Untersuchungen der Expression, Funktion und Struktur archaeeller Membrantransportproteine

Im Rahmen dieser Arbeit sollte ein effizientes heterologes Expressionssystem für Membranproteine der beiden archaeellen Organismen Ignicoccus hospitalis und Nanoarchaeum equitans identifiziert und etabliert werden. Nach bioinformatischer Analyse wurden dafür zunächst geeignete Kandidaten aus beiden Organismen ausgewählt. Die Expression sollte sowohl in prokaryotischen als auch in eukaryotischen Wirtssystemen getestet werden. Trotz verschiedener Optimierungsmaßnahmen war es nicht möglich, Membranproteine von I. hospitalis erfolgreich bzw. in hinreichender Qualität in E. coli überzuexprimieren. Auch die beiden hier verwendeten archaeellen Wirte eigneten sich nicht zur heterologen Expression. Ein Wechsel zu eukaryotischen Systemen lieferte unterschiedlichen Erfolg für die Expression des putativen MFS Transporters Iho0391. Während das Protein nur in mangelhafter Qualität in einer Insektenzelllinie rekombinant hergestellt werden konnte, führte die Expression in P. pastoris zu homologem Protein, welches mittels IMAC aufgereinigt und bereits für erste funktionelle Analysen verwendet werden konnte. Diese liefern zusammen mit dem erstellten Homologiemodell erste Hinweise auf eine mögliche ATP Transport Funktion durch Iho0391. Unter Umständen könnte durch das MFS Protein sogar ein Energietransport zu N. equitans vermittelt werden. Im Genom von I. hospitalis wurden insgesamt acht MFS Transporter identifiziert und ihnen eine mögliche Beteiligung am Energietransport in I. hospitalis selbst bzw. zu seinem Symbionten N. equitans zugesprochen. Um Aufschlüsse über die Funktion von MFS Transportern aus I. hospitalis zu erhalten, wurde parallel versucht, homologe MFS Proteine aus anderen Archaeen heterolog in E. coli zu exprimieren. Die Wahl fiel dabei auf Tko1655 aus T. kodakarensis, ein Homolog zu Iho0800. Der MFS Transporter konnte bereits exprimiert und gereinigt werden, funktionelle Analysen wurden jedoch noch nicht durchgeführt. Das erstellte Homologiemodells deutet aber auf eine eventuelle Beteiligung des Transporters am Aminosäurestoffwechsel hin. Die Expression des putativen C4-Dicarboxylat Transporters Neq014 aus N. equitans war nach Codonoptimierung problemlos in E. coli möglich. Auch die Reinigung des Proteins konnte optimiert werden und erste Kristallisationsansätze wurden bereits gemacht. Eine mögliche Funktion von Neq014 als spannungsabhängiger Anionenkanal könnte mit der Regulation des Metaboliten- bzw. Energieflusses über die Membran von N. equitans einhergehen. Auch im Falle einer Trennung von seinem Wirt I. hospitalis könnte das Protein eine wichtige Rolle spielen. Insgesamt betrachtet schaffen die in dieser Arbeit erzielten Ergebnisse die Grundlage für die Expression und Reinigung von Membranproteinen der archaeellen Organismen I. hospitalis und N. equitans und können als Startpunkt für weiterführende Untersuchungen genutzt werden

"Hope for the best, prepare for the worst": A qualitative interview study on parents' needs and fears in pediatric advance care planning.

Pediatric advance care planning is advocated by healthcare providers because it may increase the chance that patient and/or parent wishes are respected and thus improve end-of-life care. However, since end-of-life decisions for children are particularly difficult and charged with emotions, physicians are often afraid of addressing pediatric advance care planning. We aimed to investigate parents' views and needs regarding pediatric advance care planning. We performed a qualitative interview study with parents of children who had died from a severe illness. The interviews were analyzed by descriptive and evaluation coding according to Saldaña. We conducted semi-structured interviews with 11 parents of 9 children. Maximum variation was sought regarding the child's illness, age at death, care setting, and parent gender. Parents find it difficult to engage in pediatric advance care planning but consider it important. They argue for a sensitive, individualized, and gradual approach. Hope and quality of life issues are primary. Parents have many non-medical concerns that they want to discuss. Written advance directives are considered less important, but medical emergency plans are viewed as necessary in particular cases. Continuity of care and information should be improved through regular pediatric advance care planning meetings with the various care providers. Parents emphasize the importance of a continuous contact person to facilitate pediatric advance care planning. Despite a need for pediatric advance care planning, it is perceived as challenging. Needs-adjusted content and process and continuity of communication should be a main focus in pediatric advance care planning. Future research should focus on strategies that facilitate parent engagement in pediatric advance care planning to increase the benefit for the families

Quantitative Assessment of Early Diabetic Retinopathy Using Fractal Analysis

OBJECTIVE—Fractal analysis can quantify the geometric complexity of the retinal vascular branching pattern and may therefore offer a new method to quantify early diabetic microvascular damage. In this study, we examined the relationship between retinal fractal dimension and retinopathy in young individuals with type 1 diabetes

Corneal Intrastromal Implantation Surgery by means of MyoRing Corneal Implant for the Treatment of Keratoconus: A Review

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Validation of seismic hazard curves using a calibrated 14 ka lacustrine record in the Eastern Alps, Austria.

Seismic hazard maps are crucial for earthquake mitigation and mostly rely on probabilistic seismic hazard analysis (PSHA). However, the practise and value of PSHA are under debate because objective testing procedures for seismic hazard maps are scarce. We present a lacustrine turbidite record revealing 44 earthquakes over the last ~ 14 ka and use it to test seismic hazard curves in southern Austria. We derive local seismic intensities for paleo-earthquakes by applying scaling relationships between the sedimentary imprint and seismic intensity of well-documented historical earthquakes. The last ~ 2.8 ka of the record agree with a Poissonian recurrence behaviour and therefore a constant hazard rate, which is the modelling choice for standard PSHA. The lacustrine data are consistent with the intensity-frequency relationship of the local seismic hazard curve, confirming the current PSHA approach for this part of Austria. On longer timescales, distinct phases of enhanced regional seismicity occurred, indicating a potential increase of seismic hazard after large earthquakes-a factor hitherto disregarded in the PSHA of the Eastern Alps. Our new method forms an independent procedure to test hazard maps in any setting where suitable lake systems are available

Quantification of collagen organization in the peripheral human cornea at micron-scale resolution

The collagen microstructure of the peripheral cornea is important in stabilizing corneal curvature and refractive status. However, the manner in which the predominantly orthogonal collagen fibrils of the central cornea integrate with the circumferential limbal collagen is unknown. We used microfocus wide-angle x-ray scattering to quantify the relative proportion and orientation of collagen fibrils over the human corneolimbal interface at intervals of 50 μm. Orthogonal fibrils changed direction 1–1.5 mm before the limbus to integrate with the circumferential limbal fibrils. Outside the central 6 mm, additional preferentially aligned collagen was found to reinforce the cornea and limbus. The manner of integration and degree of reinforcement varied significantly depending on the direction along which the limbus was approached. We also employed small-angle x-ray scattering to measure the average collagen fibril diameter from central cornea to limbus at 0.5 mm intervals. Fibril diameter was constant across the central 6 mm. More peripherally, fibril diameter increased, indicative of a merging of corneal and scleral collagen. The point of increase varied with direction, consistent with a scheme in which the oblique corneal periphery is reinforced by chords of scleral collagen. The results have implications for the cornea's biomechanical response to ocular surgeries involving peripheral incision

Controlling the 3D architecture of Self-Lifting Auto-generated Tissue Equivalents (SLATEs) for optimized corneal graft composition and stability

Ideally, biomaterials designed to play specific physical and physiological roles in vivo should comprise components and microarchitectures analogous to those of the native tissues they intend to replace. For that, implantable biomaterials need to be carefully designed to have the correct structural and compositional properties, which consequently impart their bio-function. In this study, we showed that the control of such properties can be defined from the bottom-up, using smart surface templates to modulate the structure, composition, and bio-mechanics of human transplantable tissues. Using multi-functional peptide amphiphile-coated surfaces with different anisotropies, we were able to control the phenotype of corneal stromal cells and instruct them to fabricate self-lifting tissues that closely emulated the native stromal lamellae of the human cornea. The type and arrangement of the extracellular matrix comprising these corneal stromal Self-Lifting Analogous Tissue Equivalents (SLATEs) were then evaluated in detail, and was shown to correlate with tissue function. Specifically, SLATEs comprising aligned collagen fibrils were shown to be significantly thicker, denser, and more resistant to proteolytic degradation compared to SLATEs formed with randomly-oriented constituents. In addition, SLATEs were highly transparent while providing increased absorption to near-UV radiation. Importantly, corneal stromal SLATEs were capable of constituting tissues with a higher-order complexity, either by creating thicker tissues through stacking or by serving as substrate to support a fully-differentiated, stratified corneal epithelium. SLATEs were also deemed safe as implants in a rabbit corneal model, being capable of integrating with the surrounding host tissue without provoking inflammation, neo-vascularization, or any other signs of rejection after a 9-months follow-up. This work thus paves the way for the de novo biofabrication of easy-retrievable, scaffold-free human tissues with controlled structural, compositional, and functional properties to replace corneal, as well as other, tissuesThis study was supported by the Medical Research Council grant MR/ K017217/1, the Biotechnology and Biological Sciences Research Council, grant BB/I008187/1 and the Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I + D + I) from the Spanish Ministry of Economy and Competitiveness (Instituto de Salud Carlos III), grant FIS PI14/0955 (cofinanced by FEDER funds, European Union)

Genetic algorithm based feature selection combined with dual classification for the automated detection of proliferative diabetic retinopathy

Proliferative diabetic retinopathy (PDR) is a condition that carries a high risk of severe visual impairment. The hallmark of PDR is the growth of abnormal new vessels. In this paper, an automated method for the detection of new vessels from retinal images is presented. This method is based on a dual classification approach. Two vessel segmentation approaches are applied to create two separate binary vessel map which each hold vital information. Local morphology features are measured from each binary vessel map to produce two separate 4-D feature vectors. Independent classification is performed for each feature vector using a support vector machine (SVM) classifier. The system then combines these individual outcomes to produce a final decision. This is followed by the creation of additional features to generate 21-D feature vectors, which feed into a genetic algorithm based feature selection approach with the objective of finding feature subsets that improve the performance of the classification. Sensitivity and specificity results using a dataset of 60 images are 0.9138 and 0.9600, respectively, on a per patch basis and 1.000 and 0.975, respectively, on a per image basis

Riboflavin/UVA Collagen Cross-Linking-Induced Changes in Normal and Keratoconus Corneal Stroma

Purpose To determine the effect of Ultraviolet-A collagen cross-linking with hypo-osmolar and iso-osmolar riboflavin solutions on stromal collagen ultrastructure in normal and keratoconus ex vivo human corneas. Methods Using small-angle X-ray scattering, measurements of collagen D-periodicity, fibril diameter and interfibrillar spacing were made at 1 mm intervals across six normal post-mortem corneas (two above physiological hydration (swollen) and four below (unswollen)) and two post-transplant keratoconus corneal buttons (one swollen; one unswollen), before and after hypo-osmolar cross-linking. The same parameters were measured in three other unswollen normal corneas before and after iso-osmolar cross-linking and in three pairs of swollen normal corneas, in which only the left was cross-linked (with iso-osmolar riboflavin). Results Hypo-osmolar cross-linking resulted in an increase in corneal hydration in all corneas. In the keratoconus corneas and unswollen normal corneas, this was accompanied by an increase in collagen interfibrillar spacing (p<0.001); an increase in fibril diameter was also seen in two out of four unswollen normal corneas and one unswollen keratoconus cornea (p<0.001). Iso-osmolar cross-linking resulted in a decrease in tissue hydration in the swollen normal corneas only. Although there was no consistent treatment-induced change in hydration in the unswollen normal samples, iso-osmolar cross-linking of these corneas did result in a compaction of collagen fibrils and a reduced fibril diameter (p<0.001); these changes were not seen in the swollen normal corneas. Collagen D-periodicity was not affected by either treatment. Conclusion The observed structural changes following Ultraviolet-A cross-linking with hypo-osmolar or iso-osmolar riboflavin solutions are more likely a consequence of treatment-induced changes in tissue hydration rather than cross-linking