Assessing and treating alcohol relapse risk in liver transplantation candidates.

In Europe between 30 and 50% of all liver transplantations (LTX) are done within the context of chronic end-stage alcoholic liver disease (ALD). However, post-operatively 20-25% of these patients lapse or relapse into heavy alcohol use. Thus, assessment of alcohol relapse risk before enlisting and therapeutic follow-up during and after LTX is of utmost importance. However, as yet there are enormous differences between European countries and between transplant centers, with regard to the assessment methods and criteria and the implementation of therapeutic follow-up. Only the so-called '6-month abstinence' rule is widely used. However, there are not much scientific data validating its use in predicting relapse. Thus, there is a clear need of a more homogeneous approach, which was the focus of a symposium of the European Federation of Addiction Societies during the 14th conference of the European Society for Biomedical Research on Alcoholism, 2013 (ESBRA), entitled 'Liver transplantation: A European perspective'. In a follow-up on this symposium, the authors aim to sum up the evidence of psychiatric assessment criteria and psychiatric treatment interventions relevant in the context of patient selection and patient follow-up within ALD transplantation procedures. Based upon these findings, we propose elements of a procedure that can serve as a first step toward a model of good practice regarding addiction-specialist input within the pre- and post-transplantation period.

Topological characterization of the DnaA-oriC complex using single-molecule nanomanipuation.

In most bacteria, the timing and synchrony of initiation of chromosomal replication are determined by the binding of the AAA(+) protein DnaA to a set of high- and low-affinity sites found within the origin of chromosomal replication (oriC). Despite the large amount of information on the role and regulation of DnaA, the actual structure of the DnaA-oriC complex and the mechanism by which it primes the origin for the initiation of replication remain unclear. In this study, we have performed magnetic tweezers experiments to investigate the structural properties of the DnaA-oriC complex. We show that the DnaA-ATP-oriC complex adopts a right-handed helical conformation involving a variable amount of DNA and protein whose features fit qualitatively as well as quantitatively with an existing model based on the crystal structure of a truncated DnaA tetramer obtained in the absence of DNA. We also investigate the topological effect of oriC's DNA unwinding element.

Stability of phosphatidylethanol species in spiked and authentic whole blood and matching dried blood spots.

BACKGROUND: Phosphatidylethanol (PEth) is currently under investigation as a highly sensitive and specific marker of alcohol misuse. As its stability in blood samples has not systematically been investigated, a study was performed to determine the stability of major PEth species in spiked and authentic whole blood and also in matching dried blood spots (DBS) at different conditions. METHODS: To PEth-free blood from teetotalers, low and high concentrations of two major PEth (18:1/18:1 and 16:0/18:1) species were added chosen on the basis of concentrations determined from authentic samples which were collected from the subjects undergoing alcohol detoxification treatment. Effects of sampling (EDTA or heparinized tubes), temperature, and time (≤30 days) were investigated. Processed samples (two at each condition, respectively) were subjected to LC gradient separation using multiple reaction monitoring. Stability was assessed using the critical difference or a periodic analysis result that was within 15 % of the initial concentration. Reaction kinetics of degradation was investigated with rate constants being checked for an Arrhenius relationship. RESULTS: PEth was stable in dried blood spot (DBS) stored either at room temperature or frozen, whereas it was not stable in whole blood except in samples stored at -80 °C. Activation energies increased in the following order: spiked heparinized blood < spiked EDTA blood < authentic EDTA blood. CONCLUSIONS: PEth is a labile analyte which is predominantly degraded by hydrolysis. Only at -80 °C, stability in whole blood can be ascertained, and analysis should be performed within 30 days. EDTA should be preferred over heparin as an additive. DBS is able to stabilize PEth thus partly resolving pre-analytical difficulties of PEth measurement.

Quantitative aspects of RNA silencing in metazoans.

Small regulatory RNAs (microRNAs, siRNAs, and piRNAs) exhibit several unique features that clearly distinguish them from other known gene regulators. Their genomic organization, mode of action, and proposed biological functions raise specific questions. In this review, we focus on the quantitative aspect of small regulatory RNA biology. The original nature of these small RNAs accelerated the development of novel detection techniques and improved statistical methods and promoted new concepts that may unexpectedly generalize to other gene regulators. Quantification of natural phenomena is at the core of scientific practice, and the unique challenges raised by small regulatory RNAs have prompted many creative innovations by the scientific community.

Time-dependent hyper-viscoelastic parameter identification of human articular cartilage and substitute materials.

Numerical simulations are a valuable tool to understand which processes during mechanical stimulations of hydrogels for cartilage replacement influence the behavior of chondrocytes and contribute to the success or failure of these materials as implants. Such simulations critically rely on the correct prediction of the material response through appropriate material models and corresponding parameters. In this study, we identify hyper-viscoelastic material parameters for numerical simulations in COMSOL Multiphysics® v. 5.6 for human articular cartilage and two replacement materials, the commercially available ChondroFillerliquid and oxidized alginate gelatin (ADA-GEL) hydrogels. We incorporate the realistic experimental boundary conditions into an inverse parameter identification scheme based on data from multiple loading modes simultaneously, including cyclic compression-tension and stress relaxation experiments. We provide individual parameter sets for the unconditioned and conditioned responses and discuss how viscoelastic effects are related to the materials' microstructure. ADA-GEL and ChondroFillerliquid exhibit faster stress relaxation than cartilage with lower relaxation time constants, while cartilage has the largest viscoelastic stress contribution. The elastic response predominates in ADA-GEL and ChondroFillerliquid, while the viscoelastic response predominates in cartilage. These results will help to simulate mechanical stimulations, support the development of suitable materials with distinct mechanical properties in the future and provide parameters and insight into the time-dependent material behavior of human articular cartilage.

Traumatic epiphysiolysis of the proximal femur.

PURPOSE OF THE STUDY: Several former studies show the treatment of slipped epiphysis of the femoral head (SEFH). Its reason is rather unknown. On the other hand the rare traumatic SEFH takes place due to a real accident. According to the literature these injuries are treated like chronic SEFHs. The aim of this study is to show the differences in pathology and treatment of an acute traumatic SEFH in relationship to the chronic SEFH. PATIENTS AND METHODS: In 8 patients dislocated traumatic SEFHs were reduced anatomically and stabilized by the means of 3 to 4 Kirschner- (K-) wires or two cancellous screws. Each patient got a plaster-cast fixation for about 6 weeks of the ipsilateral hip and leg and was mobilized with two crutches and partial weight bearing for 12 weeks. The implants were removed 24 weeks after surgery. Four patients with not dislocated SEFHs were immobilized or mobilized with two crutches without weight bearing according to their pain sensation. The final examination of both groups took place 2 Vz to 15 years after the initial treatment. RESULTS: Four patients primarily under 10 years of age showed no or minimal radiological signs of a dislocated femoral head and were without any further inconvenience--the suspected SEFHs revealed as hip contusions. 8 children aged 10 years or older at the time of trauma were treated by closed reduction and internal fixation. Complications occurred in three cases--one necrosis of the femoral head because of a perforating K-wire, one subtrochanteric femur fracture after implant removal of a prophylactically stabilized contralateral femoral head and one minimally dislocated femoral head after postoperative too early full weight bearing. DISCUSSION: The traumatic SEFH is very different to the chronic one regarding the pathology and acute treatment. Technical challenges must be solved. Unilateral K-wiring or screwing for 24 weeks and reduced weight bearing for the first 12 weeks after surgery is a sufficient way of treatment of the traumatic SEFH. CONCLUSIONS: In the case of a traumatic SEFH it needs to be reduced anatomically and stabilized by surgical means in the acute phase. A prophylactic stabilization of the opposite intact side is usually not required.

Morphological and mechanical characterisation of three-dimensional gyroid structures fabricated by electron beam melting for the use as a porous biomaterial.

Additive manufactured porous biomaterials based on triply periodic minimal surfaces (TPMS) are a highly discussed topic in the literature. With their unique properties in terms of open porosity, large surface area and surface curvature, they are considered to have bone mimicking properties and remarkable osteogenic potential. In this study, scaffolds of gyroid unit cells of different sizes consisting of a Ti6Al4V alloy were manufactured additively by electron beam melting (EBM). The scaffolds were analysed by micro-computed tomography (micro-CT) to determine their morphological characteristics and, subsequently, subjected to mechanical tests to investigate their quasi-static compressive properties and fatigue resistance. All scaffolds showed an average open porosity of 71-81%, with an average pore size of 0.64-1.41 mm, depending on the investigated design. The design with the smallest unit cell shows the highest quasi-elastic gradient (QEG) as well as the highest compressive offset stress and compression strength. Furthermore, the fatigue resistance of all unit cell size (UCS) variations showed promising results. In detail, the smallest unit cells achieved fatigue strength at 106 cycles at 45% of their compressive offset stress, which is comparatively good for additively manufactured porous biomaterials. In summary, it is demonstrated that the mechanical properties can be significantly modified by varying the unit cell size, thus enabling the scaffolds to be specifically tailored to avoid stress shielding and ensure implant safety. Together with the morphological properties of the gyroid unit cells, the fabricated scaffolds represent a promising approach for use as a bone substitute material.

Hyperelastic parameter identification of human articular cartilage and substitute materials.

Numerical simulations are a valuable tool in the field of tissue engineering for cartilage repair and can help to understand which mechanical properties affect the behavior of chondrocytes and contribute to the success or failure of surrogate materials as implants. However, special attention needs to be paid when identifying corresponding material parameters in order to provide reliable numerical predictions of the material's response. In this study, we identify hyperelastic material parameters for numerical simulations in COMSOL Multiphysics® v. 5.6 for human articular cartilage and two surrogate materials, commercially available ChondroFillerliquid, and oxidized alginate-gelatin (ADA-GEL) hydrogels. We consider several hyperelastic isotropic material models and provide separate parameter sets for the unconditioned and the conditioned material response, respectively, based on previously generated experimental data including both compression and tension experiments. We compare a direct parameter identification approach assuming homogeneous deformation throughout the specimen and an inverse approach, where the experiments are simulated using a finite element model with realistic boundary conditions in COMSOL Multiphysics® v. 5.6. We demonstrate that it is important to consider both compression and tension data simultaneously and to use the inverse approach to obtain reliable parameters. The one-term Ogden model best represents the unconditioned response of cartilage, while the conditioned response of cartilage and ADA-GEL is equally well represented by the two-term Ogden and five-term Mooney-Rivlin models. The five-term Mooney-Rivlin model is also most suitable to model the unconditioned response of ADA-GEL. For ChondroFillerliquid, we suggest using the five-term Mooney-Rivlin or two-term Ogden model for the unconditioned and the two-term Ogden model for the conditioned material response. These results will help to choose appropriate material models and parameters for simulations of whole joints or to advance mechanical-stimulation assisted cartilage tissue engineering in the future.

Gastrointestinal stromal tumor in a patient with undiagnosed neurofibromatosis type I: an uncommon cause of extrahepatic cholestasis.

In this case report we present a 61-year-old patient with obstructive jaundice. Bile duct obstruction was caused by a tumor at the duodenal papilla and bile flow was restored by a plastic stent. Using endoscopic ultrasound and computed tomography imaging two additional tumors of the same morphology were found in the stomach wall and the pelvic region suggesting a multilocular gastrointestinal stroma tumor (GIST). Diagnosis of GIST was confirmed cytologically from the gastric lesion. Based on typical cutaneous manifestations (café-au-lait spots, several tiny dermal neurofibromata and Lisch nodules in the iris), a thus far unidentified neurofibromatosis type I was diagnosed which is known to promote multilocular GIST formation. Tumor resection failed because of cardiac decompensation due to a Takotsubo cardiomyopathy during induction of anesthesia. The patient has been started on imatinib instead and shows so far a stable disease over 6 months.

Complex mechanical behavior of human articular cartilage and hydrogels for cartilage repair.

The mechanical behavior of cartilage tissue plays a crucial role in physiological mechanotransduction processes of chondrocytes and pathological changes like osteoarthritis. Therefore, intensive research activities focus on the identification of implant substitute materials that mechanically mimic the cartilage extracellular matrix. This, however, requires a thorough understanding of the complex mechanical behavior of both native cartilage and potential substitute materials to treat cartilage lesions. Here, we perform complex multi-modal mechanical analyses of human articular cartilage and two surrogate materials, commercially available ChondroFillerliquid, and oxidized alginate-gelatin (ADA-GEL) hydrogels. We show that all materials exhibit nonlinearity and compression-tension asymmetry. However, while hyaline cartilage yields higher stresses in tension than in compression, ChondroFillerliquid and ADA-GEL exhibit the opposite trend. These characteristics can be attributed to the materials' underlying microstructure: Both cartilage and ChondroFillerliquid contain fibrillar components, but the latter constitutes a bi-phasic structure, where the 60% nonfibrillar hydrogel proportion dominates the mechanical response. Of all materials, ChondroFillerliquid shows the most pronounced viscous effects. The present study provides important insights into the microstructure-property relationship of cartilage substitute materials, with vital implications for mechanically-driven material design in cartilage engineering. In addition, we provide a data set to create mechanical simulation models in the future.

DNA damage by low-energy electron impact: dependence on guanine content.

Single-stranded DNA oligonucleotides (33-mers) containing different numbers of guanines (n=1-4) were tethered to a gold surface and exposed to 1 eV electrons. The electrons induced DNA damage, which was analyzed with fluorescence and infrared spectroscopy methods. The damage was identified as strand breaks and found to correlate linearly with the number of guanines in the sequence. This sequence dependence indicates that the electron capture by the DNA bases plays an important role in the damage reaction mechanism.

[Hereditary hyperferritinemia cataract syndrome--the first family in Germany]. / Das hereditäre Hyperferritinämie-Katarakt-Syndrom--die erste Familie in Deutschland.

We report on a 23-year-old woman who presented with elevated serum ferritin values at our department. She had undergone cataract surgery at the age of 14 and her family pedigree showed hereditary autosomal-dominant cataract. The combination of isolated hyperferritinemia with autosomal-dominant hereditary cataract led to the diagnosis of the hereditary hyperferritinemia cataract syndrome (HHCS) which we now describe in a German family for the first time. HHCS was confirmed by detection of a causal mutation at position 32 within the iron responsive element (IRE) of L-ferritin leading to a guanine to adenine exchange and the pathognomonic star-shaped cataract. This mutation interrupts the post-transcriptional control of L-ferritin. It prevents binding of the iron regulatory protein 1 (IRP1) to the 5alpha untranslated region of L-ferritin resulting in uncontrolled L-ferritin synthesis and high serum ferritin levels independent of the body iron stores. Premature cataract is eventually caused by deposition of L-ferritin crystals in the lens of the eye. Our family shows the typical autosomal-dominant inheritance of HHCS over four generations affecting a total of 17 family members. The causal mutation, star-shaped cataract and typical laboratory configuration were confirmed in five patients. Thus, in gastroenterological practice, HHCS should be added as a differential diagnosis of hyperferritinemia in Germany. Importantly, patients with HHCS can be spared from invasive diagnostics such as liver biopsy.

Biocompatibility and thermodynamic properties of PEGDA and two of its copolymer.

During the last years substantial effort was taken in order to provide an effective and safe pharmacotherapy that can be adjusted to the individual needs of patients. Stereolithography is a simple and accurate additive manufacturing technology. According to these characteristics, it may offer unique opportunities for the industrial fabrication of structured drug delivery systems (DDS), which can be tailored to individual needs. During the stereolithographic process photopolymerizable biomaterial is transformed, layer by layer, into the designed polymer DDS. Combined with inkjet printing in an innovative 3D building system it enables selective and precise incorporation of the drug depot into the basic body of the DDS. Poly(ethylene glycol) diacrylate (PEGDA), a hydrophilic and low-immunogenic compound, is a suitable material as drug depot in a photopolymerizable basic biomaterial for this purpose. By combination of PEGDA with other acrylates, the physical properties of the DDS can be adjusted towards the desired characteristics. Therefore, it should be possible to modify the drug release profile through the positioning of drug depots and the diffusion of the drug and adjust it for a wide range of applications. In this study we investigated basic biological and thermodynamic properties of conventionally photocured systems consisting of PEGDA and its coacrylates: 1,3-butanediol diacrylate and pentaerythritol triacrylate. Our preliminary outcomes demonstrate the hydrophilic character of the samples and the importance of a rinsing process. They also show that the addition of different amounts of co-monomers influence the glass transition temperature, which increases with increasing content of coacrylate. Therefore, PEGDA/comonomer composition can be used as a tool for the modification of drug release properties. Consequently, these materials may be regarded as interesting and promising components for DDS via novel additive manufacturing with the ability of highly controlled drug release.

Numerical simulation of the electric field distribution in an electrical stimulation device for scaffolds settled with cartilaginous cells.

Electrical stimulation is a promising approach to enhance cell viability and differentiation. We aim to develop a stimulation device for the investigation and realization of cartilaginous cell engineering. The stimulation setup is capable of applying well-defined electric fields to several scaffolds at the same time. The setup consists of a flat plate with multiple test tubes for the scaffolds. A flexible printed circuit board containing a separate pair of electrodes for each tube is fixed at the bottom of the plate. In this context, numerical simulation using Finite Element Method (FEM) is a valuable tool to gain a better understanding of the electric field distribution in such devices. The thin insulating layer of the flexible printed circuit board allows sufficient field strength to be achieved at moderate input voltages but presents challenges for modelling. In simulations, thin layers would usually require a fine discretization with many degrees of freedom (DOF). This leads to large models, which are expensive regarding memory and computation time. Based on the 'contact impedance' boundary condition available in COMSOL Multiphysics® 5.4, an alternative approach is proposed that can model thin layers in capacitively coupled setups. The resulting electric field distribution in the new stimulation setup is presented and discussed.

Effect of ketone bodies on glucose production and utilization in the miniature pig.

The effect of ketone bodies on glucose production (Ra) and utilization (Rd) was investigated in the 24-h starved, conscious unrestrained miniature pig. Infusing Na-DL-beta-OH-butyrate (Na-DL-beta-OHB) and thus shifting the blood pH from 7.40 to 7.56 resulted in a decrease of Ra by 52% and of Rd by 45%, as determined by the isotope dilution technique. Simultaneously, the concentrations of arterial insulin and glucagon were slightly enhanced, whereas the plasma levels of glucose, lactate, pyruvate, alanine, alpha-amino-N, and free fatty acids (FFA) were all reduced. Infusion of Na-bicarbonate, which yielded a similar shift in blood pH, did not mimick these effects. Infusion of equimolar amounts of the ketoacid, yielding a blood pH of 7.35, induced similar metabolic alterations with respect to plasma glucose, Ra, Rd, and insulin; however, plasma alanine and alpha-amino-N increased. Infusing different amounts of Na-DL-beta-OHB resulting in plasma steady state levels of ketones from 0.25 to 1.5 mM had similar effects on arterial insulin and glucose kinetics. No dose dependency was observed. Prevention of the Na-DL-beta-OHB-induced hypoalaninemia by simultaneous infusion of alanine (1 mumol/kg X min) did not prevent hypoglycemia. Infusion of Na-DL-beta-OHB plus insulin (0.4 mU/kg X min) showed no additive effect on the inhibition of Ra. Ketones did not inhibit the insulin-stimulated metabolic clearance rate (MCR) for glucose. Infusion of somatostatin (0.2 micrograms/kg X min) initially decreased plasma glucose, Ra, and Rd, which was followed by an increase in plasma glucose and Ra; however, on infusion of somatostatin plus Na-DL-beta-OHB, hypoglycemia and the reduced Ra were maintained. In the anaesthetized 24-h starved miniature pig, Na-DL-beta-OHB infusion decreased the hepatic exchange for glucose, lactate, and FFA, whereas the exchange for glycerol, alanine, and alpha-amino-N as well as liver perfusion rate were unaffected. Simultaneously, portal glucagon and insulin as well as hepatic insulin extraction rate were elevated. Leg exchange for glucose, lactate, glycerol, alanine, alpha-amino-N, and FFA were decreased, while ketone body utilization increased. Repeated infusion of Na-DL-beta-OHB at the fourth, fifth, and sixth day of starvation in the conscious, unrestrained mini-pig resulted in a significant drop in urinary nitrogen (N)-excretion. However, this effect was mimicked by infusing equimolar amounts of Na-bicarbonate. In contrast, when only the ketoacid was given, urinary N-excretion accelerated. To summarize: (a) Ketone bodies decrease endogenous glucose production via an insulin-dependent mechanism; in addition, ketones probably exert a direct inhibitory action on gluconeogenesis. The ketone body-induced hypoalaninemia does not contribute to this effect. (b) The counterregulatory response to hypoglycemia is reduced by ketones. (c) As a consequence of the decrease in R(a), glucose utilization declines during ketone infusion. (d)The insulin-stimulated MCR for glucose is not affected by ketones. (e) Ketones in their physiological moiety do not show a protein-sparing effect.

Plant progesterone 5beta-reductase is not homologous to the animal enzyme. Molecular evolutionary characterization of P5betaR from Digitalis purpurea.

Plants of the genus Digitalis produce cardiac glycosides, i.e. digoxin, which are widely used for congestive heart failure. Progesterone 5beta-reductase (P5betaR) is a key enzyme in the biosynthesis of these natural products. Here, we have carried out the purification and partial amino acid sequencing of the native P5betaR from foxglove (Digitalis purpurea), and isolated a cDNA encoding this enzyme. Similarly to other steroid 5beta-reductases, the recombinant P5betaR catalyzes the stereospecific reduction of the Delta(4)-double bond of several steroids with a 3-oxo,Delta(4,5) structure. The gene encoding P5betaR is expressed in all plant organs, and maximally transcribed in leaves and mature flowers. P5betaR belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, bearing no structural homology to its mammalian counterpart, which is a member of the aldo-keto reductase (AKR) superfamily. A similar situation occurs with 3beta-hydroxy-Delta(5)-steroid dehydrogenase (3betaHSD), the gene immediately preceding P5betaR in the cardenolide pathway, which suggests that the entire route has evolved independently in animals and plants. P5betaR is retained only in plants, where it is ubiquitous, and a few distantly related bacterial lineages after its diversification from the last universal common ancestor. Evolutionary conserved changes in its putative active site suggest that plant P5betaR is a member of a novel subfamily of extended SDRs, or a new SDR family.

Timing of developmentally programmed excision and circularization of Paramecium internal eliminated sequences.

Paramecium internal eliminated sequences (IESs) are short AT-rich DNA elements that are precisely eliminated from the germ line genome during development of the somatic macronucleus. They are flanked by one 5'-TA-3' dinucleotide on each side, a single copy of which remains at the donor site after excision. The timing of their excision was examined in synchronized conjugating cells by quantitative PCR. Significant amplification of the germ line genome was observed prior to IES excision, which starts 12 to 14 h after initiation of conjugation and extends over a 2- to 4-h period. Following excision, two IESs were shown to form extrachromosomal circles that can be readily detected on Southern blots of genomic DNA from cells undergoing macronuclear development. On these circular molecules, covalently joined IES ends are separated by one copy of the flanking 5'-TA-3' repeat. The similar structures of the junctions formed on the excised and donor molecules point to a central role for this dinucleotide in IES excision.

Endocultivation: continuous application of rhBMP-2 via mini-osmotic pumps to induce bone formation at extraskeletal sites.

The continuous presence of recombinant human bone morphogenetic protein 2 (rhBMP-2) inside a scaffold may be crucial to the outcome in bone tissue engineering. This study investigated whether the release of the growth factor rhBMP-2 via different continuous application schemes influences histomorphological aspects of the hard and soft tissues induced. Three-dimensionally printed hydroxyapatite scaffolds were implanted into one latissimus dorsi muscle of 42 female Lewis rats. Simultaneously implanted mini-osmotic pumps were used to provide a continuous application of rhBMP-2 over 1, 2, or 4 weeks (total dose 200µg). A reference group received rhBMP-2 at the time of implantation only, and a control group received only block implantation. Bone density and histological examinations were performed after 8 weeks. No significant difference in bone density was found between the groups; however, the blood vessel count differed significantly between the groups receiving continuous treatments and both the control group and simultaneous rhBMP-2 treatment group (P<0.0001). Soft tissue types were distributed differently among the study groups. RhBMP-2 application via mini-osmotic pumps is as suitable for inducing bone formation as a single application at the time of implantation. The time interval over which rhBMP-2 was administered had no impact on the amount of new bone formation, probably due to the study duration and low local concentrations of growth factor.