The PINE study: rationale and design of a randomised comparison of epidural injection of local anaesthetics and steroids versus care-as-usual to prevent postherpetic neuralgia in the elderly [ISRCTN32866390]

BACKGROUND: Postherpetic neuralgia (PHN) is by far the most common complication of herpes zoster (HZ) and one of the most intractable pain disorders. Since PHN is seen most often in the elderly, the number of patients with this disorder is expected to increase in our ageing society. PHN may last for months to years and has a high impact on the quality of life. The results of PHN treatment are rather disappointing. Epidural injection of local anaesthetics and steroids in the acute phase of HZ is a promising therapy for the prevention of PHN. Since randomised trials on the effectiveness of this intervention are lacking, the PINE (Prevention by epidural Injection of postherpetic Neuralgia in the Elderly) study was set up. The PINE study compares the effectiveness and cost-effectiveness of a single epidural injection of local anaesthetics and steroids during the acute phase of HZ with that of care-as-usual (i.e. antivirals and analgesics) in preventing PHN in elderly patients. METHODS / DESIGN: The PINE study is an open, multicenter clinical trial in which 550 elderly (age >/= 50 yr.) patients who consult their general practitioner in the acute phase of HZ (rash < 7 days) are randomised to one of the treatment groups. The primary clinical endpoint is the presence of HZ-related pain one month after the onset of the rash. Secondary endpoints include duration and severity of pain, re-interventions aiming to treat the existing pain, side effects, quality of life, and cost-effectiveness. CONCLUSION: The PINE study is aimed to quantify the (cost-) effectiveness of a single epidural injection during the acute phase of HZ on the prevention of PHN.     

In vitro selection of enzymatically active lipase variants from phage libraries using a mechanism-based inhibitor

The 'detergent lipase' Lipolase, from Thermomyces lanuginosa was subjected to a combinatorial protein engineering/phage display approach with the aim of identifying new enzyme variants with improved characteristics in the presence of detergents. First it was demonstrated that wild-type Lipolase could be produced in Escherichia coli retaining full activity and be displayed as an active enzyme fused to coat protein 3 on E. coli phage M13. A phagemid library designed to result in approximately two to three mutations per lipase gene was then constructed. Nine amino acids located in two regions close to the active site were targeted for randomization. Selections using a mechanism-based biotinylated inhibitor showed that phages displaying Lipolase could be specifically enriched from a population of control phages. Selections on a library phage stock in the presence of inhibitor and a commercial powder detergent resulted in a step-wise increase in the proportion of active clones. Analysis of 84 active clones revealed that they all expressed lipase activity, but with lower activities than that of a wild-type Lipolase-producing clone. In six of the seven most active clones a wild-type serine at position 83 had been replaced by threonine, a substitution known to alter the substrate chain length preference of Lipolase variants. Furthermore, the selection had enriched enzyme variants with a high degree of conservatism in one of the variegated regions, suggesting that this region is important for enzymatic activity and that the designed selection procedure was relevant. The selected variants contained primarily basic amino acid residues within the other variegated region. Taken together, the described results show that selection protocols based on enzymatic activity can be designed for this enzyme class which should be of importance for future protein engineering attempts.     

Spectral correspondence between visual spectral sensitivity and bioluminescence emission spectra in the click beetle Pyrophorus punctatissimus (Coleoptera: Elateridae)

The presence of two spectral mechanisms, near-ultraviolet and green (lambda(max)=545nm), is strongly suggested by electroretinographic visual spectral sensitivity curves obtained under dark and red chromatic adaptation conditions in the compound eyes of the click beetle Pyrophorus punctatissimus. The bioluminescence emission of the dorsal prothoracic lanterns is deep green (lambda(max)=543nm) and that of the ventral abdominal lantern is lime green (lambda(max)=556nm) in colour in P. punctatissimus. A broad green visual receptor would detect both deep green and lime green bioluminescent optical signals.     

Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein

An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product. The process has been used for production in E. coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer. The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm. A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium. This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein. Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues. The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography. The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56%. The suitability of this process also for the production of other recombinant proteins is discussed.     

Dual labeling of a binding protein allows for specific fluorescence detection of native protein

Up-Converting Phosphor Technology (UPT) is based on lanthanide-containing, submicrometer-sized, ceramic particles that can absorb infrared light and emit visible light. Biological matrices do not up-convert; hence, there is no contribution to test background from sample autofluorescence. Up-converting phosphors do not photobleach and are inert to common assay interferants such as hemoglobin. A reader called UPlink has been developed to interrogate lateral flow test strips that utilize UPT labels. The reader contains a miniaturized, 1-W, infrared laser with peak emission at 980 nm. Preliminary assays that use up-converting phosphor labels, including tests for a drugs of abuse panel and Escherichia coli O157:H7, have been developed. In a "sandwich" assay format, 10(3) org/mL E. coli O157:H7 organisms were detectable in a negative control background of 10(9) other organisms per milliliter of culture medium. Coefficients of variation in concentrations tested from 0 to 10(7) org/mL were all < or =10%. In a competitive inhibition assay format, a multiplexed test simultaneously detected amphetamine, methamphetamine, phencyclidine, and opiates in saliva. For all assays, the percent displacement at 10 ng/mL was > or =40% demonstrating performance comparable with lab-based, commercially available EIAs. All assays were complete in 10 min. The development of rapid tests using UPT creates new applications for on-site testing with sensitivity not available using other label technologies.     

Monitored whole gene in vitro evolution of an anti-hRaf-1 affibody molecule towards increased binding affinity

The use of library technologies for the generation of affinity proteins often includes an affinity maturation step, based on the construction of secondary libraries from which second generation variants with improved affinities are selected. Here, we describe for the first time the affinity maturation of affibody molecules based on step-wise in vitro molecular evolution, involving cycles of error-prone PCR (epPCR) amplification for the introduction of diversity over the entire 58-residue three-helix bundle structure and ribosome display (RD) for the selection of improved variants. The model affibody molecule for the process was Z(RAF322), binding with a 1.9μm equilibrium dissociation constant (K(D)) to human Raf-1 (hRaf-1), a protein kinase of central importance in the MAPK/ERK proliferation pathway. The molecular evolution process was followed on both gene and protein levels via DNA sequencing and a biosensor-based binding analysis of pools of selected variants. After two cycles of diversification and selection, a significant increase in binding response of selected pools was seen. DNA sequencing showed that a dominant alanine to valine substitution had been effectively enriched, and was found in 83% of all selected clones, either alone or in combination with other enriched substitutions. The evolution procedure resulted in variants showing up to 26-fold increases in affinity to the hRaf-1 target. Noteworthy, for the two variants showing the highest affinities, substitutions were also found in affibody framework positions, corresponding to regions of the protein domain not addressed by traditional affibody molecule affinity maturation strategies. Interestingly, thermal melting point (T(m)) analyses showed that an increased affinity could be associated with both higher and lower T(m) values. All investigated variants showed excellent refolding properties and selective binding to hRaf-1, as analysed using a multiplexed bead-based binding assay, making them potentially valuable affinity reagents for cell biology studies.     

Neurologic Manifestations Associated with an Outbreak of Typhoid Fever, Malawi - Mozambique, 2009: An Epidemiologic Investigation

Background

The bacterium Salmonella enterica serovar Typhi causes typhoid fever, which is typically associated with fever and abdominal pain. An outbreak of typhoid fever in Malawi-Mozambique in 2009 was notable for a high proportion of neurologic illness.

Objective

Describe neurologic features complicating typhoid fever during an outbreak in Malawi-Mozambique

Methods

Persons meeting a clinical case definition were identified through surveillance, with laboratory confirmation of typhoid by antibody testing or blood/stool culture. We gathered demographic and clinical information, examined patients, and evaluated a subset of patients 11 months after onset. A sample of persons with and without neurologic signs was tested for vitamin B6 and B12 levels and urinary thiocyanate.

Results

Between March – November 2009, 303 cases of typhoid fever were identified. Forty (13%) persons had objective neurologic findings, including 14 confirmed by culture/serology; 27 (68%) were hospitalized, and 5 (13%) died. Seventeen (43%) had a constellation of upper motor neuron findings, including hyperreflexia, spasticity, or sustained ankle clonus. Other neurologic features included ataxia (22, 55%), parkinsonism (8, 20%), and tremors (4, 10%). Brain MRI of 3 (ages 5, 7, and 18 years) demonstrated cerebral atrophy but no other abnormalities. Of 13 patients re-evaluated 11 months later, 11 recovered completely, and 2 had persistent hyperreflexia and ataxia. Vitamin B6 levels were markedly low in typhoid fever patients both with and without neurologic signs.

Conclusions

Neurologic signs may complicate typhoid fever, and the diagnosis should be considered in persons with acute febrile neurologic illness in endemic areas.     

Parallel Markov chain Monte Carlo - bridging the gap to high-performance Bayesian computation in animal breeding and genetics

Background

Most Bayesian models for the analysis of complex traits are not analytically tractable and inferences are based on computationally intensive techniques. This is true of Bayesian models for genome-enabled selection, which uses whole-genome molecular data to predict the genetic merit of candidate animals for breeding purposes. In this regard, parallel computing can overcome the bottlenecks that can arise from series computing. Hence, a major goal of the present study is to bridge the gap to high-performance Bayesian computation in the context of animal breeding and genetics.

Results

Parallel Monte Carlo Markov chain algorithms and strategies are described in the context of animal breeding and genetics. Parallel Monte Carlo algorithms are introduced as a starting point including their applications to computing single-parameter and certain multiple-parameter models. Then, two basic approaches for parallel Markov chain Monte Carlo are described: one aims at parallelization within a single chain; the other is based on running multiple chains, yet some variants are discussed as well. Features and strategies of the parallel Markov chain Monte Carlo are illustrated using real data, including a large beef cattle dataset with 50K SNP genotypes.

Conclusions

Parallel Markov chain Monte Carlo algorithms are useful for computing complex Bayesian models, which does not only lead to a dramatic speedup in computing but can also be used to optimize model parameters in complex Bayesian models. Hence, we anticipate that use of parallel Markov chain Monte Carlo will have a profound impact on revolutionizing the computational tools for genomic selection programs.     

The genomic landscape of the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV

Background

Aerobic methanotrophs can grow in hostile volcanic environments and use methane as their sole source of energy. The discovery of three verrucomicrobial Methylacidiphilum strains has revealed diverse metabolic pathways used by these methanotrophs, including mechanisms through which methane is oxidized. The basis of a complete understanding of these processes and of how these bacteria evolved and are able to thrive in such extreme environments partially resides in the complete characterization of their genome and its architecture.

Results

In this study, we present the complete genome sequence of Methylacidiphilum fumariolicum SolV, obtained using Pacific Biosciences single-molecule real-time (SMRT) sequencing technology. The genome assembles to a single 2.5 Mbp chromosome with an average GC content of 41.5%. The genome contains 2,741 annotated genes and 314 functional subsystems including all key metabolic pathways that are associated with Methylacidiphilum strains, including the CBB pathway for CO₂ fixation. However, it does not encode the serine cycle and ribulose monophosphate pathways for carbon fixation. Phylogenetic analysis of the particulate methane mono-oxygenase operon separates the Methylacidiphilum strains from other verrucomicrobial methanotrophs. RNA-Seq analysis of cell cultures growing in three different conditions revealed the deregulation of two out of three pmoCAB operons. In addition, genes involved in nitrogen fixation were upregulated in cell cultures growing in nitrogen fixing conditions, indicating the presence of active nitrogenase. Characterization of the global methylation state of M. fumariolicum SolV revealed methylation of adenines and cytosines mainly in the coding regions of the genome. Methylation of adenines was predominantly associated with 5′-^m6ACN₄GT-3′ and 5′-CC^m6AN₅CTC-3′ methyltransferase recognition motifs whereas methylated cytosines were not associated with any specific motif.

Conclusions

Our findings provide novel insights into the global methylation state of verrucomicrobial methanotroph M. fumariolicum SolV. However, partial conservation of methyltransferases between M. fumariolicum SolV and M. infernorum V4 indicates potential differences in the global methylation state of Methylacidiphilum strains. Unravelling the M. fumariolicum SolV genome and its epigenetic regulation allow for robust characterization of biological processes that are involved in oxidizing methane. In turn, they offer a better understanding of the evolution, the underlying physiological and ecological properties of SolV and other Methylacidiphilum strains.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-914) contains supplementary material, which is available to authorized users.