up in France and Ireland.
At that point you'd imagine the Lions will have dropped him a text to see if he'd fancy taking the tour to South Africa 12 months later. With that sort of lead-in imagine the level of detail he would bring to the task? Moreover for a brand that has massive appeal to fans it is constantly fighting for survival in a stuffed calendar dominated by clubs. Schmidt would be the perfect advocate, the ideal man to roll out to make your case.
As for Ireland the pressing issue now is who will take on the job of attack coach. Stuart Lancaster would do a terrific job but by then he will be inundated with offers. Bath, where director of rugby Stuart Hooper would have a relationship with Lancaster going back to their Leeds days, will be leading the charge, waving great wads of pound notes.
International Rugby Newsletter
In any case Lancaster already is doing a first rate job for Ireland via his work with Leinster, and trying to prise him away from that might not work given it might not suit Farrell. Given the stress of his commute, keeping Lancaster in Dublin beyond this season will be a hard sell.
More likely Farrell will have to find his own man rather than recreate, via swapped positions, a coaching team that would bring back bad memories of England's 2015 World Cup. Mark McCall would have the perfect profile, and Farrell worked well with him at Saracens before shifting up to the England set-up.
The silver lining on the cloud that is Joe Schmidt's departure is the lead-in time to him packing his bags. He will have lots of space in which to figure out a life without full-on rugby, and the IRFU have the same to find another coach.
Online Editors
Related Content
- Paul Kimmage: 'Rugby needs to tackle game's dirtiest secret'
- Rob Kearney says Joe Schmidt involved in IRFU 'contingency plans' to make sure Ireland succeed when he leaves


text:
Address: terror95.dyndns.org:27910
It's online most of the time. Based in Europe, Croatia
I have a dynamic IP so on quakeservers it always shows that is offline.
Finally ... Test It
Discussion about the game D-Day: Normandy.
Post Reply
1 post • Page 1 of 1
Post Reply
1 post • Page 1 of 1


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Hot off the Press
Braner USA Names New President
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Tuesday, January 3, 2012
Chuck Damore has been named president of Braner USA, Inc., Schiller Park, IL, a manufacturer of flat-rolled coil-processing equipment including slitting and cut-to-length lines. Damore previously served the company as executive vice president and sales manager; www.braner.com/.
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The Government has spent £7million on a series of adverts warning us men are four times more likely to keel over with soaring blood pressure if they have another pint.
"More likely" than who? Men who only drink one pint? Half a pint? Nothing? Men who live in a yurt and exist on a diet of rainwater and berries?
Like the "two or three units a day" safe drinking figures these sound like guesstimates plucked from thin air with no scientific basis at all. Only one fact remains indisputably solid… the Government, which claims to care so much about our drinking habits, thought 24-hour drinking licences were a good idea.


text:
Layli Pietri
Diversity Director, Balfour Beatty
Layli Pietri has 23 years of experience with Balfour Beatty Construction in various roles from Project Accounting, Purchasing, Estimating and Supplier Diversity. During her career working on Federal Project pursuits across the country, she establish Balfour Beatty's Federal Supplier Diversity Policy and using that framework helped create a national diversity program. As the Director of Diversity in the Mid-Atlantic Region, Layli's responsibilities are to oversee the supplier diversity program; train estimating, project management and purchasing personnel; identify new talent in the marketplace; project opportunities in the Public Sector and Business Development with clients who value diversity. She assists project teams with mentoring diverse Contractors and Suppliers as well as employees to cultivate an inclusive corporate culture.


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M. Alexander Shehan, MD
Articles by this author...
Out With the Skim: Research Suggests Full-Fat Milk Does the Body Better
Healthy Lifestyle
Whole milk may not cause heart disease and stroke after all. In fact, it may actually protect against such risks.
The ABCs of Getting More ZZZs
Healthy Lifestyle
When it comes to improving your sleep, what works? What doesn't?


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If you are ready to pursue a Bachelor of Chiropractic at the Australian Chiropractic College, we encourage you to apply now.
Our Admissions team is available to assist you as you go through the application process.Contact Admissions


text: On 05/07/2015 03:43 AM, Michael Niedermayer wrote: > On Thu, May 07, 2015 at 01:29:11AM +0530, Anshul wrote: >> >> On 05/06/2015 01:21 AM, Michael Niedermayer wrote: >>> On Tue, May 05, 2015 at 08:33:42PM +0530, Anshul wrote: >>> [...] >>>> and here is shorter file then previous one. >>>> http://gsocdev.ccextractor.org/~anshul/test_video/Closedcaption_atsc_rollup.ts >>> considering that this is created by FFmpeg >>> Metadata: >>> service_name : Service01 >>> service_provider: FFmpeg >>> >>> please make a smaller file >> I cant encode any video with subtitle, I used ffmpeg to cut the >> previous video in smaller video. >> if I transcode the video then cc will be lost. >>> this is still 16 times larger than the next largest file in >>> fate/sub >>> >>> the video streams bitrate should play no role in testing the subtites >> I searched for other opensource encoder that could embed cc in videos. >> but I was unable to find out. if you know about any openSource cc encoder. >> then please share. > i thought it was easy > if not then lets upload carls file, its the smallest so far, > > if that file works for your test ? Carl, can you please give me link of the sample, I would make the test case patch again. -Anshul


text:
Juliska Country Estate Winter Frolic Ruby Ramekin
Style Number CE12/73
Style:CE12/73 Dimensions: 4" W, 2.5" H
The Winter Frolic collection features cheerful scenes of snowball fights and even Santa's sleigh! Each piece is perfect for gifts or making your home uniquely festive!
- Made of Ceramic stoneware
- Oven, Microwave, Dishwasher, and Freezer Safer


text:
Most Viewed
Read other
Paramedics are not allowed to save Palestinians injured during clashes in Al-Bireh city - Israeli soldiers are apparently taught the "bleed to death" policy against Palestinians in all occurrences.
Bahar: Palestinian people will never give up RoR
Al Qassam website - First deputy speaker of the Palestinian legislative council Dr. Ahmed Bahar has asserted that the Palestinian people would never give up the right of return for millions of its refugees.
He said during a meeting with members of the Jordanian aid convoy Ansar-3 on Tuesday that the Palestinian people are adamant on their constants topped by the right of return and refuse any alternative homeland to Palestine.
He told the 76-member delegation accompanying the convoy that such convoys prove that Arab and Islamic peoples along with free people of the world were in support of the Palestinian people and their right to live in freedom and dignity.
Ahmed Al-Armuti, the head of the delegation, affirmed that the Jordanian monarch, government and people support the just Palestine cause.
He said that the Palestinian refugees are entitled to return to their cities and villages from which they were forcibly evicted, adding that Jordan supported the full liberation of all occupied Palestinian land. Source: PIC


text: The Swedish Baseball and Softball Academy will hold a couple of training camps in southern France next week. The softball squad will travel to St. Raphael from October 14 to 18, the baseball team to Toulouse from October 14 to 19. Both teams will play training matches against the French academy. The trip is financed thanks to some extra resources for international exchange by the Swedish Sports Confederation and Swedish Baseball and Softball Federation's agreement with MLB International.