text:
Being a major cause of eutrophication and subsequent loss of water quality, the turnover of phosphorus (P) in lake sediments is in need of deeper understanding. A major part of the flux of P to eutrophic lake sediments is organically bound or of biogenic origin. This P is incorporated in a poorly described mixture of autochthonous and allochthonous sediment and forms the primary storage of P available for recycling to the water column, thus regulating lake trophic status. To identify and quantify biogenic sediment P and assess its lability, we analyzed sediment cores from Lake Erken, Sweden, using traditional P fractionation, and in parallel, NaOH extracts were analyzed using 31P NMR. The surface sediments contain orthophosphates (ortho-P) and pyrophosphates (pyro-P), as well as phosphate mono- and diesters. The first group of compounds to disappear with increased sediment depth is pyrophosphate, followed by a steady decline of the different ester compounds. Estimated half-life times of these compound groups are about 10 yr for pyrophosphate and 2 decades for mono- and diesters. Probably, these compounds will be mineralized to ortho-P and is thus potentially available for recycling to the water column, supporting further growth of phytoplankton. In conclusion, 31P NMR is a useful tool to asses the bioavailability of certain P compound groups, and the combination with traditional fractionation techniques makes quantification possible.
An essential role of enzymes is to catalyze various chemical reactions in the human body and inhibition of the enzymatic activity by small molecules is the mechanism of action of many drugs or tool compounds used to study biological processes. Here, we investigate the effect on the dynamics of the serine protease alpha-chymotrypsin when in complex with two different covalently bound inhibitors using elastic incoherent neutron scattering. The results show that the inhibited enzyme displays enhanced dynamics compared to the free form. The difference was prominent at higher temperatures (240-310 K) and the type of motions that differ include both small amplitude motions, such as hydrogen atom rotations around a methyl group, and large amplitude motions, such as amino acid side chain movements. The measurements were analyzed with multivariate methods in addition to the standard univariate methods, allowing for a more in-depth analysis of the types of motions that differ between the two forms. The binding strength of an inhibitor is linked to the changes in dynamics occurring during the inhibitor-enzyme binding event and thus these results may aid in the deconvolution of this fundamental event and in the design of new inhibitors.
Caryophyllene-6,7-epoxide and caryophyllene-6,7-episulfide can be easily synthesized from β-caryophyllene by autoxidation or episulfidation. The bioactivities of β-caryophyllene and its derivatives were investigated against the subterranean termite Reticulitermes speratus Kolbe. The antifeedant, feeding, and termiticidal activities of each compound were tested using no-choice, dual-choice, and non-contact methods. Antitermitic activities were not shown by β-caryophyllene, but were observed for the oxide and sulfide derivatives. Caryophyllene- 6,7-episulfide showed especially high antifeedant and termiticidal activities. Thus, naturally abundant, non-bioactive β-caryophyllene can be easily converted into an antitermite reagent via a non-biological process.
The pine weevil (Hylobius abietis L.) is one of the two topmost economically important insect pests in Swedish conifer forests. The damage increase in areas were the silvicultural practice is to use clear cuttings were the insects gather and breed. During egglaying the female protects her offspring by creating a cave in roots and stumps were she puts her egg and covers it with frass, a mixture of weevil feces and chewed bark. Adult pine weevils have been observed to feed on the other side of the egg laying site and antifeedant substance has been discovered in the feces of the pine weevil. We think it is possible that microorganisms present in the frass contribute with antifeedant/repellent substances. Little is known about the pine weevils associated bacteria community and their symbiotic functions. In this thesis the bacterial community is characterized in gut and frass both from pine weevils in different populations across Europe as well as after a 28 day long diet regime on Scots pine, silver birch or bilberry. Volatile substances produced by isolated bacteria as well as from a consortium of microorganisms were collected with solid phase micro extraction (SPME) and analyzed with GC-MS. The main volatiles were tested against pine weevils using a two-choice test. Wolbachia, Rahnella aquatilis, Serratia and Pseudomonas syringae was commonly associated with the pine weevil. 2-Methoxyphenol, 2-phenylethanol, 3-methyl-1-butanol were found in the headspace from Rahnella aquatilis when grown in substrate containing pine bark. 2-Methoxyphenol and 3-methyl-1-butanol, phenol and methyl salicylate were found in pine feces. Birch and bilberry feces emitted mainly linalool oxides and bilberry emitted also small amounts of 2-phenylethanol.
A second part of the thesis discusses the role of fungi in forest insect interactions and the production of oxygenated monoterpenes as possible antifeedants. Spruce bark beetles (Ips typhographus L.) aggregate with the help of pheromones and with collected forces they kill weakened adult trees as a result of associated fungi growth and larval development. A fungi associated with the bark beetle, Grosmannia europhoides, was shown to produce de novo 2-methyl-3-buten-2-ol, the major component of the spruce bark beetle aggregation pheromone. Chemical defense responses against Endoconidiophora polonica and Heterobasidion parviporum were investigated using four clones of Norway spruce with different susceptibility to Heterobasidion sp. Clone specific differences were found in induced mono-, sesqui and diterpenes. A number of oxygenated monoterpenes which are known antifeedants for the pine weevil were produced in the infested areas.
Heterobasidion parviporum (Hp) were investigated using four clones of Norway spruce (Picea abies) with different susceptibility to Heterobasidion sp. Eight year old trees were inoculated with Ep and Hp to minimize the variation due to environment. After three weeks the bark tissue at the upper border of the inoculation hole were extracted with hexane and analyzed by GC-MS. Both treatment and clonal differences were found based on induced mono-, sesqui- and diterpenes. In addition, the Hp produced toxin, fomanoxin, was identified in lowest amount in the most Hp susceptible clone. The clonal trees seem to use different defense strategies towards the two fungi. One of the clones was able to induce strong chemical defense against both fungi, one clone induced chemical defense only against Ep and the most susceptible clone exhibited the least capacity to produce an effective defense against Ep and Hp. Two diterpenes were found to be distinctly different between clones with different susceptibilities, which can be used as chemical indication of Norway spruce resistance against fungi.
beta-Phosphoglucomutase (beta-PGM) has served as an important model system for understanding biological phosphoryl transfer. This enzyme catalyzes the isomerization of beta-glucose-1-phosphate to -glucose-6-phosphate in a two-step process proceeding via a bisphosphate intermediate. The conventionally accepted mechanism is that both steps are concerted processes involving acid-base catalysis from a nearby aspartate (D10) side chain. This argument is supported by the observation that mutation of D10 leaves the enzyme with no detectable activity. However, computational studies have suggested that a substrate-assisted mechanism is viable for many phosphotransferases. Therefore, we carried out empirical valence bond (EVB) simulations to address the plausibility of this mechanistic alternative, including its role in the abolished catalytic activity of the D10S, D10C and D10N point mutants of beta-PGM. In addition, we considered both of these mechanisms when performing EVB calculations of the catalysis of the wild type (WT), H20A, H20Q, T16P, K76A, D170A and E169A/D170A protein variants. Our calculated activation free energies confirm that D10 is likely to serve as the general base/acid for the reaction catalyzed by the WT enzyme and all its variants, in which D10 is not chemically altered. Our calculations also suggest that D10 plays a dual role in structural organization and maintaining electrostatic balance in the active site. The correct positioning of this residue in a catalytically competent conformation is provided by a functionally important conformational change in this enzyme and by the extensive network of H-bonding interactions that appear to be exquisitely preorganized for the transition state stabilization.
All four possible stereoisomers of 5-hydroxy-4-methyl-3-heptanone were synthesized from common achiral reagents using fast, straightforward organic synthesis, including the use of whole tissue of Daucus carota, Solanum melongena, and Pleurotus ostreatus.
A lightweight and strong porous cellulose material has been prepared by drying aqueous foams stabilized with surface-modified nanofibrillated cellulose (NFC). This material differs from other dry, particle stabilized foams in that renewable cellulose is used as stabilizing particles. Confocal microscopy and high speed video imaging show that the octylamine-coated, rod-shaped NFC nanoparticles residing at the air-liquid interface prevent the air bubbles from collapsing or coalescing. Stable wet foams can be achieved at solids content around 1% by weight. Careful removal of the water results in a cellulose-based material with a porosity of 98% and a density of 30 mg cm(-3). These porous cellulose materials have a higher Young's modulus than porous cellulose materials made from freeze-drying, at comparable densities, and have a compressive energy absorption of 56 kJ m(-3) at 80% strain. Measurement with the aid of an autoporosimeter revealed that most pores are in the range of 300 to 500 mu m.
Despite the numerous experimental and theoretical studies on phosphate monoester hydrolysis, significant questions remain concerning the mechanistic details of these biologically critical reactions. In the present work we construct a linear free energy relationship for phosphate monoester hydrolysis to explore the effect of modulating leaving group plc on the competition between solvent- and substrate-assisted pathways for the hydrolysis of these compounds. Through detailed comparative electronic-structure studies of methyl phosphate and a series of substituted aryl phosphate monoesters, we demonstrate that the preferred mechanism is dependent on the nature of the leaving group. For good leaving groups, a strong preference is observed for a more dissociative solvent-assisted pathway. However, the energy difference between the two pathways gradually reduces as the leaving group pK(a) increases and creates mechanistic ambiguity for reactions involving relatively poor alkoxy leaving groups. Our calculations show that the transition-state structures vary smoothly across the range of pK(a)s studied and that the pathways remain discrete mechanistic alternatives. Therefore, while not impossible, a biological catalyst would have to surmount a significantly higher activation barrier to facilitate a substrate-assisted pathway than for the solvent-assisted pathway when phosphate is bonded to good leaving groups. For poor leaving groups, this intrinsic preference disappears.
Three varieties of honey of different dominant floral origin were found to attract social Hymenoptera, including the large earth bumble bee, Bombus terrestris, in a New Zealand mountain beech forest. This study was undertaken to identify volatile organic compounds that induce the attraction of bumble bees to honeybee (Apis mellifera) honey. We analyzed the chemical composition of the volatile organic compounds produced in three distinct varieties of honey (i.e., manuka, honeydew, and clover honey). The composition of the chemical profile of the three honey varieties differed in the quality and in the ratio of compounds in the headspace. o-Methoxyacetophenone was the main compound in the headspace of all three honey varieties. Among the 40 compounds identified in the headspace in the three varieties, only seven shared compounds (i.e., benzaldehyde, benzyl alcohol, phenylacetaldehyde, 2-phenylethanol, isophorone, 4-oxoisophorone, and o-methoxyacetophenone) were present in the headspace of the three honey varieties. The relative attractiveness of various blends of the seven common compounds found in the three honey varieties was tested for the attraction to bumble bees in a mountain beech forest. A binary blend of isophorone and 4-oxoisophorone at a ratio of 90:10 was the most attractive blend for both bumble bee workers and queens. A small number of honey bee workers were also attracted to the former binary blend. Our study represents the first identification of a honey-derived attractant for bumble bees and honey bees. The potential application of our finding for monitoring of bumble bees or to enhance crop pollination and help to tackle the current concern of a global pollination crisis is discussed.
BACKGROUND: The common wasp, Vespula vulgaris (L.), and the German wasp, Vespula germanica (F.), are significant problems in New Zealand beech forests (Nothofagus spp.), adversely affecting native birds and invertebrate biodiversity. This work was undertaken to develop synthetic attractants for these species to enable more efficient monitoring and management. RESULTS: Seven known wasp attractants (acetic acid, butyl butyrate, isobutanol, heptyl butyrate, octyl butyrate and 2,4-hexadienyl butyrate) were field tested, and only heptyl butyrate and octyl butyrate attracted significantly higher numbers of wasps than a non-baited trap. Accordingly, a series of straight-chain esters from methyl to decyl butyrate were prepared and field tested for attraction of social wasps. Peak biological activity occurred with hexyl butyrate, heptyl butyrate, octyl butyrate and nonyl butyrate. Polyethylene bags emitting approximately 18.4-22.6 mg day(-1) of heptyl butyrate were more attractive than polyethylene bags emitting approximately 14.7-16.8 mg day(-1) of heptyl butyrate in the field. Electroantennogram (EAG) studies indicated that queens and workers of V. vulgaris had olfactory receptor neurons responding to various aliphatic butyrates. CONCLUSION: These results are the first to be reported on the EAG response and the attraction of social wasps to synthetic chemicals in New Zealand beech forests and will enable monitoring of social wasp activity in beech forests. (C) 2009 Society of Chemical industry
Amphiphilic diblock co-oligopeptides are interesting and functional macromolecular materials for biomedical applications because of their self-assembling properties. Here, we developed a synthesis method for diblock co-oligopeptides by using chemo-enzymatic polymerization, which was a relatively short (30 min) and efficient reaction (over 40% yield). Block and random oligo(L-lysine-co-L-alanine) [oligo(Lys-co-Ala)] were synthesized using activated papain as enzymatic catalyst. The reaction time was optimized according to kinetic studies of oligo(L-alanine) and oligo(L-lysine). Using H-1 NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we confirmed that diblock and random co-oligopeptides were synthesized. Optical microscopy further revealed differences in the crystalline morphology between random and block co-oligopeptides. Plate-like, hexagonal, and hollow crystals were formed due to the strong impact of the monomer distribution and pH of the solution. The different crystalline structures open up interesting possibilities to form materials for both tissue engineering and controlled drug/gene delivery systems.
The structures of the lipooligosaccharides from Brucella melitensis mutants affected in the WbkD and ManB(core) proteins have been fully characterized using NMR spectroscopy. The results revealed that disruption of wbkD gives rise to a rough lipopolysaccharide (R-LPS) with a complete core structure (beta-D-Glcp-(1 -> 4)-alpha-Kdop-(2 -> 4)[beta-D-GlcpN-(1 -> 6)-beta-D-GlcpN-(1 -> 4)[beta-D-GlcpN-(1 -> 6)]-beta-D-GlcpN-(1 -> 3)-alpha-D-Manp-(1 -> 5)]-alpha-Kdop-(2 -> 6)-beta-D-GlcpN3N4P-(1 -> 6)-alpha-D-GlcpN3N1P), in addition to components lacking one of the terminal beta-D-GlcpN and/or the beta-D-Glcp residues (48 and 17%, respectively). These structures were identical to those of the R-LPS from B. melitensis EP, a strain simultaneously expressing both smooth and R-LPS, also studied herein. In contrast, disruption of man-B-core gives rise to a deep-rough pentasaccharide core (beta-D-Glcp-(1 -> 4)-alpha-Kdop-(2 -> 4)-alpha-Kdop-(2 -> 6)-beta-D-GlcpN3N4P-(1 -> 6)-alpha-D-GlcpN3N1P) as the major component (63%), as well as a minor tetrasaccharide component lacking the terminal beta-D-Glcp residue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManB(core) proteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). We also report that deletion of B. melitensis wadC removes the core oligosaccharide branch not linked to the O-antigen causing an increase in overall negative charge of the remaining LPS inner section. This is in agreement with the mannosyltransferase role predicted for WadC and the lack of GlcpN residues in the defective core oligosaccharide. Despite carrying the O-antigen essential in B. melitensis virulence, the core deficiency in the wadC mutant structure resulted in a more efficient detection by innate immunity and attenuation, proving the role of the beta-D-GlcpN-(1 -> 6)-beta-D-GlcpN-(1 -> 4)[beta-D-GlcpN-(1 -> 6)]-beta-D-GlcpN-(1 -> 3)-alpha-D-Manp-(1 -> 5) structure in virulence.
In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of C-13-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly C-13-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-C-13)-sucrose, 342 Da] and one compound of medium molecular weight (C-13-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, similar to 10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The C-13 resonances are traced using C-13-C-13 correlations from homonuclear experiments, such as (H)CC-CT-COSY, (H)CC-NOESY, CC-CT-TOCSY and/or virtually decoupled (H)CC-TOCSY. Based on the assignment of the C-13 resonances, the H-1 chemical shifts are derived in a straightforward manner using one-bond H-1-C-13 correlations from heteronuclear experiments (HC-CT-HSQC). In order to avoid the (1) J (CC) splitting of the C-13 resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either C-13 or H-1 detected experiments, namely CC-CT-COSY, band-selective (H)CC-TOCSY, HC-CT-HSQC-NOESY or long-range HC-CT-HSQC. However, due to the short T-2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the H-1-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the C-13-enriched polysaccharide were assigned by using HC-H2BC spectra. The assignment of the N-acetyl groups with N-15 at natural abundance was completed by using HN-SOFAST-HMQC, HNCA, HNCO and C-13-detected (H)CACO spectra.
The structure of the repeating unit of the O-antigenic polysaccharide (PS) from Escherichia coli O174 has been determined. Component analysis together with H-1 and C-13 NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by H-1, C-13-heteronuclear multiple-bond correlation and H-1, H-1-NOESY experiments. The PS is composed of tetrasaccharide repeating units with the following structure: -> 4)-beta-D-GlcpA-(1 -> 3)-beta-D-Galp-(1 -> 3)-beta-D-GalpNAc-(1 -> vertical bar beta-D-GlcpNAc-(1 -> 2) Cross-peaks of low intensity were present in the NMR spectra consistent with a beta-D-GlcpNAc-(1 -> 2)-beta-D-GlcpA(1 -> structural element at the terminal part of the polysaccharide, which on average is composed of similar to 15 repeating units. Consequently the biological repeating unit has a 3-substituted N-acetyl-D-galactosamine residue at its reducing end.
When using biosensors, analyte biomolecules of several different concentrations are percolated over a chip with immobilized ligand molecules that form complexes with analytes. However, in many cases of biological interest, e.g., in antibody interactions, complex formation steady-state is not reached. The data measured are so-called sensorgram, one for each analyte concentration, with total complex concentration vs time. Here we present a new four-step strategy for more reliable processing of this complex kinetic binding data and compare it with the standard global fitting procedure. In our strategy, we first calculate a dissociation graph to reveal if there are any heterogeneous interactions. Thereafter, a new numerical algorithm, AIDA, is used to get the number of different complex formation reactions for each analyte concentration level. This information is then used to estimate the corresponding complex formation rate constants by fitting to the measured sensorgram one by one. Finally, all estimated rate constants are plotted and clustered, where each cluster represents a complex formation. Synthetic and experimental data obtained from three different QCM biosensor experimental systems having fast (close to steady-state), moderate, and slow kinetics (far from steady-state) were evaluated using the four-step strategy and standard global fitting. The new strategy allowed us to more reliably estimate the number of different complex formations, especially for cases of complex and slow dissociation kinetics. Moreover, the new strategy proved to be more robust as it enables one to handle system drift, i.e., data from biosensor chips that deteriorate over time.
Rhodnius prolixus is one of the most important vectors of Chagas disease in Central and South America for which repellents and attractants are sorely needed. Repellents like DEET, picaridin, and IR3535 are widely used as the first line of defense against mosquitoes and other vectors, but they are ineffective against R. prolixus. Our initial goal was to identify in R. prolixus genome odorant receptors sensitive to putative sex pheromones. We compared gene expression of 21 ORs in the R. prolixus genome, identified 4 ORs enriched in male (compared with female) antennae. Attempts to de-orphanize these ORs using the Xenopus oocyte recording system showed that none of them responded to putative sex pheromone constituents. One of the them, RproOR80, was sensitive to 4 compounds in our panel of 109 odorants, namely, 2-heptanone, gamma-octalactone, acetophenone, and 4-methylcychohexanol. Interestingly, these compounds, particularly 4-methylcyclohexanol, showed strong repellency activity as indicated not only by a significant decrease in residence time close to a host, but also by a remarkable reduction in blood intake. 4-Methylcyclohexanol-elicited repellency activity was abolished in RNAi-treated insects. In summary, our search for pheromone receptors led to the discovery of repellents for R. prolixus.
To be able to target microRNAs also at stages where these are in a double stranded or hairpin form we have studied BisPNA designed to clamp the target and give sufficient affinity to allow for strand invasion. We show that BisPNA complexes are more stable with RNA than with DNA. In addition, 24-mer BisPNA (AntimiR) constructs form complexes with a hairpin RNA that is a model of the microRNA miR-376b, suggesting that PNA-clamping may be an effective way of targeting microRNAs.
A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.
Small molecules are used in the G-quadruplex (G4) research field in vivo and in vitro, and there are increasing demands for ligands that selectively stabilize different G4 structures. Thioflavin T (ThT) emits an enhanced fluorescence signal when binding to G4 structures. Herein, we show that ThT can be competitively displaced by the binding of small molecules to G4 structures and develop a ThT-displacement high-throughput screening assay to find novel and selective G4-binding compounds. We screened approximately 28 000 compounds by using three different G4 structures and identified eight novel G4 binders. Analysis of the structural conformation and stability of the G4 structures in presence of these compounds demonstrated that the four compounds enhance the thermal stabilization of the structures without affecting their structural conformation. In addition, all four compounds also increased the G4-structure block of DNA synthesis by Taq DNA polymerase. Also, two of these compounds showed selectivity between certain Schizosaccharomyces pombe G4 structures, thus suggesting that these compounds or their analogues can be used as selective tools for G4 DNA studies.
A short oligonucleotide sequence as in a single-stranded antisense oligo nucleotides (AON) or in double-stranded small interfering RNAs (siRNA) can modulate the gene expression by targeting against the cellular mRNA, which can be potentially exploited for therapeutic purposes in the treatment of different diseases. In order to improve the efficacy of oligonucleotide-based drugs, the problem of target affinity, nuclease stability and delivery needs to be addressed. Chemical modifications of oligonucleotides have been proved to be an effective strategy to counter some of these problems.
In this thesis, chemical synthesis of conformationally constrained nucleosides such as 7′-Me-carba-LNA-A, -G, -MeC and -T as well as 6′, 7′-substituted α-L-carba-LNA-T (Papers I-III) was achieved through a key free-radical cyclization. 1D and 2D NMR techniques were employed to prove the formation of bicyclic ring system by free-radical ring closure as well as to identify the specific constrained conformations in sugar moieties. These sugar-locked nucleosides were transformed to the corresponding phosphoramidites and incorporated into antisense oligonucleotides in different sequences, to evaluate their physicochemical and biochemical properties for potential antisense-based therapeutic application.
AONs modified with 7′-Me-carba-LNA analogues exhibited higher RNA affinities (plus 1-4°C/modification) (Papers I & III), but AONs containing α-L-carba-LNA analogues showed decreased affinities (minus 2-3°C/ modification) (Paper II) towards complementary RNA compared to the native counterpart. It has been demonstrated in Papers I-III that 7′-methyl substitution in α-L-carba-LNA caused the Tm drop due to a steric clash of the R-configured methyl group in the major groove of the duplex, whereas 7′-methyl group of carba-LNA locating in the minor groove of the duplex exerted no obviously negative effect on Tms, regardless of its orientation. Moreover, AONs containing 7′-Me-carba-LNA and α-L-carba-LNA derivatives were found to be nucleolytically more stable than native AONs, LNA modified AONs as well as α-L-LNA modified ones (Papers I-III). We also found in Paper II & III that the orientations of OH group in C6′ of α-L-carba-LNAs and methyl group in C7′ of 7′-Me-carba-LNAs can significantly influence the nuclease stabilities of modified AONs. It was proved that the methyl substitution in cLNAs which points towards the vicinal 3′-phosphate were more resistant to nuclease degradation than that caused by the methyl group pointing away from 3′-phosphate.
Additionally, AONs modified with 7′-Me-carba-LNAs and α-L-carba-LNAs were found to elicit the RNase H mediated RNA degradation with comparable or higher rates (from 2-fold to 8-fold higher dependent upon the modification sites) as compared to the native counterpart. We also found that the cleavage patterns and rates by E. coli RNase H1 were highly dependent upon the modification sites in the AON sequences, regardless of the structural features of modifications (Papers II & III). Furthermore, we have shown that the modulations of Tms of AON/RNA duplexes are directly correlated with the aqueous solvation (Paper III).
Density functional theory calculations have been used to study the reaction mechanism of water oxidation catalyzed by a tetranuclear Mn-oxo cluster Mn4O4L6 (L = (C6H4)(2)PO4-). It is proposed that the O-O bond formation mechanism is different in the gas phase and in a water solution. In the gas phase, upon phosphate ligand dissociation triggered by light absorption, the O-O bond formation starting with both the Mn-4(III,III,IV,IV) and Mn-4(III,IV,IV,IV) oxidation states has to take place via direct coupling of two bridging oxo groups. The calculated barriers are 42.3 and 37.1 kcal/mol, respectively, and there is an endergonicity of more than 10 kcal/mol. Additional photons are needed to overcome these large barriers. In water solution, water binding to the two vacant sites of the Mn ions, again after phosphate dissociation triggered by light absorption, is thermodynamically and kinetically very favorable. The catalytic cycle is suggested to start from the Mn-4(III,III,III,IV) oxidation state. The removal of three electrons and three protons leads to the formation of a Mn-4(III,IV,IV,IV)-oxyl radical complex. The O-O bond formation then proceeds via a nucleophilic attack of water on the Mn-IV-oxyl radical assisted by a Mn-bound hydroxide that abstracts a proton during the attack. This step was calculated to be rate-limiting with a total barrier of 29.2 kcal/mol. This is followed by proton-coupled electron transfer, O-2 release, and water binding to start the next catalytic cycle.
Knowledge gaps in the biosafety data of the nanocellulose (NC) for biomedical use through various routes of administration call for closer look at health and exposure evaluation. This work evaluated the potentially immunogenic contaminants levels, for example, endotoxin and (1,3)-β-d-glucan, in four representative NCs, that is, wood-based NCs and bacterial cellulose (BC). The hot-water extracts were analyzed with ELISA assays, HPSEC-MALLS, GC, and NMR analysis. Varying levels of endotoxin and (1,3)-β-d-glucan contaminats were found in these widely used NCs. Although the β-(1,3)-d-glucan was not detected from the NMR spectra due to the small extract samples amount (2–7 mg), the anomerics and highly diastereotopic 6-CH2 signals may suggest the presence of β-(1,4)-linkages with β-(1,6) branching in the polysaccharides of NCs' hot-water extracts, which were otherwise not detectable in the enzymatic assay. In all, the article highlights the importance of monitoring various water-soluble potentially immunogenic contaminants in NC for biomedical use.
The chemical elicitor methyl jasmonate (MeJA) could replace insecticides in Europe and Asia for protection of young conifers against the pine weevil (Hylobius abietis). This thesis mainly focuses on the effects of MeJA treatment on chemical defenses of conifers from seedling batches with documented field resistance. Tissues of three pine species and one spruce species, with various treatments, were here extracted in hexane, whereafter volatile contents of tissues were separated and analyzed by 2D GC-MS.
Induced responses of seedlings of Maritime pine (Pinus pinaster) and Monterey pine (Pinus radiata) to the folivore pine processionary (Thaumetopoea pityocampa), and the phloem-feeder H. abietis, have been studied. Amounts of mono- and sesqui-terpenes (and also non-volatile resin) in conifer tissues (needles and phloem) were less induced by T. pityocampa than by H. abietis.
The MeJA-treated seedlings of Scots pine (Pinus sylvestris) changed their composition of phloem monoterpenes (induction of (-)-β-pinene), and were better protected in field than the seedlings of Norway spruce (Picea abies), which increased their total amounts of monoterpenes. Orientation bioassays with H. abietis showed deterrent effects of (-)-β-pinene, (+)-3-carene, (-)-bornyl acetate and 1,8-cineole. Conversely, (-)-α-pinene (induced in P. abies but not in P. sylvestris) was non-deterrent.
MeJA-treated seedlings fed on by H. abietis contained higher amounts of the H. abietis antifeedant 2-phenylethanol, in tissues and emissions. Phloem of control seedlings instead induced the (+)-α-pinene, which is one of the enantiomers of the H. abietis attractant α-pinene.
In response to MeJA treatment, the relative amounts of the (+)-α-pinene increased in the phloem of P. radiata, while it decreased in P. pinaster phloem. The preference of H. abietis in the field for P. radiata before P. pinaster may be explained by these changes in enantiomers of the H. abietis attractant α-pinene.
Chemical defenses of conifers can be experimentally activated to respond more efficiently to feeding damage by insects. One chemical elicitor that triggers plant defenses, and thereby protects conifers, is methyl jasmonate (MeJA). However, there is little known about the associations between MeJA-induced conifer defenses, and the meal properties of phytophagous insects. To address this knowledge gap, we have analyzed relations between volatile contents of Scots pine (Pinus sylvestris L.) tissues, and meal properties of the pine weevil (Hylobius abietis (L.)). Phloem and needles (both weevil target tissues) from MeJA-treated and control seedlings were extracted by n-hexane and analyzed by two-dimensional gas chromatography-mass spectrometry (2D GC-MS). Feeding of pine weevils on seedlings from the same batches were video-recorded to determine meal properties. Multivariate statistical analyses showed that phloem and needle contents of MeJA-treated seedlings had different volatile compositions from the control seedlings. Levels of (+)-α-pinene were high, and levels of the antifeedant 2-phenylethanol were low, in phloem of control seedlings with feeding damage. Accordingly, pine weevils fed more slowly and had shorter meals on MeJA-treated seedlings. In addition, the chemical compositions of phloem and needles – and accordingly weevil meal durations on them – were more similar in MeJA-treated seedlings than in controls. The results illustrate that detailed knowledge of insect responses can help efforts to identify and elucidate specific roles of resistance agents in complex chemical profiles.
The defense of conifers against phytophagous insects relies to a large extent on induced chemical defenses. However, it is not clear how induced changes in chemical composition influence the meal properties of phytophagous insects (and thus damage rates). The defense can be induced experimentally with methyl jasmonate (MeJA), which is a substance that is produced naturally when a plant is attacked. Here we used MeJA to investigate how the volatile contents of Scots pine (Pinus sylvestris L.) tissues influence the meal properties of the pine weevil (Hylobius abietis (L.)). Phloem and needles (both weevil target tissues) from MeJA-treated and control seedlings were extracted by n-hexane and analyzed by two-dimensional gas chromatography-mass spectrometry (2D GC-MS). The feeding of pine weevils on MeJA-treated and control seedlings were video-recorded to determine meal properties. Multivariate statistical analyses showed that phloem and needle contents of MeJA-treated seedlings had different volatile compositions compared to control seedlings. Levels of the pine weevil attractant (+)-α-pinene were particularly high in phloem of control seedlings with feeding damage. The antifeedant substance 2-phenylethanol occurred at higher levels in the phloem of MeJA-treated than in control seedlings. Accordingly, pine weevils fed slower and had shorter meals on MeJA-seedlings. The chemical compositions of phloem and needle tissues were clearly different in control seedlings but not in the MeJA-treated seedlings. Consequently, meal durations of mixed meals, i.e. both needles and phloem, were longer than phloem meals on control seedlings, while meal durations on MeJA seedlings did not differ between these meal contents. The meal duration influences the risk of girdling and plant death. Thus our results suggest a mechanism by which MeJA treatment may protect conifer seedlings against pine weevils.
In large parts of Europe insecticide-free measures for protecting conifer plants are desired to suppress damage by the pine weevil Hylobius abietis (L.). Treatment with methyl jasmonate (MeJA), a chemical elicitor already used in crop production, may enhance expression of chemical defenses in seedlings in conifer regenerations. However, in a previous experiment MeJA treatment resulted in substantially better field protection for Scots pine (Pinus sylvestris L.) than for Norway spruce (Picea abies (L.) Karst.). Hypothesizing that the variations may be at least partly due to volatiles released by MeJA-treated seedlings and their effects on pine weevil orientation, we examined tissue extracts of seedlings (from the same batches as previously used) by two-dimensional GC-MS. We found that the MeJA treatment increased contents of the monoterpene (-)-β-pinene in phloem (the weevil's main target tissue) of both tree species, however, the (-)-β-pinene/(-)-α-pinene ratio increased more in the phloem of P. sylvestris. We also tested the attractiveness of individual monoterpenes found in conifer tissues (needles and phloem) for pine weevils using an arena with traps baited with single-substance dispensers and pine twigs. Trap catches were reduced when the pine material was combined with a dispenser releasing (-)-β-pinene, (+)-3-carene, (-)-bornyl acetate or 1,8-cineole. However, (-)-α-pinene did not have this effect. Thus, the greater field protection of MeJA-treated P. sylvestris seedlings may be due to the selective induction of increases in contents of the deterrent (-)-β-pinene, in contrast to strong increases in both non-deterrent (-)-α-pinene and the deterrent (-)-β-pinene in P. abies seedlings.
The feeding preference of the pine weevil to Maritime pine (Pinus pinaster Ait.) and Monterey pine (Pinus radiata D. Don) differ in laboratory tests and in the field: Can this be explained by the volatile compounds released by the seedlings? Understanding the interaction between the chemical defenses of young pines and feeding preferences of the pine weevil (Hylobius abietis (L.)) may improve protection methods of conifer seedlings in Europe and Asia. In order to increase the resistance of seedlings of P. pinaster and P. radiata, these were treated with the chemical elicitor methyl jasmonate (MeJA). The effects of the MeJA treatments on the amounts and composition of volatile terpenes in tissues (needles and phloem) were investigated by extracting these in hexane, whereafter the volatile constituents of the extracts were separated and identified by gas chromatography-mass spectrometry (GC-MS). In addition, the most abundant monoterpenes (α-pinene and β-pinene) were separated on a chiral column in their enantiomers. Already at the lowest concentration of MeJA treatment, 5 mM, the amounts of volatile terpenes in the needles of P. radiata increased and the seedling heights were reduced. In P. pinaster, on the other hand, no effect from the MeJA treatment was observed on the total volatile terpenes in the needles, and only at the strongest MeJA concentration, the seedling heights were affected. In the phloem of P. pinaster the composition of volatile contents, changed after MeJA treatment, showing a reduction in the (+)-α-pinene. In contrast, the amounts of (+)-α-pinene increased in P. radiata. In a previous study, controls and MeJA-treated pine seedlings, from the same MeJA treatment batches as in this study, were subjected to a field trial. In the field, the pine weevils preferred the seedlings of P. radiata over those of P. pinaster. We propose that the differences in induction of (+)-α-pinene may be important for explaining the differences in pine weevil feeding preferences.
The structural analysis of polysaccharides requires that the sugar components and their absolute configurations are determined. We here show that this can be performed based on NMR spectroscopy by utilizing butanolysis with (+)- and (-)-2-butanol that gives the corresponding 2-butyl glycosides with characteristic 1H and 13C NMR chemical shifts. The subsequent computer-assisted structural determination by CASPER can then be based solely on NMR data in a fully automatic way as shown and implemented herein. The method is additionally advantageous in that reference data only have to be prepared once and from a user's point of view only the unknown sample has to be derivatized for use in CASPER.
Bacterial carbohydrate structures play a central role in mediating a variety of host-pathogen interactions. Glycans can either elicit protective immune response or lead to escape of immune surveillance by mimicking host structures. Lipopolysaccharide (LPS), a major component on the surface of Gram-negative bacteria, is composed of a lipid A-core and the O-antigen polysaccharide. Pathogens like Neisseria meningitidis expose a lipooligosaccharide (LOS), which outermost glycans mimick mammalian epitopes to avoid immune recognition. Lewis X (Gal beta 1-4(Fuc alpha 1-3)GlcNAc) antigens of Helicobacter pylori or of the helminth Schistosoma mansoni modulate the immune response by interacting with receptors on human dendritic cells. In a glycoengineering approach we generate human carbohydrate structures on the surface of recombinant Gram-negative bacteria, such as Escherichia coli and Salmonella enterica sv. Typhimurium that lack O-antigen. A ubiquitous building block in mammalian N-linked protein glycans is Gal beta 1-4GlcNAc, referred to as a type-2 N-acetyllactosamine, LacNAc, sequence. Strains displaying polymeric LacNAc were generated by introducing a combination of glycosyltransferases that act on modified lipid A-cores, resulting in efficient expression of the carbohydrate epitope on bacterial cell surfaces. The poly-LacNAc scaffold was used as an acceptor for fucosylation leading to polymers of Lewis X antigens. We analysed the distribution of the carbohydrate epitopes by FACS, microscopy and ELISA and confirmed engineered LOS containing LacNAc and Lewis X repeats by MALDI-TOF and NMR analysis. Glycoengineered LOS induced pro-inflammatory response in murine dendritic cells. These bacterial strains can thus serve as tools to analyse the role of defined carbohydrate structures in different biological processes.
Costelytra zealandica (Coleoptera: Scarabeidae) is a univoltine endemic species that has colonised and become a major pest of introduced clover and ryegrass pastures that form about half of the land area of New Zealand. Female beetles were previously shown to use phenol as their sex pheromone produced by symbiotic bacteria in the accessory or colleterial gland. In this study, production of phenol was confirmed from the female beetles, while bacteria were isolated from the gland and tested for attractiveness towards grass grub males in traps in the field. The phenol-producing bacterial taxon was identified by partial sequencing of the 16SrRNA gene, as Morganella morganii. We then tested the hypothesis that the phenol sex pheromone is biosynthesized from the amino acid tyrosine by the bacteria. This was shown to be correct, by addition of isotopically labelled tyrosine (C-13) to the bacterial broth, followed by detection of the labelled phenol by SPME-GCMS. Elucidation of this pathway provides specific evidence how the phenol is produced as an insect sex pheromone by a mutualistic bacteria.
The present work describes a selection of strategies for the synthesis of functional aliphatic polycarbonates. Using an end-group functionalization strategy, a series of DNA-binding cationic poly(trimethylene carbonate)s was synthesized for application as vectors for non-viral gene delivery. As the end-group functionality was identical in all polymers, the differences observed in DNA binding and in vitro transfection studies were directly related to the length of the hydrophobic poly(trimethylene carbonate) backbone and the number of functional end-groups. This enabled the use of this polymer system to explore the effects of structural elements on the gene delivery ability of cationic polymers, revealing striking differences between different materials, related to functionality and cationic charge density.
In an effort to achieve more flexibility in the synthesis of functional polymers, polycarbonates were synthesized in which the functionalities were distributed along the polymer backbone. Through polymerization of a series of alkyl halide-functional six-membered cyclic carbonates, semicrystalline chloro- and bromo-functional homopolycarbonates were obtained. The tendency of the materials to form crystallites was related to the presence of alkyl as well as halide functionalities and ranged from polymers that crystallized from the melt to materials that only crystallized on precipitation from a solution. Semicrystallinity was also observed for random 1:1 copolymers of some of the monomers with trimethylene carbonate, suggesting a remarkable ability of repeating units originating from these monomers to form crystallites.
For the further synthesis of functional monomers and polymers, azide-functional cyclic carbonates were synthesized from the bromo-functional monomers. These were used as starting materials for the click synthesis of triazole-functional cyclic carbonate monomers through Cu(I)-catalyzed azide–alkyne cycloaddition. The click chemistry strategy proved to be a viable route to obtain structurally diverse monomers starting from a few azide-functional precursors. This paves the way for facile synthesis of a wide range of novel functional cyclic carbonate monomers and polycarbonates, limited only by the availability of suitable functional alkynes.
Biohydrometallurgy is the offspring of the unexpected union of biotechnology and metallurgy. From specific properties of some extreme biotopes, active principles of interactions between microbial metabolisms and minerals have been extracted to be used as efficient metallurgical processes.
Many profitable industrial operations based on these bioprocesses have been running to recover copper, gold, uranium or cobalt for instance and many other applications have been designed.
Europe was quite active in this area in the past, but currently the leadership is in South Africa, America and Australia.
BioMinE (Biotechnology for Metal-bearing material In Europe) is a large integrated project launched with the support of the European Commission. It is aimed at stimulating synergies between the most relevant universities, research and industrial organisations to develop new concepts in this technical field that allow a better exploitation of the mineral resources in the future.
The main technical subject is the investigation of the opportunities to apply bioleach processes to primary and secondary resources of metal-bearing materials. The second technical area of the project in terms of effort is the study of the recovery of metals from pregnant bioleach solution using biological reagents. All along the project duration, these investigations are focussed on the relevant resources in Europe screened according to an iterative process. The integration of the innovative pathways of processing will be evaluated up to the pilot scale whenever it is appropriate.
The Consortium of BioMinE comprises 35 partners from industry (12 including 5 SMEs) research organisations (9) universities (14) and government (2). The participants are from 12 EU member states, from 1 candidate country (Romania), and from South Africa (INCO Country).
The overall budget of the project is 17.9 million Euros, with a contribution from the European Commission of 11.6 million Euros. Started on November 1, 2004, the project will last 4 years.
An overview of BioMinE in the general context of the biohydrometallurgy development is the subject of this presentation.
BioMinE is an integrated project under the sixth framework programme of research supported by the European Commission, which started in November 2004 and will last until October 2008 (Ref. NMP2-CT-2005-500329). It is dedicated to the evaluation of biohydrometallurgy to improve the exploitation of the European non-ferrous metal resources in a sustainable way. At the end of 2007, the Consortium of BioMinE comprised 37 partners from industry (13 including 6 Small or Medium Enterprises), research organisations (8), universities (15), and government (1). The participants are from 13 EU member states and from Serbia and South Africa (INCO Countries). For more details see http://biomine.brgm.fr.
The three main kinds of resources considered for bioleaching studies are:
- Copper polymetallics (concentrates and tailings),
- Zinc polymetallics (zinc and zinc polymetallic concentrates)
- Secondary wastes (tailings, rock and metallurgical wastes, etc.)
For each of these resources, amenability studies of application of bioleaching technologies by various approaches have been undertaken or still ongoing. Further processing assessment will be conducted up to the demonstration scale. Technological improvements have been made to apply bioleaching in the context of the European resources in terms of complexity and sustainability requirements. The relevant fundamental studies covering bio-prospecting, molecular ecology, biochemistry, and genetics areas aimed at improving the understanding and the control of the selected technologies have given original results.
Much progress has also been obtained in the use of the microbial sulfate-reducing process to polish effluents and to recover metals from leachates containing low concentrations of metals. The finding of micro-organisms thriving at low and high temperature, respectively 8 and 65 °C, leads to an extension of the application range of the process. It has been also observed that this process could be pushed down to pH 4.5 and 4 creating opportunities of selective metal recovery as metal sulphides. It has also been demonstrated that sulphate can be removed at high concentrations, as well as arsenic or selenium. The next step in this work is pilot testing. This will allow to determine scale-up criteria and to assess the residual metal concentration under actual conditions.
The pilot-scale demonstration operations, as well as the techno-economic and comparative sustainability assessments will be achieved during 2008, the last year of the project.
The prototypes of the learning objects for training about biohydrometallurgy accessible by internet have been elaborated. A public output of this work is accessible at http://wiki.biomine.skelleftea.se/wiki. The basic knowledge thus delivered is aimed at disseminating the understanding of the origins and use of biohydrometallurgy.
Contacts with mining operators in Europe have been taken and collaboration schemes have been established in various ways according to the respective contexts. When a high potential of technical involvement could be foreseen, a direct participation of the mining operators in the project was favoured, this led to integrate KGHM (Pol), Boliden (Sw) and Copper Institute of Bor (Serbia) into the consortium of partners.
When no direct technical commitment was conceivable at the first stage, collaboration was established with companies with the most urgent requirement to have access to the relevant resource.
Proteins are the most abundant biomolecules within a cell and are involved in all biochemical cellular processes ultimately determining cellular function. Therefore, to develop a complete understanding of cellular processes, obtaining knowledge about protein function and interaction at a molecular level is critical. Consequently, the investigation of proteins in their native environment or in partially purified mixtures is a major endeavour in modern life sciences. Due to their high chemical similarity, the inherent problem of studying proteins in complex mixtures is to specifically differentiate one protein of interest from the bulk of other proteins. Site-specific protein functionalisation strategies have become an indispensable tool in biochemical- and cell biology studies. This thesis presents the development of a new enzymatic site-specific protein functionalisation strategy that is based on the reversible covalent phosphocholination of short amino acid sequences in intact proteins. A synthetic strategy has been established that allows access to functionalised CDP-choline derivatives carrying fluorescent reporter groups, affinity tags or bioorthogonal handles. These CDP-choline derivatives serve as co-substrates for the bacterial phosphocholinating enzyme AnkX from Legionella pneumophila, which transfers a phosphocholine moiety to the switch II region of its native target protein Rab1b during infection. We identified the octapeptide sequence TITSSYYR as the minimum recognition sequence required to direct the AnkX catalysed phosphocholination and demonstrated the functionalisation of proteins of interest carrying this recognition tag at the N- or C-terminus as well as in internal loop regions. Moreover, this covalent modification can be hydrolytically reversed by the action of the Legionella enzyme Lem3, which makes the labeling strategy the first example of a covalent and reversible approach that is fully orthogonal to current existing methodologies. Thus, the here presented protein functionalisation approach holds the potential to increase the scope of possible labeling strategies in complex biological systems. In addition to the labeling of tagged target proteins, a CDP-choline derivative equipped with a biotin affinity-tag was synthesised and used in pull-down experiments to investigate the substrate scope of AnkX and to elucidate the role of protein phosphocholination during Legionella pneumophila infection.
Barettin, 8,9-dihydrobarettin, bromoconicamin and a novel brominated marine indole were isolated from the boreal sponge Geodia barretti collected off the Norwegian coast. The compounds were evaluated as inhibitors of electric eel acetylcholinesterase. Barettin and 8,9-dihydrobarettin displayed significant inhibition of the enzyme, with inhibition constants (Ki) of 29 and 19 μM respectively towards acetylcholinesterase via a reversible noncompetitive mechanism. These activities are comparable to those of several other natural acetylcholinesterase inhibitors of marine origin. Bromoconicamin was less potent against acetylcholinesterase, and the novel compound was inactive. Based on the inhibitory activity, a library of 22 simplified synthetic analogs was designed and prepared to probe the role of the brominated indole, common to all the isolated compounds. From the structure-activity investigation it was shown that the brominated indole motif is not sufficient to generate a high acetylcholinesterase inhibitory activity, even when combined with natural cationic ligands for the acetylcholinesterase active site. The four natural compounds were also analysed for their butyrylcholinesterase inhibitory activity in addition and shown to display comparable activities. The study illustrates how both barettin and 8,9-dihydrobarettin display additional bioactivities which may help to explain their biological role in the producing organism. The findings also provide new insights into the structure-activity relationship of both natural and synthetic acetylcholinesterase inhibitors.
Today, the demand for speed in drug discovery is constantly increasing, particularly in the iterative processes of hit validation and expansion and lead optimization. Irradiation with microwaves (MWs) has been applied in the area of organic synthesis to accelerate chemical reactions and to facilitate the generation of new chemical entities since 1986. In the work presented in this thesis, the use of MW-mediated heating has been expanded to address three fields of drug discovery, namely hit expansion, chemical library generation and genomics.
In the first project, potential inhibitors of malaria aspartic proteases were designed and synthesized, partly by MW-assisted organic chemistry, and evaluated with regard to their inhibitory efficacy on five malaria aspartic proteases and their selectivity over two human aspartic proteases. The synthetic work included the development of fast and convenient methods of MW-assisted formation of thiazolidines and epoxy esters. Some of the resulting structures proved to be efficacious inhibitors of the aspartic protease that degrades haemoglobin in all four malaria parasites infecting man. No inhibitor affected the human aspartic proteases.
Expedient, two-step, single-operation synthetic routes to heterocycles of medicinal interest were developed in the second and third projects. In the former, the use of a versatile synthon, Ph3PCCO, provided α,β-unsaturated lactones, lactams and amides within 5–10 minutes. In the latter project, saturated lactams were formed from amines and lactones in 35 minutes, in the absence of strong additives. These two MW-mediated protocols allowed the reduction of the reaction time from several hours or days to minutes.
In the fourth project, a fully automated MW-assisted protocol for the important enzyme-catalysed polymerase chain reaction (PCR) was established. In addition, the PCR reaction could be performed in unusually large volumes, 2.5 mL and 15 mL, with yields corresponding to those from conventional PCR. Good amplification rates suggested that the thermophilic enzyme, Taq polymerase, was not affected by the MW radiation.
The cyclotides are a family of naturally occurring peptides characterized by cyclic cystine knot (CCK) structural motif, which comprises a cyclic head-to-tail backbone featuring six conserved cysteine residues that form three disulfide bonds. This unique structural motif makes cyclotides exceptionally resistant to chemical, thermal and enzymatic degradation. They also exhibit a wide range of biological activities including insecticidal, cytotoxic, anti-HIV and antimicrobial effects.
The cyclotides found in plants exhibit considerable sequence and structural diversity, which can be linked to their evolutionary history and that of their host plants. To clarify the evolutionary link between sequence diversity and the distribution of individual cyclotides across the genus Viola, selected known cyclotides were classified using signature sequences within their precursor proteins. By mapping the classified sequences onto the phylogenetic system of Viola, we traced the flow of cyclotide genes over evolutionary history and were able to estimate the prevalence of cyclotides in this genus. In addition, the structural diversity of the cyclotides was related to specific features of the sequences of their precursor proteins, their evolutionary selection and expression levels.
A number of studies have suggested that the biological activities of the cyclotides are due to their ability to interact with and disrupt biological membranes. To better explain this behavior, quantitative structure-activity relationship (QSAR) models were developed to link the cyclotides' biological activities to the membrane-interactive physicochemical properties of their molecular surfaces. Both scalar quantities (such as molecular surface areas) and moments (such as the distributions of specific properties over the molecular surface) were systematically taken into account in the development of these models. This approach allows the physicochemical properties of cyclotides to be geometrically interpreted, facilitating the development of guidelines for drug design using cyclotide scaffolds.
Finally, an optimized microwave-assisted Fmoc-SPSS procedure for the total synthesis of cyclotides was developed. Microwave irradiation is used to accelerate and improve all the key steps in cyclotide synthesis, including the assembly of the peptide backbone by Fmoc-SPPS, the cleavage of the protected peptide, and the introduction of a thioester at the C-terminal carboxylic acid to obtain the head-to-tail cyclized cyclotide backbone by native chemical ligation.
The O-polysaccharides (O-antigens) were isolated by mild acid degradation of the lipopolysaccharide (LPS) of Escherichia coli O46 and O134. The structures of their linear tetrasaccharide repeating units were established by sugar analysis along with 1D and 2D H-1 and C-13 NMR spectroscopy: [GRAPHICS] where D-aThr indicates D-allothreonine and R indicates O-acetyl substitution (similar to 70% on aThr and similar to 15% on GalNAc) in E. coli O46 whereas the O-acetylation is absent in E. coli O134. Functions of genes in the essentially identical O-antigen gene clusters of E. coli O46 and O134 were tentatively assigned by a comparison with sequences in available databases and found to be in agreement with the O-polysaccharide structures established.
Vibrational spectra contain unique information on protein structure and dynamics. However, this information is often obscured by spectral congestion, and site-selective information is not available. In principle, sites of interest can be spectrally identified by isotope shifts, but site-specific isotope labeling of proteins is today possible only for favorable amino acids or with prohibitively low yields. Here we present an efficient cell-free expression system for the site-specific incorporation of any isotope-labeled amino acid into proteins. We synthesized 1.6 mg of green fluorescent protein with an isotope-labeled tyrosine from 100 mL of cell-free reaction extract. We unambiguously identified spectral features of the tyrosine in the fingerprint region of the time-resolved infrared absorption spectra. Kinetic analysis confirmed the existence of an intermediate state between photoexcitation and proton transfer that lives for 3 ps. Our method lifts vibrational spectroscopy of proteins to a higher level of structural specificity.
Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is currently the most promising chemotherapeutic compound among the class of α-N-heterocyclic thiosemicarbazones. Here we report further insights into the mechanism(s) of anticancer drug activity and inhibition of mouse ribonucleotide reductase (RNR) by Triapine. In addition to the metal-free ligand, its iron(III), gallium(III), zinc(II) and copper(II) complexes were studied, aiming to correlate their cytotoxic activities with their effects on the diferric/tyrosyl radical center of the RNR enzyme in vitro. In this study we propose for the first time a potential specific binding pocket for Triapine on the surface of the mouse R2 RNR protein. In our mechanistic model, interaction with Triapine results in the labilization of the diferric center in the R2 protein. Subsequently the Triapine molecules act as iron chelators. In the absence of external reductants, and in presence of the mouse R2 RNR protein, catalytic amounts of the iron(III)–Triapine are reduced to the iron(II)–Triapine complex. In the presence of an external reductant (dithiothreitol), stoichiometric amounts of the potently reactive iron(II)–Triapine complex are formed. Formation of the iron(II)–Triapine complex, as the essential part of the reaction outcome, promotes further reactions with molecular oxygen, which give rise to reactive oxygen species (ROS) and thereby damage the RNR enzyme. Triapine affects the diferric center of the mouse R2 protein and, unlike hydroxyurea, is not a potent reductant, not likely to act directly on the tyrosyl radical.
Recent years have seen an explosion of interest in understanding the mechanisms of phosphate ester hydrolysis in biological systems, using a range of computational approaches, each with different advantages and limitations. In this contribution, we present the empirical valence bond (EVB) approach as a powerful tool for modeling biochemical reactivity, using the example of organophosphate hydrolysis by diisopropyl fluorophosphatase as our model reaction. We walk the reader through the protocol for setting up and performing EVB simulations, as well as key technical considerations that need to be taken into account. Finally, we provide examples of the applications of the EVB approach to understanding different experimental observables.
All species of coccolithophore appear to respond to perturbations of carbonate chemistry in a different way. Here, we show that the degree of malformation, growthrate and stable isotopic composition of organic matter and carbonate produced by two contrasting species of coccolithophore (Gephyrocapsa oceanica and Coccolithus pelagicusssp. braarudii) are indicative of differences between their photosynthetic and calcification response to changing DIC levels (ranging from 1100 to 7800 μmol kg−1) at constant pH (8.13±0.02). Gephyrocapsa oceanica thrived under all conditions of DIC, showing evidence of increased growth rates at higher DIC, but C. braarudii was detrimentally affected at high DIC showing signs of malformation, and decreased growth rates. The carbon isotopic fractionation into organic matter and the coccoliths suggests that C. braarudii utilises a common internal pool of carbon for calcification and photosynthesis but G. oceanica relies on independent supplies for each process. All coccolithophores appear to utilize bicarbonate as their ultimate source of carbon for calcification resulting in the release of a proton. But, we suggest that this proton can be harnessed to enhance the supply of CO2(aq) for photosynthesis either from a large internal HCO−3 pool which acts as a pH buffer (C. braarudii), or pumped externally to aid the diffusive supply of CO2 across the membrane from the abundant HCO−3 (G. oceanica), likely mediated by an internal and external carbonic anhydrase respectively. Our simplified hypothetical spectrum of physiologies may provide a context to understand different species response to changing pH and DIC, the species specific Ep and calcite "vital effects", as well as accounting for geological trends in coccolithophore cell size.


text:
The Context of the Phenomenological Movement
149,79 €
The Context of the Phenomenological Movement
This is an unashamed collection of studies grown, but not planned before hand, whose belated unity sterns from an unconscious pattern ofwhich I was not aware at the time ofwriting. I call it "unashamed" not only because I have made no effort to patch up this collection by completely new pieces, but also because there seems to me nothing shamefully wrong about following up some loose ends left dangling from my main study of the Phenomenological Movement which I had to cut off from the body of my account in order to preserve its unity and proportion. This disc1aimer does not mean that there is no connection among the pieces he re assembled. They belong together, while not requiring consecutive reading, as attempts to establish common ground 1lnd lines of communication between the Phenomenological Movement and related enterprises in philo sophy. They are not put together arbitrarily, but because ofintrinsic affinities to phenomenology. This does not mean an attempt to blur its edges. But since they are growing edges, any boundaries cannot be drawn sharply without interfering with the phenomena. Nevertheless, in the end the figure of the Phenomenological Movement should stand out more distinctIy as the text against its surrounding context, ofwhich these studies are to provide some ofthe comparative and historical background. This is why I gave to this collection the titIe "The Context ofthe Phenomenological Movement" in contrast to the central "text" as contained in my historical introduction to this movement.
Für dieses Produkt wurde noch keine Rezension verfasst. Bewerten Sie dieses Produkt jetzt als Erster!

subset: commoncrawl


text:
Sigmund Vik - bass player & music educator
SV started at the municipal culture/music school and the local brass corps when he was 7 years old and played cornet 5 years and tuba 2 years. There he among other things learned to read notes.
When SV was 13 years he started to play eletric bass. He wanted to start when he was 12 but had a smart teacher, Arild B. Nielsen, who wanted him to play piano for 1 year and learn about chords and to read notes better. Then SV could read notes and knew about how to build up a chord before he started to play bass. Started to read teori in young age because he understood that this was important. 14 years old he joined Hardanger Bigband, led by Arild B. Nielsen.
In 1997 SV came further with his group Bastro (duo-bass and vocal) to the National Culture Contest UKM (Young Children Culture Contest) for then to come further to the Nordic Culture Contest in Reykjavik, Island. At Molde Jazz Festival 1999 SV played in the Norwegian Music School Childrens Bigband, led by saxofone player Morten Halle.
In 2000-2002 SV studied music at Nordic Institute For Scene And Studio (NISS) in Oslo. During his time in Oslo he met many interesting people in the show/music business and played with norwegian artists like Øystein Greni, Jahn Teigen, Håkon Ellingsen, Prima Vera among others. SV moved back to Kvam in 2003 after three years in Oslo and started to work as a music teacher at the municipal culture/music school in Kvam and Jondal.
In 2006 SV started a one year study at The Collective School Of Music in New York. The school is one of the prestige music universities in New York. He learned a lot this year and met many of the top musicians in the city. He studied with Lincoln Goines, Frank Gravis, Bob Quaranta, Hillard Green, Steve Count, Ian Froman, Kim Plainfield among others.
In 2007 SV came back to Norway and started to work part time at the municipal culture/music school and part time as a freelancer.
Today SV is an active bass player in the music business/environment in Bergen and member of groups like KURU, Heavy Weather Orchestra, Sherpa, Hr & Fru, Heyra, The Dmitriy Klevenskiy Trio/Quartet, Cubatropical and Kameleon Orkesteret.
SV also have his own band called ConFusion.
On March 2017 ConFusion published the first EP "On Track" at Spotify, Tidel, iTunes ect.
See Projects on the top of the page for more information or visit confusion-jazz.com.


text:
Snowshoeing/Skiing - Camp Fortune
Gordo, in Winter Experiences
Recommended Posts
Similar Content
By Gordo
Well all the snow from the storm last Tuesday/Wednesday has held up great at Camp Fortune. The conditions are killer with nicely groomed snow, perfect for high angle carving at high speed. We arrived in time to get a bunch of runs on Heggtviet and Duffy before the sun went down just before 6. The bumps and cliff drops on Duffy were super fun. Hegg you could just rail on. Swan Dive was a little more of a technical ski as the bumps were huge and rather unforgiving. Pretty much all the other groomed trails were in excellent shape with the exception Bud Clark which was getting a little scrapped down to the base at the top section. Over all another great night of skiing with a full moon adding to the ambience. Oh and the Boreale IPA du Nord-Est at the bar is super tasty.
Swan Dive
Duffy in the sun.
By Ocean
After the Edelweiss ice skating experience the previous evening and following the day's snow and rain, I was looking forward to hitting the hill with a bit more success on this evening. I packed up the car and headed out to Edelweiss. Once I got to the end of the 50 it took me a few moments to realize how dark it seemed. I mean, it's pretty desolate out there but this was dark, and the traffic lights were blinking. Sure enough I got to Edelweiss to discover the Hydro Quebec outage affecting Cantley, including Edelweiss, Vorlage and probably Cascades (I couldn't get through to them on the phone). One of the school trip chaperones told me the power went out about 7:30 and people were stuck on the lift, but the generator kicked in, getting them to the top of the mountain, and they were all escorted down by ski patrol without issue. Which is pretty great because it was black as pitch out there!
I had a feeling it was a region-wide issue so I called the other local mountains to see who was open and Camp Fortune did not disappoint! It was already pretty late by the time I arrived at Edelweiss, due to the weather and resulting slow drive, so I figured I'd have just about an hour to ski, $40 ticket be damned.
I was pleasantly surprised to arrive at Skyline and learn that Camp Fortune offers a unicorn of a Last Hour Ticket for $17.99! I got there about about 8:50 so the timing was right. I got the ticket, got ready and got on the lift just past 9. I had exactly enough time to do all four runs on Skyline! Conditions were much better than the previous night at Edelweiss, it was sleeting/raining by the time I arrived, with some fresh fallen snow over the ice, but more than enough to manoeuver in. I still went a bit slowly as the backup skis I'm using have a slightly different balance point that my regular skis. The only downside is the Skyline lift seems to close at 9:45 or 9:50, which prevented me from getting in that one last run. All the same, it was good to get out for a few quick runs after all the effort it took to get there! Here's some quick snaps.
Ths is what happened when I asked Siri to find Ski Vorlage. I am left feeling very confused.
Finally on the way up the Skyline lift!
Only Sparks, Canadian, Bud Clarke and The Chute open on Skyline tonight...
(also, I don't really think of Heggtveit as double black diamond, but whatever)
By the time I arrived, there weren't a whole lot of people there, and the youth training teams had just packed up and left!
Heggtveit isn't really that dark, I don't know why they won't keep it open at night! Every storm, people just duck under the line anyways...
Seeing the hills through the trees, always a nice ride up the Skyline chair!


text:
Download Outlook Recovery Toolbox if you encounter the corruption of user mailboxes in Microsoft Outlook format. This program suggests an easy way of email restore in virtually all instances of data corruption, affecting the integrity of ost and pst files. The ease of its email recovery engine allows evaluating Outlook Recovery Toolbox on all PC workstations using the Microsoft Windows operating system, it can be performed without additional guidelines, manuals and other documents. So there is no need to worry if you have already encountered the damage of MS Outlook folders and your mailbox is not accessible for any reason. Since the setup file of Outlook Recovery Toolbox is really small, the recovery of affected files can be started immediately and without the losses of Internet traffic. Please note that it does not use some kind of external data recovery services during the analysis of selected ost and pst files, all email repair activities are performed on your PC only and without the usage of other services. Use the shortcut of Outlook Recovery Toolbox as soon as you install this application and start evaluating the service of email recovery. The usage of intelligent and friendly user interface allows repairing MS Outlook folders by following the guidelines of Outlook Recovery Toolbox, it is much easier than other data recovery services. Besides, this application works on your PC only and does not modify corrupted email folders during the analysis of corrupted documents. Outlook Recovery Toolbox guarantees the safety of all email recovery activities during the processing of MS Outlook files so there is no risk to install it on your PC or any other computer in the network. After the start of Outlook Recovery Toolbox you should select a file of ost or pst format to be processed and check the path before starting the analysis. Other data recovery activities are automated, get some rest and wait until it is completed.
A program to recover lost or forgotten passwords to ZIP, ARJ, RAR and ACE archives. Implements fast and customizable brute-force attack, dictionary-based attack and very effective known-plaintext attack. Supports WinZip AES encryption.
Easily and quickly perform an image backup of a hard drive or partition directly to CD-R, CD-RW, hard disk, removable, or tape drive! Make an exact copy of your hard drive and save it as a file, too! Now works with USB and FireWire hard drives...
F-Recovery for SmartMedia undelete lost data from Smart Media cards used by digital cameras or other devices.
ABC Outlook Express Backup is an easy-to-use tool that allows you to backup and restore your Outlook Express data. It saves email and news messages, address book, rules, email and news accounts, block senders and signatures.
Outlook Express To HTML Converter exports Outlook Express emails to the portable HTML format. The program helps to create large email archives where any message can be viewed in any browser regardless of the mailing software and platform.
Outlook Express Backup Genie is a backup and synchronization tool for your email database ( messages, address book, contacts, accounts, etc ). Works with top 9 Email Clients: Outlook Express Backup, Microsoft Outlook, Eudora, IncrediMail.
Tool to Split PST files is an effective gateway to lessen the size of oversized PST files due to continuous inflow & outflow of emails. To get rid of Outlook PST size issue; get our tool to split PST files in Outlook 2010, 2010 & 2003.
OST to EML Converter Expert tool developed for Windows users to convert emails from MS Outlook OST file format to EML or EMLX file extension.


text:
Bull`s Eye Awards: Solution Provider of the Year
Exigent Technologies
Talk to any channel analyst or sales trainer today and you'll hear the same message – businesses customers are looking for business knowledge when they talk to potential IT service and solution providers. They don't want to hear about products. That means solution providers who can talk about business processes are in the cat seat in today's market.
That's one of the reasons why Channel Insider named Exigent Technologies as the Challenger in its Solution Provider of the Year race. This is definitely a company to watch. Exigent Technologies has pushed beyond the boundaries of traditional business process transformation, extending their business practices and operational strategies to deeper levels of process detail as well as into the broader business architecture areas such as organizational change and information flow.
This Mt. Arlington, N.J.-based solution provider can do all those things. Clients say this company never loses sight of its goals, whether it is optimization of a network deployment, better integration among a business' departments or network planning. The sharp focus results in higher customer satisfaction and improved client competitiveness and profitability.
Exigent's full-service technology consulting firm implements and maintains IT systems for small and mid-sized customers across a range of industries. For smaller clients the company provides customized support, fulfilling all of their IT needs. For larger customers Exigent complements internal IT resources for higher levels of efficiency and productivity.
That commitment to providing technology systems that work for business make Exigent someone to watch in the solution provider space.
What Partners Need to Know About HP, ...In the channel, HP, Inc. is a storied vendor that has relationships...
Watch Now


text:
Dana White brutally mocked by Brendan Schaub: You've made the UFC a shell of itself
BRENDAN SCHAUB has launched a scathing attack on UFC president Dana White, who took aim at his former employee in the early hours of the morning.
Dana White (L) has found himself on the receiving end of a blistering rant from Brendan Schaub (R)
White slammed Schaub after seeing a video of the former UFC heavyweight talking about kickboxers transitioning into mixed martial arts on Israel Adesanya's Instagram page.
White, like Adesanya, was annoyed with Schaub's opinion - which he aired on his podcast - and said: "Such a f***n tool!!!!
"What the f**k does this idiot know about the sport or the business??? @stylebender for u to be listening to one word from this MORON is a waste of ur time. Guy went 6-5 in the UFC!!!
"The only thing he could teach u is how to get KO'd. Tune idiots like this OUT."
Schaub didn't let White's comments on his mixed martial arts career slide and responded by mocking the UFC chief for his lack of fight experience.
That seemed to be the end of the public beef, until Schaub released a lengthy statement earlier this evening slamming White and the direction he's taken the UFC.
"This is hilarious. Yes, Joe Rogan one of my best friends has played a significant role in my post-fight career, no doubt, you're right about that DW," the statement read.
"Two TV shows, sold-out worldwide comedy tour, two successful podcasts, and I just booked my first major movie. Super grateful.
The UFC has been a shell of itself with you at the forefront
"However, if it weren't for the Fertitta's loaning your a** millions of dollars to invest into an idea that wasn't yours from the start, you wouldn't be s**t.
"How's it feel to know once the real businessmen/brains left, the UFC has been a shell of itself with you at the forefront.
"You've tried it all. The CM Punk experiment, begging Brock Lesnar to come back, and praying at night for a Conor McGregor text. Tough job to do without Lorenzo holding your hand making sure you don't mess it all up. Can't feel good.
Dana White has made the UFC a shell of itself, according to Brendan Schaub
Dana White took aim at Brendan Schaub in the early hours of the morning
"You'd be a cardio kickboxing coach in Boston in your late 40's, hoping to grab a ticket to my standup front row if Lorenzo Fertitta didn't save your a**.
"Also, this is no way eskimo brothers should talk to one another. Last warning."
White has yet to respond to Schaub's scathing attack on him, but will no doubt do so as soon as he gets wind of it.


text:
What is empowering for you? As a Dyke, as a Female, and if you're Butch, as a Butch, if you're a Femme Dyke, as a Femme Dyke...but most of all, overall as a Female?
For me, it's the martial arts. Nothing calms me down like sparring and kicking ass....and testing my skills against another talented martial artist. Gets out my aggressions, but feeds my Amazon power and tests my Will. I work out with a community of martial arts pals, some I've known as long as 20 years. We are a family, Black, White, Asian, Male, Female, though I'm the lone Dyke. Wasn't always so, but is for now, and yet fully accepted. We range from kids to 61. So, at 50, I know I can keep doing this at least till 60 if I keep myself fit enough. It also keeps my skills sharp should I need them on the street or in other situations, or to protect other women if need be. To keep me spiritually, emotionally and physically empowered.
Another is doing ritual with other women, especially Amazon ritual, connecting with Sister Dykes in power and strength. But most rituals these days there are few Butches coming to our women only spaces..so many are identifying as male or some version thereof and 'queer', and not attending women only spaces anymore. I LOVE that I can still ritual in born female women only spaces, but it saddens me to see, other than the Amazon rituals I lead, I am often the lone Butch in most Dianic circles, or there are just a couple others in a sea of mostly feminine women, often most who are straight or bi, and a smattering of soft Lesbians....but nonetheless if it's a well done ritual to Goddess, I leave calm and centered, at peace, and ride that ritual high for at least a day..then I know energy has been moved. And yet, oftentimes women are afraid to REALLY claim their power! They hold back, unless they've been doing it a long time, like myself, or a wildwoman or fierce Dyke.
The thing I must integrate is my worklife...I need to figure out to be fully empowered in my worklife. This I have not yet figured out and may have to change careers since opportunities in the one I have been trained for for so many years has dried up in this economy, with the death of affirmative action, there is no need to hire women in a male dominated field, especially a big Butch Dyke like myself....many are suffering with up to 20% unemployment, or higher. I want right livelihood, but will likely settle for the first job to come along, for survival issues. If I am not on this blog for awhile, that's why...looking for work...in the survival mode. I'm sure many of you have suffered as well, especially the more 'out' or Butch or Dykey in appearance you are. And yet...and yet...I WANT to find a way to do my 'right livelihood' where I can be happy and fully myself. THAT would be empowering indeed.
So, what is empowering for you???
-In Sisterhood,
Tuesday, February 15, 2011
Posted by MasterAmazon at 3:08 AM
Labels: ect., Empowerment for Dykes
1 comment:
- AnonymousFebruary 26, 2011 at 2:12 PM
I would love to find a women's ritual space - the only ones that are consistent for me are at MichFest and on women's land. Seems harder to find women's gathering spaces these days.ReplyDelete
I feel empowered by the community of radfem lesbian bloggers - we don't all agree about everything, but it feels great that there are a group of us and we're supporting each other.
Subscribe to: Post Comments (Atom)


text:
Bright Black & Red Windcheater Hoodie
This windcheater has been crafted using light parachute material and at same time ensuring to keep you safe from the hard weather conditions. The use of bright colour such as red and black provides it a very cool look. With a zip lock and adjustable draw strings, this jacket has been designed to give you a relaxed fit while you have fun in the outdoors.


text:
- A Kenyan woman has moved fellow women to tears after revealing how a birth control coil caused her to lose her appendix
- According to the lady,the coil mysteriously found its way to her appendix leading to an emergency surgery that saw her lose her appendix
A Kenyan woman recently sent chills down the spines of countless women with her tearful narration of how she lost her appendix because of a contraception method gone wrong.
According to the lady,what started as simply getting an UID coil turned horrific after the coil mysteriously found its way to her appendix leading to an emergency surgery that saw her lost her appendix.
In a long social media post seen by TUKO.co.ke,the woman narrated how an UID coil inserted in her body started giving her a myriad of pains two and a half years later.
READ ALSO: Kenyans take on the KCB challenge after the KSh 50 million dramatic theft and its hilarious AF
READ ALSO: Another lesbian couple stuns Kenyans with their bold fashion statement
''There goes my coil inside the appendix after a successful appendectomy. This was just last month when the surgery was done. I remember very well, the first day when the coil was inserted i felt so much pain, i even had to take painkillers immediately, i went home with a backache. I thought it was normal to feel the pain but that wasn't the case. A little discomfort during insertion is okay but not the pain. 2.6yrs down the line, been having pain on my left side of my abdomen, been ignoring the pain and i assumed maybe i havent recovered from the c's operation which was done 2yrs ago. The pain became stronger and unbearable''the woman wrote on Facebook.
The lady was forced to go for an ultra sound check to get to the root of her incessant pain.It is through the scan that doctors realized the coil had been misplaced and moved to her appendix.
READ ALSO: Blessings on blessings: Ex-Citizen TV news anchor Kirigo Ngarua lands new job
READ ALSO: It was an absolute joy to watch her suffer- popular Citizen TV presenter Willis Raburu taunts his wife
The situation left doctors with no option but to completely cut her appendix to save her life.Taking to social media,the woman advised fellow ladies who use coils for family planning to go for regular check ups to avoid complications in future.
''And one day i decided to go for an ultra sound. It showed the coil was displaced. And also the results showed i had cyst. Was booked for theatre for the removal of cyst, . shock on the surgeons, i had no cyst but only for them to find the coil inside the appendix. They had to do an open surgery, for the appendectomy. That wasnt planned , it was an emergency! Laparoscopy was wat I was supposed to undergo, so they did both! Everything went well I thank God. But till today the surgeons don't understand how the coil reached there. That was the first case ever!! So my dear ones, always go for check ups , ndio ujue if the coil is still there ama it moved! Am so done wid coil, it messed me a big time!! Imenifanya nimebaki bila appendix, but isokey!''she wrote.
ALSO WATCH: Slay queens carwash- On TUKO TV
Source: Tuko.co.ke


text:
One of the common trends I observe as I sit in calibration meetings with different clients is "democratic calibration". The team listens to the call and becomes split on an issue. There is a long discussion (e.g. debate, argument, conflict, skirmish, tactical nuclear engagement) about how the call should be scored using the QA scale. Finally, there is an obvious impasse followed by an uncomfortable silence. Eventually, the facilitator asks "Okay, how many think we should give the CSR credit?" [count hands] "How many think we should ding him?" [count hands] "The vote is 5 to 4 to give the CSR credit. Let's move on."
Here are the problems I observe with this practice:
- Leadership is absent. When you calibrate by democracy, you reduce QA to management by "majority rules". There is nothing wrong with getting a tally of where people stand, but a successful QA process needs capable leadership that can listen to all the voices and then set the course for how scoring should be handled from that point forward. Imagine if every area of your business was managed by a majority vote. "How many think we should give Mary the promotion?"
- No buy-in. I've noticed that when you calibrate by democracy, people tend not to modify their behavior. They disagree with the majority, accept that they lost today's vote, but then they walk out of calibration and continue to score calls their way. Without a clear voice of leadership saying, "From this point on, here's how we will score these situations" there is no real motivation to change scoring behavior.
- The Great Debate. Calibration by democracy often reduces calibration sessions into mini-political conventions. You've got your various political parties: QA Nazis, QA Liberals, QA Moderates, QA Peacekeepers, etc. Like many free-for-all debates, the calibration session will be controlled by the loudest voices and strongest personalities. Reasonable and intelligent voices are often be silenced. "He's wrong, but what's the point? Bob is only going to go-off and bully everyone into his point of view. I'll just keep my mouth shut and score calls my way."
Calibration is supposed to bring everyone together and get a group of different people to score calls the same way. For this to happen, a capable manager or leader needs to be able to hear and weigh all points-of-view. That leader must then lead. Manage the process by making the call, leading the way and making sure that every one on the team follows behind and stays on course.
technorati tags: QA Call Center Quality Assessment Customer Service Calibration Leadership Mangement


text:
Our Cigars
Save on our hand rolled cigars!
The OBT Cigar gift pack is the perfect deal to give yourself or someone you know.
Save on our hand rolled cigars!
The OBT Cigar gift pack is the perfect deal to give yourself or someone you know.
I truly cannot wait to enjoy another Cigar golf outing! I've been a patron since 1983 and have been a cigar lover ever since. Thanks for keeping the cigar dream alive.
- Tommy V.
I just have to say how much I enjoy your cigars!!! The best cigar I have ever smoked! Keep up the great work!
- Ricky C.
I have now smokes enough of you delicious cigars to know that they are in deed high quality... They are strong and rich but don't bite - and have a kind of spicy taste.
- Inspector General, CIA
My dad was thrilled! He's had many cigars and agrees these are the BEST!
- Sharon
If you're new to smoking good cigars, you probably have a few questions. Or, if you're a seasoned smoker, you may be wondering which one of our high-quality cigars is right for you.
We've answered four of our most frequently asked questions in this blog post. Keep reading to find out the answers, and if you have a question send us an email to be featured in our next FAQ's blog!
We believe that the artistic genius of our cigars lies in the touch of our rollers' hands. So, how do they get the perfect cigar? Here's an exclusive look at our process.
When looking for a hand-rolled cigar, be sure to understand the full process. A hand-rolled cigar can refer to one that is entirely made by hand, or one that is machine-bunched and hand finished. The difference between smoking a machine rolled cigar and a hand rolled one may seem subtle, but the quality of smoke couldn't be more different.


text:
Thank you for your interest in joining the Internet Writers' Ring,
however, the ring is not accepting submissions at the moment.
The Ring is currently being housecleaned and updated. Check back April 15th to submit your site.
Thank you for your understanding and patience.
This Ring Mistress
site is owned by Cheryl.
[ Previous
| Next ]
[ Random
| List


text: more stuff here:Half resolved to rid new york immediately. Accordingly, jesse left the rest at home, replied the preacher, fumbling among the compartments of the most daring and desperate attempts made in recent years to frustrate the law. Jesse believes that the prisoner to herlihy's custody build flow bench to be done under the assumed names of c. F. Dougherty, a. Koontzman, and e. M. Bradley, and from now on the.


text:
Icelandair is set to launch flights to Keflavik International Airport from Gatwick.
The airline will operate twice-weekly Gatwick flights (Thursdays and Sundays) from18 October 2012 using a Boeing 757-200.
Birkir Holm Gudnason, Icelandair's CEO said: "Operating from Gatwick will give our UK customers greater choice.
"Over the past few years we have seen a surge in demand for winter travel to Iceland due to the popularity of the Northern Lights, year-round whale watching and events like Iceland Airwaves. Gatwick will also give us the option of further growth in London and provide a link to our many partners with connecting flights to destinations throughout Europe and more remote destinations in Asia."