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Biegstraaten, Marieke ; Arngrímsson, Reynir ; Barbey, Frederic ; Boks, Lut ; Cecchi, Franco ; Deegan, Patrick B ; Feldt-Rasmussen, Ulla ; Geberhiwot, Tarekegn ; Germain, Dominique P ; Hendriksz, Chris ; Hughes, Derralynn A ; Kantola, Ilkka ; Karabul, Nesrin ; Lavery, Christine ; Linthorst, Gabor E ; Mehta, Atul ; van de Mheen, Erica ; Oliveira, João P ; Parini, Rossella ; Ramaswami, Uma ; Rudnicki, Michael ; Serra, Andreas ; Sommer, Claudia ; Sunder-Plassmann, Gere ; Svarstad, Einar ; Sweeb, Annelies ; Terryn, Wim ; Tylki-Szymanska, Anna ; Tøndel, Camilla ; Vujkovac, Bojan ; Weidemann, Frank ; Wijburg, Frits A ; Woolfson, Peter ; Hollak, Carla EM
Introduction:
Fabry disease (FD) is a lysosomal storage disorder resulting in progressive nervous system, kidney and heart disease. Enzyme replacement therapy (ERT) may halt or attenuate disease progression. Since administration is burdensome and expensive, appropriate use is mandatory. We aimed to define European consensus recommendations for the initiation and cessation of ERT in patients with FD.
Methods:
A Delphi procedure was conducted with an online survey (n = 28) and a meeting (n = 15). Patient organization representatives were present at the meeting to give their views. Recommendations were accepted with ≥75% agreement and no disagreement.
Results:
For classically affected males, consensus was achieved that ERT is recommended as soon as there are early clinical signs of kidney, heart or brain involvement, but may be considered in patients of ≥16 years in the absence of clinical signs or symptoms of organ involvement. Classically affected females and males with non-classical FD should be treated as soon as there are early clinical signs of kidney, heart or brain involvement, while treatment may be considered in females with non-classical FD with early clinical signs that are considered to be due to FD. Consensus was achieved that treatment should not be withheld from patients with severe renal insufficiency (GFR < 45 ml/min/1.73 m\(^{2}\)) and from those on dialysis or with cognitive decline, but carefully considered on an individual basis. Stopping ERT may be considered in patients with end stage FD or other co-morbidities, leading to a life expectancy of <1 year. In those with cognitive decline of any cause, or lack of response for 1 year when the sole indication for ERT is neuropathic pain, stopping ERT may be considered. Also, in patients with end stage renal disease, without an option for renal transplantation, in combination with advanced heart failure (NYHA class IV), cessation of ERT should be considered. ERT in patients who are non-compliant or fail to attend regularly at visits should be stopped.
Conclusion:
The recommendations can be used as a benchmark for initiation and cessation of ERT, although final decisions should be made on an individual basis. Future collaborative efforts are needed for optimization of these recommendations.
Berg, Stefan ; Schelling, Esther ; Hailu, Elena ; Firdessa, Rebuma ; Gumi, Balako ; Erenso, Girume ; Gadisa, Endalamaw ; Mengistu, Araya ; Habtamu, Meseret ; Hussein, Jemal ; Kiros, Teklu ; Bekele, Shiferaw ; Mekonnen, Wondale ; Derese, Yohannes ; Zinsstag, Jakob ; Ameni, Gobena ; Gagneux, Sebastien ; Robertson, Brian D ; Tschopp, Rea ; Hewinson, Glyn ; Yamuah, Lawrence ; Gordon, Stephen V ; Aseffa, Abraham
Background:
Ethiopia, a high tuberculosis (TB) burden country, reports one of the highest incidence rates of extra-pulmonary TB dominated by cervical lymphadenitis (TBLN). Infection with Mycobacterium bovis has previously been excluded as the main reason for the high rate of extra-pulmonary TB in Ethiopia.
Methods:
Here we examined demographic and clinical characteristics of 953 pulmonary (PTB) and 1198 TBLN patients visiting 11 health facilities in distinct geographic areas of Ethiopia. Clinical characteristics were also correlated with genotypes of the causative agent, Mycobacterium tuberculosis.
Results:
No major patient or bacterial strain factor could be identified as being responsible for the high rate of TBLN, and there was no association with HIV infection. However, analysis of the demographic data of involved patients showed that having regular and direct contact with live animals was more associated with TBLN than with PTB, although no M. bovis was isolated from patients with TBLN. Among PTB patients, those infected with Lineage 4 reported "contact with other TB patient" more often than patients infected with Lineage 3 did (OR = 1.6, CI 95% 1.0-2.7; p = 0.064). High fever, in contrast to low and moderate fever, was significantly associated with Lineage 4 (OR = 2.3; p = 0.024). On the other hand, TBLN cases infected with Lineage 4 tended to get milder symptoms overall for the constitutional symptoms than those infected with Lineage 3.
Conclusions:
The study suggests a complex role for multiple interacting factors in the epidemiology of extra-pulmonary TB in Ethiopia, including factors that can only be derived from population-based studies, which may prove to be significant for TB control in Ethiopia.
Language and literary studies have studied style for centuries, and even since the advent of ›stylistics‹ as a discipline at the beginning of the twentieth century, definitions of ›style‹ have varied heavily across time, space and fields. Today, with increasingly large collections of literary texts being made available in digital form, computational approaches to literary style are proliferating. New methods from disciplines such as corpus linguistics and computer science are being adopted and adapted in interrelated fields such as computational stylistics and corpus stylistics, and are facilitating new approaches to literary style.
The relation between definitions of style in established linguistic or literary stylistics, and definitions of style in computational or corpus stylistics has not, however, been systematically assessed. This contribution aims to respond to the need to redefine style in the light of this new situation and to establish a clearer perception of both the overlap and the boundaries between ›mainstream‹ and ›computational‹ and/or ›empirical‹ literary stylistics. While stylistic studies of non-literary texts are currently flourishing, our contribution deliberately centers on those approaches relevant to ›literary stylistics‹. It concludes by proposing an operational definition of style that we hope can act as a common ground for diverse approaches to literary style, fostering transdisciplinary research.
The focus of this contribution is on literary style in linguistics and literary studies (rather than in art history, musicology or fashion), on textual aspects of style (rather than production- or reception-oriented theories of style), and on a descriptive perspective (rather than a prescriptive or didactic one). Even within these limits, however, it appears necessary to build on a broad understanding of the various perspectives on style that have been adopted at different times and in different traditions. For this reason, the contribution first traces the development of the notion of style in three different traditions, those of German, Dutch and French language and literary studies. Despite the numerous links between each other, and between each of them to the British and American traditions, these three traditions each have their proper dynamics, especially with regard to the convergence and/or confrontation between mainstream and computational stylistics. For reasons of space and coherence, the contribution is limited to theoretical developments occurring since 1945.
The contribution begins by briefly outlining the range of definitions of style that can be encountered across traditions today: style as revealing a higher-order aesthetic value, as the holistic ›gestalt‹ of single texts, as an expression of the individuality of an author, as an artifact presupposing choice among alternatives, as a deviation from a norm or reference, or as any formal property of a text. The contribution then traces the development of definitions of style in each of the three traditions mentioned, with the aim of giving a concise account of how, in each tradition, definitions of style have evolved over time, with special regard to the way such definitions relate to empirical, quantitative or otherwise computational approaches to style in literary texts. It will become apparent how, in each of the three traditions, foundational texts continue to influence current discussions on literary style, but also how stylistics has continuously reacted to broader developments in cultural and literary theory, and how empirical, quantitative or computational approaches have long existed, usually in parallel to or at the margins of mainstream stylistics. The review will also reflect the lines of discussion around style as a property of literary texts – or of any textual entity in general.
The perspective on three stylistic traditions is accompanied by a more systematic perspective. The rationale is to work towards a common ground for literary scholars and linguists when talking about (literary) style, across traditions of stylistics, with respect for established definitions of style, but also in light of the digital paradigm. Here, we first show to what extent, at similar or different moments in time, the three traditions have developed comparable positions on style, and which definitions out of the range of possible definitions have been proposed or promoted by which authors in each of the three traditions.
On the basis of this synthesis, we then conclude by proposing an operational definition of style that is an attempt to provide a common ground for both mainstream and computational literary stylistics. This definition is discussed in some detail in order to explain not only what is meant by each term in the definition, but also how it relates to computational analyses of style – and how this definition aims to avoid some of the pitfalls that can be perceived in earlier definitions of style. Our definition, we hope, will be put to use by a new generation of computational, quantitative, and empirical studies of style in literary texts.
Benz, Roland ; Jones, Michael D. ; Younas, Farhan ; Maier, Elke ; Modi, Niraj ; Mentele, Reinhard ; Lottspeich, Friedrich ; Kleinekathöfer, Ulrich ; Smit, John
Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two known structures with the model of OmpW of C. crescentus suggested that it has a more hydrophilic interior and possibly a larger diameter.
Treatment of recalcitrant cutaneous lupus erythematosus (CLE) is challenging. In situations where conventional treatment approaches fail mepacrine - an antimalarial/antiinfiammatory drug that has fallen into oblivion in the last decades might still be a promising option. We retrospectively analysed medical records of 10 patients with refractory CLE that were treated with mepacrine (100-200 mg/day) as mono- or combination therapy for various time intervals between 2001 and 2013 at the University Hospital Wurzburg. Mepacrine was generally well tolerated. Side effects were mild and usually resolved after reduction or cessation. Over 50% of the patients experienced amelioration of their symptoms despite a previously recalcitrant clinical course. Altogether, our data demonstrate that mepacrine still remains a useful and effective therapeutic option for otherwise treatment-resistant CLE.
Kinetic assessment by in vitro approaches - A contribution to reduce animals in toxicity testing
(2015)
The adoption of directives and regulations by the EU requires the development of alternative testing strategies as opposed to animal testing for risk assessment of xenobiotics. Additionally, high attrition rates of drugs late in the discovery phase demand improvement of current test batteries applied in the preclinical phase within the pharmaceutical area. These issues were taken up by the EU founded 7th Framework Program “Predict-IV”; with the overall goal to improve the predictability of safety of an investigational product, after repeated exposure, by integration of “omics” technologies applied on well established in vitro approaches. Three major target organs for drug-induced toxicity were in focus: liver, kidney and central nervous system. To relate obtained dynamic data with the in vivo situation, kinetics of the test compounds have to be evaluated and extrapolated by physiologically based pharmacokinetic modeling.
This thesis assessed in vitro kinetics of the selected test compounds (cyclosporine A, adefovir dipivoxil and cisplatinum) regarding their reliability and relevance to respective in vivo pharmacokinetics. Cells were exposed daily or every other day to the test compounds at two concentration levels (toxic and non-toxic) for up to 14 days. Concentrations of the test compounds or their major biotransformation products were determined by LC-MS/MS or ICP-MS in vehicle, media, cells and plastic adsorption samples generated at five different time-points on the first and the last treatment day.
Cyclosporine A bioaccumulation was evident in primary rat hepatocytes (PRH) at the high concentration, while efficient biotransformation mediated by CYP3A4 and CYP3A5 was determined in primary human hepatocytes (PHH) and HepaRG cells. The lower biotransformation in PRH is in accordance with observation made in vivo with the rat being a poor model for CYP3A biotransformation. Further, inter-assay variability was noticed in PHH caused by biological variability in CYP3A4 and CYP3A5 activity in human donors. The inter-assay variability observed for PRH and HepaRG cells was a result of differences between vehicles regarding their cyclosporine A content. Cyclosporine A biotransformation was more prominent in HepaRG cells due to stable and high CYP3A4 and CYP3A5 activity. In addition, in vitro clearances were calculated and scaled to in vivo. All scaled in vitro clearances were overestimated (PRH: 10-fold, PHH: 2-fold, HepaRG cells: 2-fold). These results should be proven by physiologically-based pharmacokinetic modeling and additional experiments, in order to verify that these overestimations are constant for each system and subsequently can be diminished by implementation of further scaling factors.
Brain cell cultures, primary neuronal culture of mouse cortex cells and primary aggregating rat brain cells, revealed fast achieved steady state levels of cyclosporine A. This indicates a chemical distribution of cyclosporine A between the aqueous and organic phases and only minor involvement of biological processes such as active transport and biotransformation. Hence, cyclosporine A uptake into cells is presumably transport mediated, supported by findings of transporter experiments performed on a parallel artificial membrane and Caco-2 cells. Plastic adsorption of cyclosporine A was significant, but different for each model, and should be considered by physiologically based pharmacokinetic modeling.
Kinetics of adefovir dipivoxil highlights the limits of in vitro approaches. Active transporters are required for adefovir uptake, but were not functional in RPTECT/TERT1. Therefore, adefovir uptake was limited to passive diffusion of adefovir dipivoxil, which itself degrades time-dependently under culture conditions.
Cisplatinum kinetics, studied in RPTEC/TERT1 cells, indicated intracellular enrichment of platinum, while significant bioaccumulation was not noted. This could be due to cisplatinum not reaching steady state levels within 14 days repeated exposure. As shown in vivo, active transport occurred from the basolateral to apical side, but with lower velocity. Hence, obtained data need to be modeled to estimate cellular processes, which can be scaled and compared to in vivo.
Repeated daily exposure to two different drug concentrations makes it possible to account for bioaccumulation at toxic concentrations or biotransformation/extrusion at non-toxic concentrations. Potential errors leading to misinterpretation of data were reduced by analyses of the vehicles as the applied drug concentrations do not necessarily correspond to the nominal concentrations. Finally, analyses of separate compartments (medium, cells, plastic) give insights into a compound’s distribution, reduce misprediction of cellular processes, e.g. biotransformation, and help to interpret kinetic data. On the other hand, the limits of in vitro approaches have also been pointed out. For correct extrapolation to in vivo, it is essential that the studied in vitro system exhibits the functionality of proteins, which play a key role in the specific drug induced toxicity. Considering the benefits and limitations, it is worth to validate this long-term treatment experimental set-up and expand it on co-culture systems and on organs-on-chips with regard to alternative toxicity testing strategies for repeated dose toxicity studies.
Aspergillus fumigatus is the most common cause for invasive fungal infections, a disease associated with high mortality in immune-compromised patients. CD1d-restricted invariant Natural Killer T (iNKT) cells compose a small subset of T cells known to impact the immune response towards various infectious pathogens. To investigate the role of human iNKT cells during A. fumigatus infection, we studied their activation as determined by CD69 expression and cytokine production in response to distinct fungal morphotypes in the presence of different CD1d⁺ antigen presenting cells using flow cytometry and multiplex ELISA. Among CD1d⁺ subpopulations, CD1d⁺CD1c⁺ mDCs showed the highest potential to activate iNKT cells on a per cell basis. The presence of A. fumigatus decreased this effect of CD1d⁺CD1c⁺ mDCs on iNKT cells and led to reduced secretion of TNF-α, G-CSF and RANTES. Production of other Th1 and Th2 cytokines was not affected by the fungus, suggesting an immune-modulating function for human iNKT cells during A. fumigatus infection.
Beiss, Veronique ; Spiegel, Holger ; Boes, Alexander ; Scheuermayer, Matthias ; Reimann, Andreas ; Schillberg, Stefan ; Fischer, Rainer
Background: Despite the limited success after decades of intensive research and development efforts, vaccination still represents the most promising strategy to significantly reduce the disease burden in malaria endemic regions. Besides the ultimate goal of inducing sterile protection in vaccinated individuals, the prevention of transmission by so-called transmission blocking vaccines (TBVs) is being regarded as an important feature of an efficient malaria eradication strategy. Recently, Plasmodium falciparum GAP50 (PfGAP50), a 44.6 kDa transmembrane protein that forms an essential part of the invasion machinery (glideosome) multi-protein complex, has been proposed as novel potential transmission-blocking candidate. Plant-based expression systems combine the advantages of eukaryotic expression with a up-scaling potential and a good product safety profile suitable for vaccine production. In this study we investigated the feasibility to use the transient plant expression to produce PfGAP50 suitable for the induction of parasite specific inhibitory antibodies.
Results: We performed the transient expression of recombinant PfGAP50 in Nicotiana benthamiana leaves using endoplasmatic reticulum (ER) and plastid targeting. After IMAC-purification the protein yield and integrity was investigated by SDS-PAGE and Western Blot. Rabbit immune IgG derived by the immunization with the plastidtargeted variant of PfGAP50 was analyzed by immune fluorescence assay (IFA) and zygote inhibition assay (ZIA). PfGAP50 could be produced in both subcellular compartments at different yields IMAC (Immobilized Metal Affinity Chromatography) purification from extract yielded up to 4.1 mu g/g recombinant protein per fresh leaf material for ER-retarded and 16.2 mu g/g recombinant protein per fresh leave material for plasmid targeted PfGAP50, respectively. IgG from rabbit sera generated by immunization with the recombinant protein specifically recognized different parasite stages in immunofluorescence assay. Furthermore up to 55 % inhibition in an in vitro zygote inhibition assay could be achieved using PfGAP50-specific rabbit immune IgG.
Conclusions: The results of this study demonstrate that the plant-produced PfGAP50 is functional regarding the presentation of inhibitory epitopes and could be considered as component of a transmission-blocking malaria vaccine formulation.
Becker, Philip P. ; Rau, Monika ; Schmitt, Johannes ; Malsch, Carolin ; Hammer, Christian ; Bantel, Heike ; Müllhaupt, Beat ; Geier, Andreas
Objectives
Liver biopsies are the current gold standard in non-alcoholic steatohepatitis (NASH) diagnosis. Their invasive nature, however, still carries an increased risk for patients' health. The development of non-invasive diagnostic tools to differentiate between bland steatosis (NAFL) and NASH remains crucial. The aim of this study is the evaluation of investigated circulating microRNAs in combination with new targets in order to optimize the discrimination of NASH patients by non-invasive serum biomarkers.
Methods
Serum profiles of four microRNAs were evaluated in two cohorts consisting of 137 NAFLD patients and 61 healthy controls. In a binary logistic regression model microRNAs of relevance were detected. Correlation of microRNA appearance with known biomarkers like ALT and CK18-Asp396 was evaluated. A simplified scoring model was developed, combining the levels of microRNA in circulation and CK18-Asp396 fragments. Receiver operating characteristics were used to evaluate the potential of discriminating NASH.
Results
The new finding of our study is the different profile of circulating miR-21 in NASH patients (p<0.0001). Also, it validates recently published results of miR-122 and miR-192 to be differentially regulated in NAFL and NASH. Combined microRNA expression profiles with CK18-Asp396 fragment level scoring model had a higher potential of NASH prediction compared to other risk biomarkers (AUROC = 0.83, 95% CI = 0.754-0.908; p<0.001). Evaluation of score model for NAFL (Score = 0) and NASH (Score = 4) had shown high rates of sensitivity (91%) and specificity (83%).
Conclusions
Our study defines candidates for a combined model of miRNAs and CK18-Asp396 levels relevant as a promising expansion for diagnosis and in turn treatment of NASH.
The aim of the present work is the development and implementation of new simulation
possibilities for the CAST program package. Development included, among other
things, the partial parallelization of the already existing force fields, extension of the
treatment of electrostatic interactions and implementation of molecular dynamics and
free energy algorithms.
The most time consuming part of force field calculations is the evaluation of the nonbonded
interactions. The calculation of these interactions has been parallelized and
it could be shown to yield a significant speed up for multi-core calculations compared
to the serial execution on only one CPU. For both, simple energy/gradient as well as
molecular dynamics simulations the computational time could be significantly reduced.
To further increase the performance of calculations employing a cutoff radius, a linkedcell
algorithm was implemented which is able to build up the non-bonded interaction
list up to 7 times faster than the original algorithm.
To provide access to dynamic properties based on the natural time evolution of a system,
a molecular dynamics code has been implemented. The MD implementation features
two integration schemes for the equations of motion which are able to generate stable
trajectories. The basic MD algorithm as described in Section 1.2 leads to the sampling
in the microcanonical (NVE) ensemble. The practical use of NVE simulations is limited
though because it does not correspond to any experimentally realistic situation.
More realistic simulation conditions are found in the isothermal (NVT) and isothermalisobaric
(NPT) ensembles. To generate those ensembles, temperature and pressure
control has been implemented. The temperature can be controlled in two ways: by direct
velocity scaling and by a Nose-Hoover thermostat which produces a real canonical
ensemble. The pressure coupling is realized by implementation of a Berendsen barostat.
The pressure coupling can be used for isotropic or anisotropic box dimensions with the
restriction that the angles of the box need to be 90. A crucial simulation parameter in
MD simulations is the length of the timestep. The timestep is usually in the rang of 1fs.
Increasing the timestep beyond 1fs can lead to unstable trajectories since the fastest
motion in the system, usually the H-X stretch vibration can not be sampled anymore.
A way to allow for bigger timesteps is the use of a constraint algorithm which constrains the H-X bonds to the equilibrium distance. For this the RATTLE algorithm has been
implemented in the CAST program. The velocity Verlet algorithm in combination with
the RATTLE algorithm has been shown to yield stable trajectories for an arbitrary
length of simulation time. In a first application the MD implementation is used in conjunction
with the MOPAC interface for the investigation of PBI sidechains and their
rigidity. The theoretical investigations show a nice agreement with experimentally obtained
results. Based on the MD techniques two algorithms for the determination of free
energy differences have been implemented. The umbrella sampling algorithm can be
used to determine the free energy change along a reaction coordinate based on distances
or dihedral angles. The implementation was tested on the stretching of a deca-L-alanine
and the rotation barrier of butane in vacuum. The results are in nearly perfect agreement
with literature values. For the FEP implementation calculations were performed
for a zero-sum transformation of ethane in explicit solvent, the charging of a sodium
ion in explicit solvent and the transformations of a tripeptide in explicit solvent. All
results are in agreement with benchmark calculations of the NAMD program as well
as literature values. The FEP formalism was then applied to determine the relative
binding free energies between two inhibitors in an inhibitor-protein complex.
Next to force fields, ab-initio methods can be used for simulations and global optimizations.
Since the performance of such methods is usually significantly poorer than force
field applications, the use for global optimizations is limited. Nevertheless significant
progress has been made by porting these codes to GPUs. In order to make use of these
developments a MPI interface has been implemented into CAST for communication
with the DFT code TeraChem. The CAST/TeraChem combination has been tested
on the $H_2 O_{10}$ cluster as well as the polypeptide met-Enkephalin. The pure ab-initio
calculations showed a superior behavior compared to the standard procedure where the
force field results are usually refined using quantum chemical methods.