Practical implementation of the planar and spatial rotator in a complex tissue: the brain.

In neuroscience, application of widely used stereological local volume estimators, including the planar rotator, is challenged by the combination of a complex tissue organisation and an estimator requirement of either isotropic or vertical sections, i.e. randomly oriented tissue. The spatial rotator is applicable with any tissue orientation but is sensitive to projection artefacts. The challenge is thus to select the most appropriate method for individual analyses. In this study, agreement between estimates of mean cell volume acquired with the vertical planar and the spatial rotator is assessed for two brain regions with different types of cytoarchitecture (motor cortex and hippocampal cornu ammonis 1). The possibility of using the planar rotator in tissues cut in an arbitrary direction is explored and requirements for a theoretically unbiased result as well as histological considerations are provided. LAY DESCRIPTION: Cells may change volume both during disease and with advancing age. Assessment of the volume of individual cells can therefore serve as a useful indicator of general tissue state. Most available methods to estimate cell volume in tissue sections, however, require that the tissue analysed has random orientation. Particularly for complex tissues such as the brain this is a challenge as identification, delineation and subdivision of many brain areas rely heavily on the use of anatomical atlases where illustrations depict the tissue in a few well-known orientations. In this study, the practical application of two different methods for estimating mean cell volumes in tissues cut in a preferred orientation is evaluated. Requirements for the feasibility of cell volume estimation without random tissue orientation as well as histological considerations are provided.

[Systematic Review of the Application of Complementary and Alternative Medicine and their Potential Therapeutic Benefits in the Treatment of Ophthalmology Patients]. / Ergebnisse eines systematischen Reviews zu Einsatz und berichtetem therapeutischem Nutzen komplementärmedizinischer Methoden in der Augenheilkunde.

Purpose A systematic review was carried out of the reported therapeutic effects of complementary and alternative medicine methods as supplementary or primary treatments for patients suffering from glaucoma, cataract or age-related macular degeneration (AMD). Material and Methods For the years 1990 to 2013, the following databases were screened for reports of the application of complementary and alternative treatments: PubMed, Cochrane Library, EMBASE, CAMbase and AMED. Both randomised and prospective non-randomised patient trials were included in the review; results were evaluated in the following classes: "phytotherapy", "acupuncture/acupressure", "biofeedback" and "other alternative treatments". The studies were evaluated by measures of clinical effect, statistical significance (p value and/or confidence interval) and the underlying trial design. Results 30 clinical trials were included, including 13 on glaucoma, 5 on cataract and 12 on AMD patients. These trials were based on patient numbers of 6 - 332, 27 - 157 and 6 - 328 patients, respectively. Phytotherapy was applied in 14 trials, including 6 on glaucoma patients (all 6 with a controlled design, and 3 of which reporting statistically significant results); 5 trials were on cataract patients (3 with a controlled design and 2 with a significant result) and 3 on AMD patients (only 1 with a controlled design, with a significant result). Acupuncture/acupressure was investigated in 9 trials, 5 on glaucoma patients (3 with a controlled design, 1 with a significant result); no acupuncture/acupressure trial was found in cataract patients, but 4 trials in AMD patients (none with a controlled design). Biofeedback was studied in 4 trials, all on AMD patients (only one with a controlled design, without statistically significant findings). Conclusion Despite its rigorous inclusion criteria, this review identified several clinical trials on complementary and alternative medicine in ophthalmological patients. Phytotherapeutic methods gave significant results in half of the reported controlled trials, whereas there were few significant benefits with acupuncture or acupressure.

Thalidomide in angiodysplasia-related bleeding.

Gastrointestinal haemorrhage from angiodysplastic lesions is not only difficult to identify, but often refractory to endoscopic intervention. Patients often require substantial transfusion support. Thalidomide has emerged as a promising medical strategy in angiodysplasia-related bleeding. We present our experience and report the findings from a review of the literature. Despite its side-effect profile, thalidomide remains the therapeutic modality with the best evidence in this difficult clinical scenario.

[Conception and Content Validation of a Questionnaire Relating to the Potential Need for Information of Visually Impaired Persons with Regard to Services and Contact Persons]. / Konzeption und Inhaltsvalidierung eines Fragebogens zu Informationsbedürfnissen bezüglich Dienstleistungen und Ansprechpartnern von sehbehinderten Menschen (FIDAS).

A questionnaire was drafted to identify the needs of visually impaired persons and to optimize their access to non-medical support and services. Subjects had to rate a list of 15 everyday activities that are typically affected by visual impairment (for example, being able to orient themselves in the home environment), by indicating the degree to which they perceive each activity to be affected, using a four-stage scale. They had to evaluate these aspects by means of a relevance assessment. The needs profile derived from this is then correlated with individualized information for assistance and support. The questionnaire shall be made available for use by subjects through advisers in some ophthalmic practices and via the internet. The validity of the content of the proposed tool was evaluated on the basis of a survey of 59 experts in the fields of medical, optical and psychological care and of persons involved in training initiatives. The experts were asked to rate the activities by relevance and clarity of the wording and to propose methods to further develop and optimize the content. The validity of the content was quantified according to a process adopted in the literature, based on the parameters Interrater Agreement (IRA) and Content Validity Index (CVI). The results of all responses (n = 19) and the sub-group analysis suggest that the questionnaire adequately reflects the potential needs profile of visually impaired persons. Overall, there was at least 80% agreement among the 19 experts for 93% of the proposed parameterisation of the activities relating to the relevance and clarity of the wording. Individual proposals for optimization of the design of the questionnaire were adopted.

The spatial rotator.

This paper presents a new local volume estimator, the spatial rotator, which is based on measurements on a virtual 3D probe, using computer assisted microscopy. The basic design of the probe builds upon the rotator principle which requires only a few manual intersection markings, thus making the spatial rotator fast to use. Since a 3D probe is involved, it is expected that the spatial rotator will be more efficient than the the nucleator and the planar rotator, which are based on measurements in a single plane. An extensive simulation study shows that the spatial rotator may be more efficient than the traditional local volume estimators. Furthermore, the spatial rotator can be seen as a further development of the Cavalieri estimator, which does not require randomization of sectioning or viewing direction. The tissue may thus be sectioned in any arbitrary direction, making it easy to identify the specific tissue region under study. In order to use the spatial rotator in practice, however, it is necessary to be able to identify intersection points between cell boundaries and test rays in a series of parallel focal planes, also at the peripheral parts of the cell boundaries. In cases where over- and underprojection phenomena are not negligible, they should therefore be corrected for if the spatial rotator is to be applied. If such a correction is not possible, it is needed to avoid these phenomena by using microscopy with increased resolution in the focal plane.

Improving efficiency in stereology: a study applying the proportionator and the autodisector on virtual slides.

Cell counting in stereology is time-consuming. The proportionator is a new stereological sampling method combining automatic image analysis and non-uniform sampling. The autodisector on virtual slides combines automatic generation of disector pairs with the use of digital images. The aim of the study was to investigate the time efficiency of the proportionator and the autodisector on virtual slides compared with traditional methods in a practical application, namely the estimation of osteoclast numbers in paws from mice with experimental arthritis and control mice. Tissue slides were scanned in a digital slide scanner and the autodisector was applied on the obtained virtual tissue slides. Every slide was partitioned into fields of view, and cells were counted in all of them. Based on the original exhaustive data set comprising 100% of fields of view and covering the total section area, a proportionator sampling and a systematic, uniform random sampling were simulated. We found that the proportionator was 50% to 90% more time efficient than systematic, uniform random sampling. The time efficiency of the autodisector on virtual slides was 60% to 100% better than the disector on tissue slides. We conclude that both the proportionator and the autodisector on virtual slides may improve efficiency of cell counting in stereology.

Humanized theta-defensins (retrocyclins) enhance macrophage performance and protect mice from experimental anthrax infections.

Retrocyclins are humanized versions of the -defensin peptides expressed by the leukocytes of several nonhuman primates. Previous studies, performed in serum-free media, determined that retrocyclins 1 (RC1) and RC2 could prevent successful germination of Bacillus anthracis spores, kill vegetative B. anthracis cells, and inactivate anthrax lethal factor. We now report that retrocyclins are extensively bound by components of native mouse, human, and fetal calf sera, that heat-inactivated sera show greatly enhanced retrocyclin binding, and that native and (especially) heat-inactivated sera greatly reduce the direct activities of retrocyclins against spores and vegetative cells of B. anthracis. Nevertheless, we also found that retrocyclins protected mice challenged in vivo by subcutaneous, intraperitoneal, or intranasal instillation of B. anthracis spores. Retrocyclin 1 bound extensively to B. anthracis spores and enhanced their phagocytosis and killing by murine RAW264.7 cells. Based on the assumption that spore-bound RC1 enters phagosomes by "piggyback phagocytosis," model calculations showed that the intraphagosomal concentration of RC1 would greatly exceed its extracellular concentration. Murine alveolar macrophages took up fluorescently labeled retrocyclin, suggesting that macrophages may also acquire extracellular RC1 directly. Overall, these data demonstrate that retrocyclins are effective in vivo against experimental murine anthrax infections and suggest that enhanced macrophage function contributes to this property.

The saucor, a new stereological tool for analysing the spatial distributions of cells, exemplified by human neocortical neurons and glial cells.

The 3D spatial arrangement of particles or cells, for example glial cells, with respect to other particles or cells, for example neurons, can be characterized by the radial number density function, which expresses the number density of so-called 'secondary' particles as a function of their distance to a 'primary' particle. The present paper introduces a new stereological method, the saucor, for estimating the radial number density using thick isotropic uniform random or vertical uniform random sections. In the first estimation step, primary particles are registered in a disector. Subsequently, smaller counting windows are drawn with random orientation around every primary particle, and the positions of all secondary particles within the windows are recorded. The shape of the counting windows is designed such that a large portion of the volume close to the primary particle is examined and a smaller portion of the volume as the distance to the primary object increases. The experimenter can determine the relation between these volumina as a function of the distance by adjusting the parameters of the window graph, and thus reach a good balance between workload and obtained information. Estimation formulae based on the Horvitz-Thompson theorem are derived for both isotropic uniform random and vertical uniform random designs. The method is illustrated with an example where the radial number density of neurons and glial cells around neurons in the human neocortex is estimated using thick vertical sections for light microscopy. The results indicate that the glial cells are clustered around the neurons and the neurons have a tendency towards repulsion from each other.

[Glaucoma in inpatient care in Germany in 2015]. / Glaukome im stationären Leistungsgeschehen in Deutschland in 2015.

AIM: Analysis of structural characteristics of inpatient treatment of glaucoma in clinical units run by affiliated physicians and those run by hospital physicians in Germany are described. The frequency and distribution of glaucoma diagnoses as the main and secondary diagnosis at different departments and glaucoma diagnoses, number of cases, surgeries and procedures and patient characteristics of the four glaucoma-specific diagnosis-related groups (DRG) are presented. METHOD: Secondary data analysis using the G­DRG browser from 2015/2016 provided by the German Institute for Remuneration Systems in Hospitals. RESULTS: In 30% of the 156,524 cases glaucoma was classified as the main diagnosis, in 24% as secondary diagnosis in ophthalmological departments and 46% as secondary diagnosis in other departments. Primary open angle glaucoma was reported most frequently (40%), whilst non-ophthalmological departments mainly coded as "other" or "unspecified types of glaucoma". Glaucoma was coded in 20 DRGs as the main diagnosis and in all ophthalmic DRGs as secondary diagnosis. The number of cases and procedures differed among the four glaucoma-specific DRGs (C06Z, C07A, C07B, C64Z), the diagnostic spectrum, however, was similar. Patients were mainly women, older and with few comorbidities or complications. The C64Z was mainly characterized by cases with "suspected glaucoma" and only conservative procedures. Only 6% of the glaucoma patients were treated in clinical units run by affiliated physicians. In contrast to units run by hospital physicians surgical DRGs and operations prevailed but the diagnostic spectrum and patient characteristics were not different. DISCUSSION AND CONCLUSION: With 20% of the main diagnoses, glaucoma was highly relevant for inpatient ophthalmological care. Glaucoma as a secondary diagnosis was also common in other medical specialties, though no diagnostic differentiation was made here. Patients with glaucoma are mainly treated in clinical units run by hospital physicians. Nevertheless, patients of affiliated physicians showed the same characteristics.

Spinal cord compression through extraosseous extension of a vertebral low-grade hemangioendothelioma with histiocytoid differentiation.

Herein, we report the case of a 47-year-old man clinically presenting a slow progressive loss of lower extremity functions within 8 weeks followed by an acute neurogenic bladder dysfunction. The patient exhibited high-grade paralysis of both legs with reduced sensation from dermatome Th11 downwards as well as marked spasticity of the lower extremity. Neuroradiological examinations revealed a protruding spinal tumor with extraosseous-intraspinal extension. The resected tumor mass exhibited a highly vascularized tumor with architectural complexity and high cellularity finally leading to the diagnosis of a hemangioendothelioma. Interesting was the fact that the tumor vasculature exhibited many CD68-positive cells protruding into the lumen and, therefore, being part of a partially histiocytoid differentiation which is all the more uncommon in hemangioendothelioma. The time frame of 3 hours between embolization and tumor resection is too short to explain a monocytic intravascular reaction. Usually, hemangioendotheliomas arise from the soft tissue, lungs or liver, but intraspinal manifestations are only rarely observed. Furthermore, the clinical course with a progressive development of a paraparesis due to a hemangioendothelioma is very uncommon.

[Employed and self-employed specialists in outpatient ophthalmological care : Comparison with other specialist fields]. / Angestellte und selbstständige Fachärzte in der vertragsärztlichen ophthalmologischen Versorgung : Ein Vergleich mit anderen Fachgebieten.

AIM: The aim of the study was to evaluate the contribution to outpatient care and the perspectives of employed and self-employed ophthalmologists in isolation and in comparison to other professional groups. METHOD: Based on statistical data (source: statistical information provided by the National Association of Statutory Health Insurance Physicians) from 31 December 2015, newly admitted physicians in 2015 and their contribution differentiated by employment relationships were evaluated according to head count and working time equivalents (using "planning of demand equivalents"). The results were subdivided into type of outpatient facility, e.g. single practice, group practice and medical care centers (MVZ). RESULTS: The proportion of employed ophthalmologists (head count) in outpatient care (23%) and newly admitted physicians (81%) is above the interdisciplinary average (16% and 65%, respectively). In all medical professional groups the contribution according to time equivalents of employed physicians compared to self-employed physicians is lower (utilization rate: 65% in total and 70% in ophthalmology). In ophthalmology employment is seen in all types of outpatient practices: the number of ophthalmologists working in group practices is above average, compared to other groups they are underrepresented in MVZ and are working in single practices as frequently as all other specialties. DISCUSSION AND CONCLUSION: Employment of physicians is more common in all types of outpatient ophthalmological facilities than in other specialized professional groups. Possible reasons are the working conditions in ophthalmology attracting physicians preferring employment and the competitive situation for practice takeover. Since employed physicians contribute less time to medical care than self-employed physicians, ophthalmology has a higher risk of a shortage in medical care despite an increasing number of physicians. An increasing number of employed physicians has an impact on the structures of outpatient care; however, the intensity of concentration in healthcare structures can only be partially evaluated due to insufficient data. Based on the available data there is no end in sight for the trend towards employment in outpatient ophthalmological care.

Activating KIR Haplotype Influences Clinical Outcome Following HLA-Matched Sibling Hematopoietic Stem Cell Transplantation.

Natural killer cells are thought to influence the outcome of hematopoietic stem cell transplant (HSCT), impacting on relapse, overall survival, graft versus host disease and the control of infection, in part through the complex interplay between the large and genetically diverse killer immunoglobulin-like receptor (KIR) family and their ligands. This study examined the relationship between KIR gene content and clinical outcomes including the control of opportunistic infections such as cytomegalovirus in the setting of human leucocyte antigen (HLA)-matched sibling HSCT in an Australian cohort. The presence of the KIR B haplotype which contain more activating receptors in the donor, in particular centromeric B haplotype genes (Cen-B), was associated with improved overall survival of patients with acute myeloid leukemia (AML) undergoing sibling HSCT and receiving myeloablative conditioning. Donor Cen-B haplotype was also associated with reduced acute graft versus host disease grades II-IV whereas donor telomeric-B haplotype was associated with decreased incidence of CMV reactivation. In contrast, we were not able to demonstrate a reduced rate of relapse when the donor had KIR Cen-B, however relapse with a donor Cen-A haplotype was a competing risk factor to poor overall survival. Here we show that the presence of donor activating KIR led to improved outcome for the patient, potentially through reduced relapse rates and decreased incidence of acute GvHD translating to improved overall survival. This article is protected by copyright. All rights reserved.

Binding of TmHU to single dsDNA as observed by optical tweezers.

Optical tweezers are employed to study the action of the histone-like protein from Thermotoga maritima (TmHU) on DNA at a single molecule level. Binding and disruption of TmHU to and from DNA are found to take place in discrete steps of 4-5 nm length and a net binding enthalpy of about 16kBT. This is in reasonable agreement with a microscopic model that estimates the extension of the binding sites of the protein and evaluates the energetics mainly for bending of the DNA in the course of interaction.

Selection of RNase-resistant RNAs.

All RNA types are susceptible to ribonuclease (RNase) digestion, which might be a serious problem for several in vitro and in vivo applications. RNase resistance can be reached through chemical modifications or the selection of stable secondary structures via SELEX (systematic evolution of ligands by exponential enrichment). This chapter focuses on the selection of natural RNase-resistant RNAs, enriched by a selection process in the presence of RNase T1. Results of these investigations led to the identification of a particular structural motif, the tetraloop. Further applications could be the advised use of such motifs in order to reach higher stability of RNA molecules.

The semantics of procedures and diseases in SNOMED CT.

OBJECTIVES: SNOMED CT is emerging as a reference terminology for the entire health care process. It claims to be founded on logic-based modelling principles. In this article, we analyze a special encoding scheme for diseases and procedures in SNOMED CT, the so-called relationship groups (RGs), which had been devised to avoid ambiguities in definitions. METHODS: We reformulate SNOMED CT's relationship groups in the format of description logics in order to check whether RGs serve the needs they were designed for. RESULTS: We show that a considerable proportion of relationship groups represent hidden mereological relations. We also report discrepancies encountered between the defined semantics of many SNOMED CT terms and their intuitive meaning, as well as inconsistencies detected between the definition of various complex composed terms and the definition of their top-level parents. CONCLUSIONS: We formulate recommendations for improving SNOMED CT by replacing most occurrences of relation groups by formally more adequate "part-of" relations.

Synthesis and kinetic study of transition state analogs for ribonuclease T1.

Based on the proposal that ribonucleases cleave the RNA phosphodiester bond with a mechanism involving pentacovalent phosphorous as transition state, complexes of guanosine and inosine with vanadate-(IV, V), molybdate-(VI), tungstate-(VI), chromate-(VI) and hexacyanochromate-(III) were synthesized and probed as inhibitors of recombinant ribonuclease T1, obtained from an E. coli. overproducing strain. The apparent dissociation constants of these inhibitors and RNase T1, as determined by Michaelis-Menten kinetics, vary between 0.5-0.9 microM and indicate very strong binding, 100- to 1000-fold stronger than the binding of guanosine (Kd = 545 microM) and inosine (Kd = 780 microM), and 50-100-fold stronger than the binding of the product 3' GMP (Kd = 55 microM). Therefore the synthesized inhibitors may be considered as genuine transition state analogs for the enzyme.

Thermodynamic analysis of the equilibrium, association and dissociation of 2'GMP and 3'GMP with ribonuclease T1 at pH 5.3.

Fluorescence titrations and temperature-jump relaxation experiments were performed as a function of temperature on ribonuclease T1 with the inhibitors 2'GMP and 3'GMP to obtain information on the energetics and molecular events controlling the binding of those inhibitors. Results from the titration and temperature-jump experiments were in agreement concerning the equilibrium constant. The larger equilibrium constant for 2'GMP is enthalpic in origin and is due to both a higher on rate and a lower off rate as compared to 3'GMP. On rates for both inhibitors appear to be below the diffusion controlled limit, apparently due to conformational changes in the portion of the active site responsible for recognition of the guanine base. Comparison of the measured enthalpic and entropic terms associated with the equilibrium constant determined from the fluorescence titrations are in disagreement with those calculated from the on and off rates indicating the presence of an induced conformational change in the 2'GMP-enzyme complex. This second conformational change appears to be due to additional interactions between 2'GMP and the catalytic portion of the active site, which may also be responsible for the differences in the binding constant, the on rate and the off rate between 2'GMP and 3'GMP.

Conformation of thermally denatured RNase T1 with intact disulfide bonds: a study by small-angle X-ray scattering.

Small-angle X-ray scattering of RNase T1 with intact disulfide bonds was measured at 20 degrees and 60 degrees C in order to get insight into the structural changes of the protein caused by thermal denaturation. The radius of gyration increases from R(G)= 1.43 nm to R(G) = 2.21 nm. The conformations of the molecules at 60 degrees C are similar to those of ring-shaped random walk chains. However, the molecules are more compact than one would expect under theta conditions due to attractive interactions between the chain segments. The volume needed for free rotation of the thermally unfolded protein molecules about any axis in solution is five times greater than in the native state whereas the hydrodynamic effective volume is increasing only two times.

Stability and folding kinetics of ribonuclease T1 are strongly altered by the replacement of cis-proline 39 with alanine.

The refolding of ribonuclease T1 involves two major slow processes that exhibit properties of prolyl isomerization reactions. A comparison of the wild-type protein and a designed variant where the cis Ser54-Pro55 bond was replaced by a Gly54-Asn55 bond indicated that the faster of these reactions is the isomerization of Pro55. Here we report the replacement of the other cis proline of ribonuclease T1 at position 39 by alanine. The Pro39Ala variant is similar to the wild-type protein in secondary and tertiary structure, and the enzymatic activity towards RNA and a dinucleotide substrate remains almost unchanged. The fluorescence emission of the single Trp59 is lowered by the Pro39Ala substitution, probably because Trp59 is in close contact to Pro39 in wild-type ribonuclease T1. Unlike the substitution of cis Pro55, the Pro39Ala mutation is strongly destabilizing and reduces the Gibbs free energy of the folded protein by about 20 kJ/mol. Pro39 is buried in native RNase T1 and located near the active site. The observed destabilization could originate from the presence of a cis alanyl bond in the Pro39Ala variant or from a local distortion caused by the incorporation of a trans alanyl peptide bond in the interior of the protein. In the refolding kinetics the replacement of Pro39 leads to a disappearance of the fast-refolding species. Refolding still involves two consecutive slow steps. The first and faster step could be the isomerization of the remaining cis Pro55. The second, very slow step is a novel reaction that appears to have no counterpart in the refolding of the wild-type protein. All mutant molecules must undergo this reaction before reaching the native state. These major changes in the folding kinetics strongly indicate that cis-Pro39 is indeed of major importance for the folding of the wild-type protein. They indicate, moreover, that some new feature of protein folding kinetics is observed in these studies of the Pro39Ala variant.

Destabilization of a protein helix by electrostatic interactions.

Electrostatic interactions between charged residues and the helix dipole in a protein were investigated by protein engineering methods. In ribonuclease T1, two surface-exposed acidic residues (Glu28 and Asp29) are located near the carboxyl terminus of the alpha-helix between residues 13 and 29. They were replaced, individually and in concert, by the uncharged amides Gln28 and Asn29, and the stabilities of the wild-type protein and its variants were determined as a function of pH. The effects of the two mutations are additive. Either one leads to a marginal destabilization by 0.7 kJ/mol at pH 2 but to a strong stabilization by about 3.2 kJ/mol at pH 7. This suggests that the deprotonations of Glu28 and Asp29 reduce the free energy of stabilization of folded ribonuclease T1 by about 4 kJ/mol each. This destabilization is probably caused by unfavorable electrostatic interactions of Glu28 and Asp29 with the negative end of the helix dipole. The activation energies for the unfolding of the different variants of ribonuclease T1 change in parallel with the differences in the thermodynamic stability when the pH is varied. This indicates that the unfavorable electrostatic interactions of Glu28 and Asp29 are lost very early in unfolding, and are not present in the activated state of unfolding.