Switch and template pattern formation in a discrete reaction-diffusion system inspired by the Drosophila eye.

We examine a spatially discrete reaction-diffusion model based on the interactions that create a periodic pattern in the Drosophila eye imaginal disc. This model is known to be capable of generating a regular hexagonal pattern of gene expression behind a moving front, as observed in the fly system. In order to better understand the novel "switch and template" mechanism behind this pattern formation, we present here a detailed study of the model's behavior in one dimension, using a combination of analytic methods and numerical searches of parameter space. We find that patterns are created robustly, provided that there is an appropriate separation of timescales and that self-activation is sufficiently strong, and we derive expressions in this limit for the front speed and the pattern wavelength. Moving fronts in pattern-forming systems near an initial linear instability generically select a unique pattern, but our model operates in a strongly nonlinear regime where the final pattern depends on the initial conditions as well as on parameter values. Our work highlights the important role that cellularization and cell-autonomous feedback can play in biological pattern formation.

Engineering a stable and selective peptide blocker of the Kv1.3 channel in T lymphocytes.

Kv1.3 potassium channels maintain the membrane potential of effector memory (T(EM)) T cells that are important mediators of multiple sclerosis, type 1 diabetes mellitus, and rheumatoid arthritis. The polypeptide ShK-170 (ShK-L5), containing an N-terminal phosphotyrosine extension of the Stichodactyla helianthus ShK toxin, is a potent and selective blocker of these channels. However, a stability study of ShK-170 showed minor pH-related hydrolysis and oxidation byproducts that were exacerbated by increasing temperatures. We therefore engineered a series of analogs to minimize the formation of these byproducts. The analog with the greatest stability, ShK-192, contains a nonhydrolyzable phosphotyrosine surrogate, a methionine isostere, and a C-terminal amide. ShK-192 shows the same overall fold as ShK, and there is no evidence of any interaction between the N-terminal adduct and the rest of the peptide. The docking configuration of ShK-192 in Kv1.3 shows the N-terminal para-phosphonophenylalanine group lying at the junction of two channel monomers to form a salt bridge with Lys(411) of the channel. ShK-192 blocks Kv1.3 with an IC(50) of 140 pM and exhibits greater than 100-fold selectivity over closely related channels. After a single subcutaneous injection of 100 microg/kg, approximately 100 to 200 pM concentrations of active peptide is detectable in the blood of Lewis rats 24, 48, and 72 h after the injection. ShK-192 effectively inhibits the proliferation of T(EM) cells and suppresses delayed type hypersensitivity when administered at 10 or 100 microg/kg by subcutaneous injection once daily. ShK-192 has potential as a therapeutic for autoimmune diseases mediated by T(EM) cells.

Evaluation of the cost-effectiveness of root canal treatment using conventional approaches versus replacement with an implant.

AIM: To evaluate the cost-effectiveness of root canal treatment for a maxillary incisor tooth with a pulp infection, in comparison with extraction and replacement with a bridge, denture or implant supported restoration. METHODOLOGY: A Markov model was built to simulate the lifetime path of restorations placed on the maxillary incisor following the initial treatment decision. It was assumed that the goal of treatment was the preservation of a fixed platform support for a crown without involving the adjacent teeth. Consequently, the model estimates the lifetime costs and the total longevity of tooth and implant supported crowns at the maxillary incisor site. The model considers the initial treatment decisions, and the various subsequent treatment decisions that might be taken if initial restorations fail. RESULTS: Root canal treatment extended the life of the tooth at an additional cost of pound5-8 per year of tooth life. Provision of orthograde re-treatment, if the root canal treatment fails returns further extension of the expected life of the tooth at a cost of pound12-15 per year. Surgical re-treatment is not cost-effective; it is cheaper, per year, to extend the life of the crown by replacement with a single implant restoration if orthograde endodontic treatment fails. CONCLUSION: Modelling the available clinical and cost data indicates that, root canal treatment is highly cost-effective as a first line intervention. Orthograde re-treatment is also cost-effective, if a root treatment subsequently fails, but surgical re-treatment is not. Implants may have a role as a third line intervention if re-treatment fails.

Stress (Takotsubo) Cardiomyopathy During Liver Transplantation: Case Study and Literature Review.

A case of stress (takotsubo) cardiomyopathy (TC) that occurred intraoperatively during liver transplantation surgery was identified by transesophageal echocardiography. Only a few cases of TC occurring during liver transplantation have been reported to date. Unlike other cases reported, TC occurred during the anhepatic stage of the liver transplantation, with subsequent complete recovery. Notwithstanding the large number of cases of TC in the perioperative settings reported worldwide, the exact reasons of this syndrome occurring intraoperatively as well as precipitating factors and conditions remain mostly unknown.

Insensitivity to pain induced by a potent selective closed-state Nav1.7 inhibitor.

Pain places a devastating burden on patients and society and current pain therapeutics exhibit limitations in efficacy, unwanted side effects and the potential for drug abuse and diversion. Although genetic evidence has clearly demonstrated that the voltage-gated sodium channel, Nav1.7, is critical to pain sensation in mammals, pharmacological inhibitors of Nav1.7 have not yet fully recapitulated the dramatic analgesia observed in Nav1.7-null subjects. Using the tarantula venom-peptide ProTX-II as a scaffold, we engineered a library of over 1500 venom-derived peptides and identified JNJ63955918 as a potent, highly selective, closed-state Nav1.7 blocking peptide. Here we show that JNJ63955918 induces a pharmacological insensitivity to pain that closely recapitulates key features of the Nav1.7-null phenotype seen in mice and humans. Our findings demonstrate that a high degree of selectivity, coupled with a closed-state dependent mechanism of action is required for strong efficacy and indicate that peptides such as JNJ63955918 and other suitably optimized Nav1.7 inhibitors may represent viable non-opioid alternatives for the pharmacological treatment of severe pain.

Synthesis and structural characterisation of analogues of the potassium channel blocker charybdotoxin.

Charybdotoxin is a 37-residue polypeptide toxin from scorpion venom, which acts by blocking voltage-gated and Ca(2+)-activated K+ channels. We have synthesized charybdotoxin and three mono-substituted analogues using an Fmoc-tBu protocol. The Phe-2 --> Tyr analogues was chosen to introduce a site for Tyr iodination which was distinct from the K+ channel binding surface, while the Glu-12 --> Gln and Arg-19 --> His analogues were studied to probe the roles of charged residues at these positions in the structure and activity of the toxin. The synthetic native molecule was equipped with natural toxin in inhibiting the human erythrocyte Ca(2+)-dependent K+ channel. The affinities of all three analogues for the erythrocyte K+ channel were slightly reduced, with the Arg-19 --> His analogue showing the greatest increase in IC50 (2.30-fold). Two-dimensional 1H-NMR studies of these analogues showed that the Glu-12 to Gln substitution, which appeared to destabilise the N-terminal half of the alpha-helix, possibly due to the weakening of an N-terminal helix capping interaction which is apparent from our NMR data. His-21 has a pKa more than one unit below the value for a non-interacting histidine. Possible reasons for this are that the imidazolium side chain is partly buried and is located near positively charged moieties. Thus, His-21 would be neutral at physiological pH, where charybdotoxin binds to the potassium channel.

Auto-inactivation by cleavage within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease.

An autolysis site of functional and structural significance has been mapped within the dimer interface of Kaposi's sarcoma-associated herpesvirus protease. Cleavage 27 residues from the C terminus of the 230 amino acid residue, 25 kDa protein was observed to cause a loss of dimerization and proteolytic activity, even though no active site moieties were lost. Gel-filtration chromatography and analytical ultracentrifugation were used to analyze the changes in oligomerization upon autolysis. The selective auto-disruption of this essential protein-protein interface by proteolytic cleavage resulted in a 60 % loss in mean residue ellipticity by circular dichroism as well as a 20 % weaker, 10 nm red-shifted intrinsic protein fluorescence emission spectrum. These apparent conformational changes induced a strict inhibition of enzymatic activity. An engineered substitution at the P1' position of this cleavage site attenuated autolysis by the enzyme and restored wild-type dimerization. In addition to retaining full proteolytic activity in a continuous fluorescence-based enzyme assay, this protease variant allowed the determination of the enzyme's dimerization dissociation constant of 1.7 (+/-0.9) microM. The structural perturbations observed in this enzyme may play a role in viral maturation, and offer general insight into the allosteric relationship between the dimer interface and active site of herpesviral proteases. The functional coupling between oligomerization and activity presented here may allow for a better understanding of such phenomena, and the design of an enzyme variant stabilized to autolysis should further the structural and mechanistic characterization of this viral protease.

Three-dimensional structure in solution of the calcium channel blocker omega-conotoxin.

The 27 amino acid residue polypeptide omega-conotoxin GVIA, from venom of the cone shell Conus geographus, blocks neuronal voltage-activated calcium channels at picomolar concentrations. The three-dimensional structure in aqueous solution of synthetic omega-conotoxin has been determined from two-dimensional 1H n.m.r. data recorded at 600 MHz. Structural constraints consisting of interproton distances inferred from NOEs and dihedral angles from spin-spin coupling constants were used as input for distance geometry calculations with the program DSPACE. The structures were then refined using back-calculation of NOESY spectra. The family of structures obtained in this way is well defined by the n.m.r. data, the best 12 structures having pairwise root-mean-square differences of 0.68 (+/- 0.15) A over the backbone heavy atoms (N, C alpha and C) and 1.15 (+/- 0.17) A over all heavy-atoms. The molecule adopts a compact structure consisting of a small, triple-stranded, anti-parallel beta-sheet and several reverse turns. All three tyrosine residues are located on the molecular surface, which is noteworthy for its abundance of side-chain hydroxyl groups. There is no negatively charged group in conotoxin, but the five positively charged groups are distributed in three small patches on the surface, one of which, made up of the ammonium moieties of the N terminus and Lys2, may contribute to the receptor-binding surface of the molecule. An isomer of conotoxin with the same amino acid sequence, but different disulfide pairings, has also been investigated. Its structure is less well ordered than that of native conotoxin and it shows significant heterogeneity, probably as a result of cis-trans isomerism preceding hydroxyproline residues.

Lifetime cost effectiveness of different brands of prosthesis used for total hip arthroplasty: a study using the NJR dataset.

There is little evidence on the cost effectiveness of different brands of hip prostheses. We compared lifetime cost effectiveness of frequently used brands within types of prosthesis including cemented (Exeter V40 Contemporary, Exeter V40 Duration and Exeter V40 Elite Plus Ogee), cementless (Corail Pinnacle, Accolade Trident, and Taperloc Exceed) and hybrid (Exeter V40 Trilogy, Exeter V40 Trident, and CPT Trilogy). We used data from three linked English national databases to estimate the lifetime risk of revision, quality-adjusted life years (QALYs) and cost. For women with osteoarthritis aged 70 years, the Exeter V40 Elite Plus Ogee had the lowest risk of revision (5.9% revision risk, 9.0 QALYs) and the CPT Trilogy had the highest QALYs (10.9% revision risk, 9.3 QALYs). Compared with the Corail Pinnacle (9.3% revision risk, 9.22 QALYs), the most commonly used brand, and assuming a willingness-to-pay of £20,000 per QALY gain, the CPT Trilogy is most cost effective, with an incremental net monetary benefit of £876. Differences in cost effectiveness between the hybrid CPT Trilogy and Exeter V40 Trident and the cementless Corail Pinnacle and Taperloc Exceed were small, and a cautious interpretation is required, given the limitations of the available information. However, it is unlikely that cemented brands are among the most cost effective. Similar patterns of results were observed for men and other ages. The gain in quality of life after total hip arthroplasty, rather than the risk of revision, was the main driver of cost effectiveness. Cite this article: Bone Joint J 2015;97-B:762-70.

Thirteen UDPglucuronosyltransferase genes are encoded at the human UGT1 gene complex locus.

The original novel UGT1 complex locus previously shown to encode six different UDP-glucuronosyltransferase (transferase) genes has been extended and demonstrated to specify a total of 13 isoforms. The genes are designated UGT1A1 through UGT1A13p with four pseudo ones. UGT1A2p and UGT1A11p through UGT1A13p have either nucleotide deletions or flawed TATA boxes and are therefore pseudo. In the 5' region of the locus, the 13 unique exons 1 are arranged in a tandem array with each having its own proximal TATA box element and, in turn, are linked to four common exons to allow for the independent transcriptional initiation to generate overlapping primary transcripts. Only the lead exon in the nine viable primary transcripts is predicted to undergo splicing to the four common exons generating mRNAs with identical 3' ends and transferase isozymes with an identical carboxyl terminus. The unique amino terminus specifies acceptor-substrate selection, and the common carboxyl terminus apparently specifies the interaction with the common donor substrate, UDP-glucuronic acid. In the extended region, the viable TATA boxes are either A(A)TgA(AA)T or AT14AT; in the original locus the element for UGT1A1 is A(TA)7A and TAATT/CAA(A) for all of the other genes. UGT1A1 specifies the critically important bilirubin transferase isoform. The relationships of the exons 1 to each other are as follows: UGT1A2p through UGT1A5 comprises a cluster A that is 87-92% identical, and UGT1A7 through UGT1A13p comprises a cluster B that is 67-91% identical. For the two not included in a cluster, UGT1A1 is more identical to cluster A at 60-63%, whereas UGT1A6 is identical by between 48% and 56% to all other unique exons. The locus was expanded from 95 kb to 218 kb. Extensive probing of clones beyond 218 kb with coding nucleotides for a highly conserved amino acid sequence present in all transferases was unable to detect other exons 1. The mRNAs are differentially expressed in hepatic and extrahepatic tissues. This locus is indeed novel, indicating the least usage of exon sequences in specifying different transferase isozymes that have an expansive substrate range.

Synthesis and characterization of a disulfide bond isomer of omega-conotoxin GVIA.

Solid phase peptide synthesis and air oxidation of omega-conotoxin GVIA yielded, in addition to the desired product, an isomeric peptide which could be completely separated from the native toxin by repeated HPLC. A chymotrypsin-trypsin digest of this peptide, when subjected to HPLC peptide mapping, provided peptides identical with synthetic disulfide containing peptides predicted for the omega-conotoxin isomer containing C1-C2, C3-C5, C4-C6 cystinyl pairings. The 'shaking' potency (ED50 = 1500 pmoles/kg, i.c.v.) of the isomeric peptide upon cannulated rats was 1.3% of the potency of native conotoxin (ED50 = 20 pmoles/kg). Considering that all three disulfide pairings in the isomer are different from the native toxin, its retention of biological activity is of interest.

Evaluation of TiCl4-mediated reduction of methionine sulfoxide in peptides with oxidizable or reducible residues.

Reduction of methionine sulfoxide with TiCl4/NaI is very rapid for simple methionine-containing peptides. The utility of this oxido/reduction system has been evaluated for three model peptides that contain oxidation/reduction-sensitive components such as a disulfide bond and/or a tryptophan residue. Completely specific reduction of methionine sulfoxide without some reduction of the disulfide bond was not possible with TiCl4/NaI. Reduction of the methionine sulfoxide residue in these model peptides yielded the desired product as the major component (yield ca. 70%) when a reaction time of four minutes was used. Methionine sulfoxide appears to be the most readily reducible species by low valent titanium. The competing side reactions observed were disulfide bond reduction by low valent titanium and/or tryptophan oxidation by the I2 generated by reduction of the TiCl4 with NaI. These side reactions became a serious problem when longer reaction times were used. The levels of contaminants generated by these side reactions were observed to increase with time, reducing the yield of the desired product.

Synthesis of a fluorogenic interleukin-1 beta converting enzyme substrate based on resonance energy transfer.

Interleukin 1 beta converting enzyme (ICE) is responsible for processing an inactive 31-kDa precursor to the active, mature 17-kDa Il-1 beta with cleavage occurring between the Asp116-Ala117 amide bond. We have prepared a peptide substrate that contains the protease cleavage site situated between two fluorophores located at the termini of the molecule. Upon cleavage of DABCYL-Tyr-Val-Ala-Asp-Ala-Pro-Val-EDANS (DABCYL-ICE-EDANS), an increase in fluorescence is observed at the EDANS emission wavelength of 490 nm, permitting a continuous assay of ICE that is useful in the screening of inhibitory compounds. The Km and kcat results for hydrolysis of DABCYL-ICE-EDANS by ICE were 11.4 +/- 1.6 microM and 0.79 +/- 0.4 s-1. The second order rate constant for hydrolysis of this substrate (kcat/Km = 7.0 +/- 1.3 x 10(4) M-1 s-1) is comparable to that for the cleavage of the previously described fluorogenic substrate, Ac-Tyr-Val-Ala-Asp-AMC (6.4 x 10(4) M-1 s-1).

Ionisation behaviour and solution properties of the potassium-channel blocker ShK toxin.

The effects of pH, temperature and polypeptide concentration on the solution structure and side chain interactions of ShK toxin, a potassium-channel-blocking polypeptide from the sea anemone Stichodactyla helianthus, have been investigated by means of one-dimensional and two-dimensional 1H-NMR spectroscopy. Resonance assignments have been obtained for most protons in the molecule, and for the alpha and beta carbon atoms. The lack of concentration dependence of the 1H chemical shifts and linewidths indicates that self-association is not significant and cannot account for the sheet-like structure near the N terminus. The structure is stable to high temperature, showing little change even at 353 K. This stability allowed backbone-amide temperature coefficients to be interpreted, and the correlation of these values with hydrogen bonds observed in the structures and with solvent exchange rates is discussed. pKa values have been measured for Asp5, His19 and Tyr23, and the contributions to these pKa values from other residues investigated using the analogues R11Q (denoting substitution of Argll with Gln), R11E, H19K, K22A, Y23A and K30A. These results show that Asp5 (pKa 2.8) makes an electrostatic interaction with Lys30, which may be partially responsible for the importance of these side chains in the folding of synthetic toxin. The phenolic pKa of Tyr23 is reduced to 8.7 in the native toxin, as a result of interactions with the positively charged side chains of Arg11 and to a lesser extent Lys22. Several hydrogen bonds between the Arg11 guanidino group and the Tyr23 phenolic group are found in the solution structures. As these three residues are implicated in the tight binding of ShK toxin to the T-lymphocyte voltage-gated potassium channel Kv1.3, their close interactions should be taken into account in models of binding of this toxin to the pore and vestibule of this and other potassium channels.

Synthesis and biological activity of six monosubstituted analogs of a sea anemone polypeptide neurotoxin.

Monosubstituted analogs of Stichodactyla helianthus neurotoxin I (ShI), a sea anemone polypeptide, were synthesized by solid-phase methods. The analogs were selected to probe the importance of charged residues in the amino-terminal region; ionizable side chains were replaced with a corresponding neutral isosteric amino acid side chain. Following oxidation of the three disulfide bonds and refolding, the analogs were purified by gel, ion-exchange and reversed-phase chromatography. Circular dichroism and fluorescence spectral analyses indicate that the analogs have folded structures very similar to the native toxin. Crustacean paralytic bioassays and neuronal receptor binding assays of ShI analogs show that the anionic triad Asp 6-Asp 7-Glu 8 is essential for activity, as replacement of any of these side chains with the corresponding amide reduces toxicity and binding at least 1000-fold. Substitution at Lys 4 or Asp 11 reduces biological activity at least 10-fold; these residues are apparently not as critical as the polyanionic region. Replacing the N-terminal Ala residue with a Tyr residue has only a slight effect upon crab toxicity and axolemma binding. Our results suggest that several amino acid side chains near the N-terminus of this sea anemone neurotoxin are important for binding to crab neuronal sodium channels.