Long wavelength multiphoton excitation is advantageous for intravital kidney imaging.

Intravital multiphoton microscopy is a powerful tool to study kidney physiology in living animals. However, certain technical issues have curbed its usage to date, including limited depth of tissue penetration and high background emission of endogenous signals. Most previous studies have used the excitation range 700­1000 nm. Since newer longer wavelength excitation lasers may provide solutions to these problems we constructed a microscope coupled to a laser tunable up to 1300 nm and optimized for kidney imaging. This set-up offers substantial advantages for intravital studies, especially when coupled with newly available far-red probes. First, the background at longer wavelengths is markedly reduced, thus increasing the signal to background ratio. Second, the depth of tissue penetration is significantly increased, enabling detailed imaging of previously inaccessible structures, such as deeper glomeruli. Third, using a combination of two- and three-photon excitation, multiple different fluorescent probes can be imaged simultaneously in the same animal, with clear spectral separation. Application of these techniques helped visualize pathological aspects of tubular cell function in a well-established model of acute kidney injury (maleate toxicity). Thus, utilizing long wavelength excitation offers substantial advantages for intravital kidney imaging, which together enhance the capabilities of this powerful and increasingly used research technique.

Improved osteogenic vector for non-viral gene therapy.

Therapeutic compensation of deficient bone regeneration is a challenging task and a topic of on-going search for novel treatment strategies. One promising approach for improvement involves non-viral gene delivery using the bone morphogenetic protein-2 (BMP-2) gene to provide transient, local and sustained expression of the growth factor. However, since efficiency of non-viral gene delivery is low, this study focused on the improvement of a BMP-2 gene expression system, aiming for compensation of poor transfection efficiency. First, the native BMP-2 gene sequence was modified by codon optimisation and altered by inserting a highly truncated artificial intron (96 bp). Transfection of multiple cell lines and rat adipose-derived mesenchymal stem cells with plasmids harbouring the improved BMP-2 sequence led to a several fold increased expression rate and subsequent osteogenic differentiation. Additionally, comparing expression kinetics of elongation factor 1 alpha (EF1α) promoter with a state of the art CMV promoter revealed significantly higher BMP-2 expression when under the influence of the EF1α promoter. Results obtained by quantification of bone markers as well as osteogenic assays showed reduced sensitivity to promoter silencing effects of the EF1α promoter in rat adipose-derived mesenchymal stem cells. Finally, screening of several protein secretion signals using either luciferase or BMP-2 as reporter protein revealed no superior candidates for potential replacement of the native BMP-2 secretion signal. Taken together, by enhancing the exogenous BMP-2 expression system, low transfection efficiencies in therapeutic applications can be compensated, making safe non-viral systems even more suitable for tissue regeneration approaches.

Densification and strain hardening of a metallic glass under tension at room temperature.

The deformation of metallic glasses involves two competing processes: a disordering process involving dilatation, free volume accumulation, and softening, and a relaxation process involving diffusional ordering and densification. For metallic glasses at room temperature and under uniaxial loading, disordering usually dominates, and the glass can fail catastrophically as the softening process runs away in a localized mode. Here we demonstrate conditions where the opposite, unexpected, situation occurs: the densifying process dominates, resulting in stable plastic deformation and work hardening at room temperature. We report densification and hardening during deformation in a Zr-based glass under multiaxial loading, in a notched tensile geometry. The effect is driven by stress-enhanced diffusional relaxation, and is attended by a reduction in exothermic heat and hardening signatures similar to those observed in the classical thermal relaxation of glasses. The result is significant, stable, plastic, extensional flow in metallic glasses, which suggest a possibility of designing tough glasses based on their flow properties.

[Revised consensus classification. Histopathological classification of diseases associated with joint endoprostheses]. / Erweiterte Konsensusklassifikation. Histopathologische Klassifikation von Gelenkendoprothesen-assoziierten Erkrankungen.

The revised classification of the periprosthetic membrane (synovial-like interface membrane SLIM) encompasses all pathological alterations which can occur as a result of endoprosthetic replacement of major joints and lead to a reduction in durability of prostheses. This also includes the established consensus classification of SLIM by which aseptic and septic prosthetic loosening can be subdivided into four histological types and histopathological criteria for additional pathologies: endoprosthesis-associated arthrofibrosis, immunological/allergic alterations and osseous pathologies. This revision represents the foundation for the histopathological diagnostics of the total spectrum of diseases associated with joint prostheses, is a suitable basis for a standardized diagnostic procedure and etiological clarification of endoprosthesis failure and also as a data standard for endprosthesis registers, in particular for registers based on routine data (e.g. German endoprosthesis register).

Nanoscale Dynamics of Streptococcal Adhesion to AGE-Modified Collagen.

The adhesion of initial colonizers such as Streptococcus mutans to collagen is critical for dentinal and root caries progression. One of the most described pathological and aging-associated changes in collagen-including dentinal collagen-is the generation of advanced glycation end-products (AGEs) such as methylglyoxal (MGO)-derived AGEs. Despite previous reports suggesting that AGEs alter bacterial adhesion to collagen, the biophysics driving oral streptococcal attachment to MGO-modified collagen remains largely understudied. Thus, the aim of this work was to unravel the dynamics of the initial adhesion of S. mutans to type I collagen in the presence and absence of MGO-derived AGEs by employing bacterial cell force spectroscopy with atomic force microscopy (AFM). Type I collagen gels were treated with 10 mM MGO to induce AGE formation, which was characterized with microscopy and enzyme-linked immunosorbent assay. Subsequently, AFM cantilevers were functionalized with living S. mutans UA 159 or Streptococcus sanguinis SK 36 cells and probed against collagen surfaces to obtain force curves displaying bacterial attachment in real time, from which the adhesion force, number of events, Poisson analysis, and contour and rupture lengths for each individual detachment event were computed. Furthermore, in silico computer simulation docking studies between the relevant S. mutans UA 159 collagen-binding protein SpaP and collagen were computed, in the presence and absence of MGO. Overall, results showed that MGO modification increased both the number and adhesion force of single-unbinding events between S. mutans and collagen, without altering the contour or rupture lengths. Both experimental and in silico simulations suggest that this effect is due to increased specific and nonspecific forces and interactions between S. mutans UA 159 and MGO-modified collagen substrates. In summary, these results suggest that collagen alterations due to aging and glycation may play a role in early bacterial adherence to oral tissues, associated with conditions such as aging or chronic hyperglycemia, among others.

Nanomechanical and Molecular Characterization of Aging in Dentinal Collagen.

Methylglyoxal (MGO) is an important molecule derived from glucose metabolism with the capacity of attaching to collagen and generating advanced glycation end products (AGEs), which accumulate in tissues over time and are associated with aging and diseases. However, the accumulation of MGO-derived AGEs in dentin and their effect on the nanomechanical properties of dentinal collagen remain unknown. Thus, the aim of the present study was to quantify MGO-based AGEs in the organic matrix of human dentin as a function of age and associate these changes with alterations in the nanomechanical and ultrastructural properties of dentinal collagen. For this, 12 healthy teeth from <26-y-old and >50-y-old patients were collected and prepared to obtain crown and root dentin discs. Following demineralization, MGO-derived AGEs were quantified with a competitive ELISA. In addition, atomic force microscopy nanoindentation was utilized to measure changes in elastic modulus in peritubular and intertubular collagen fibrils. Finally, principal component analysis was carried out to determine aging profiles for crown and root dentin. Results showed an increased presence of MGO AGEs in the organic matrix of dentin in the >50-y-old specimens as compared with the <26-y-old specimens in crown and root. Furthermore, an increase in peritubular and intertubular collagen elasticity was observed in the >50-y-old group associated with ultrastructural changes in the organic matrix as determined by atomic force microscopy analysis. Furthermore, principal component analysis loading plots suggested different "aging profiles" in crown and root dentin, which could have important therapeutic implications in restorative and adhesive dentistry approaches. Overall, these results demonstrate that the organic matrix of human dentin undergoes aging-related changes due to MGO-derived AGEs with important changes in the nanomechanical behavior of collagen that may affect diagnostic and restorative procedures in older people.

Modulatory Effect of Glycated Collagen on Oral Streptococcal Nanoadhesion.

Biofilm-mediated oral diseases such as dental caries and periodontal disease remain highly prevalent in populations worldwide. Biofilm formation initiates with the attachment of primary colonizers onto surfaces, and in the context of caries, the adhesion of oral streptococci to dentinal collagen is crucial for biofilm progression. It is known that dentinal collagen suffers from glucose-associated crosslinking as a function of aging or disease; however, the effect of collagen crosslinking on the early adhesion and subsequent biofilm formation of relevant oral streptococci remains unknown. Therefore, the aim of this work was to determine the impact of collagen glycation on the initial adhesion of primary colonizers such as Streptococcus mutans UA159 and Streptococcus sanguinis SK 36, as well as its effect on the early stages of streptococcal biofilm formation in vitro. Type I collagen matrices were crosslinked with either glucose or methylglyoxal. Atomic force microscopy nanocharacterization revealed morphologic and mechanical changes within the collagen matrix as a function of crosslinking, such as a significantly increased elastic modulus in crosslinked fibrils. Increased nanoadhesion forces were observed for S. mutans on crosslinked collagen surfaces as compared with the control, and retraction curves obtained for both streptococcal strains demonstrated nanoscale unbinding behavior consistent with bacterial adhesin-substrate coupling. Overall, glucose-crosslinked substrates specifically promoted the initial adhesion, biofilm formation, and insoluble extracellular polysaccharide production of S. mutans, while methylglyoxal treatment reduced biofilm formation for both strains. Changes in the adhesion behavior and biofilm formation of oral streptococci as a function of collagen glycation could help explain the biofilm dysbiosis seen in older people and patients with diabetes. Further studies are necessary to determine the influence of collagen crosslinking on the balance between acidogenic and nonacidogenic streptococci to aid in the development of novel preventive and therapeutic treatment against dental caries in these patients.

A prospective evaluation of the medical consultation system CADIAG-II/RHEUMA in a rheumatological outpatient clinic.

To evaluate the performance of CADIAG-II/RHEUMA as consultant in the primary evaluation of patients visiting a rheumatological outpatient clinic, a CADIAG-II/RHEUMA consultation was done for 54 patients and the list of generated diagnostic hypotheses was compared to each clinical discharge diagnosis. For 26 of a total of 126 rheumatological discharge diagnoses, no matching CADIAG-II/RHEUMA diagnosis was available. 94% of all other discharge diagnoses were found in the list of CADIAG-II/RHEUMA hypotheses, 82% among the first third of the list of hypotheses and 48% among the first five hypotheses. We identified the following factors limiting the ability of CADIAG-II/RHEUMA to generate a comprehensive and correctly ranked list of diagnostic hypotheses: (1) a large percentage of patients with early stages of not clearly identified rheumatological conditions; (2) the limited number of CADIAG-II/RHEUMA diagnoses compared to the large number of known rheumatological conditions; (3) the fact that rheumatological diseases are rarely characterized by a single pathognomonic feature but are usually diagnosed by combinations of rather unspecific findings.

Medical pioneers in cyberspace: German practice owners advertising on the WWW.

BACKGROUND: In the last few years, the number of Internet users has increased explosively. In the same way the number of Internet users has exploded, the costs in the public health sector have also increased. This resulted cost saving efforts by those responsible people in politics and medical administration. These economy measures have impacted in particular the established physicians. The current German practice owners are faced with an unknown economic situation and are forced to think and act like businessmen. Doubts arise concerning the age-old tradition of the advertising prohibition. Now advertisement is recognized as an important necessity. OBJECTIVES: This study was conducted to answer the following questions: Who are the pioneers among the German practice owners presenting themselves to the public with their own website? How do they differ from their colleagues not advertising on the WWW? What motives and expectations do they associate with their website? METHODS: Built on a detailed analysis of the relevant German and international literature, hypotheses were developed which were empirically checked in the further course of the work. For this purpose, an online survey was conducted on the WWW among established German physicians with their own websites. RESULTS: 194 physicians participated and 159 valid questionnaires were included in the analysis. The study revealed the following results: The age and sex distribution as well as the distribution of medical specialties in the examined group correspond to the expectations. A high percentage of the respondents participated in a medical professional organization. The median time in practice for practice age of the respondents was a little more than ten years. Many of the websites have been online less than one year. The following hypotheses could only partly be confirmed by the results of the survey: Physicians from different specialties deal with their own website differently. The Internet Familiarity of the physicians is responsible for the importance they attach to advertisement on the web, particularly to their own website. Surprisingly, the attitude towards the advertising prohibition in Germany, apparently results less from economic considerations than from age-conditioned opinions. The size of a medical practice did not influence the attitude of the physicians towards their own website. However, the type of practice in which a physician works played a crucial role in this context. CONCLUSIONS: At present, the importance of the Internet for recruiting new patients is still small, but we anticipate it will continue to expand in the future.

Decreased food intake is a risk factor for mortality in hospitalised patients: the NutritionDay survey 2006.

BACKGROUND & AIMS: Malnutrition is a known risk factor for the development of complications in hospitalised patients. We determined whether eating only fractions of the meals served is an independent risk factor for mortality. METHODS: The NutritionDay is a multinational one-day cross-sectional survey of nutritional factors and food intake in 16,290 adult hospitalised patients on January 19th 2006. The effect of food intake and nutritional factors on death in hospital within 30 days was assessed in a competing risk analysis. RESULTS: More than half of the patients did not eat their full meal provided by the hospital. Decreased food intake on NutritionDay or during the previous week was associated with an increased risk of dying, even after adjustment for various patient and disease related factors. Adjusted hazard ratio for dying when eating about a quarter of the meal on NutritionDay was 2.10 (1.53-2.89); when eating nothing 3.02 (2.11-4.32). More than half of the patients who ate less than a quarter of their meal did not receive artificial nutrition support. Only 25% patients eating nothing at lunch receive artificial nutrition support. CONCLUSION: Many hospitalised patients in European hospitals eat less food than provided as regular meal. This decreased food intake represents an independent risk factor for hospital mortality.

Quantitative insight into dislocation nucleation from high-temperature nanoindentation experiments.

Nanoindentation has become ubiquitous for the measurement of mechanical properties at ever-decreasing scales of interest, including some studies that have explored the atomic-level origins of plasticity in perfect crystals. With substantial guidance from atomistic simulations, the onset of plasticity during nanoindentation is now widely believed to be associated with homogeneous dislocation nucleation. However, to date there has been no compelling quantitative experimental support for the atomic-scale mechanisms predicted by atomistic simulations. Our purpose here is to significantly advance the quantitative potential of nanoindentation experiments for the study of dislocation nucleation. This is accomplished through the development and application of high-temperature nanoindentation testing, and the introduction of statistical methods to quantitatively evaluate data. The combined use of these techniques suggests an unexpected picture of incipient plasticity that involves heterogeneous nucleation sites, and which has not been anticipated by atomistic simulations.

Pressure-induced transformation plasticity of H(2)O ice.

Transformation plasticity is a deformation mechanism which occurs during phase transformation of an externally stressed material. Pressure-induced transformation plasticity of ice could be relevant to the geology of the moons of the outer solar system, and has long been postulated to occur in olivine in the earth's interior. In the present work, ice specimens were subjected to isostatic pressure cycling between 0 and 300 MPa to induce reversible polymorphic transformation between ice I and II at 230 K. When a small uniaxial compressive stress was applied during cycling, the specimens exhibited a compressive strain (as large as 18.5% after a single cycle) proportional to the applied stress, in agreement with observations and theory for transformation plasticity of metals induced by polymorphic thermal cycling.

Bovine cysticercosis: demonstration in experimentally infected calves of serum IgG antibodies reactive with neutral glycolipids of Taenia saginata and T. crassiceps metacestodes.

The immunoreactivity of Taenia saginata and T. crassiceps metacestode neutral glyco(sphingo)lipids towards IgG antibodies derived from the sera of calves with experimental cysticercosis has been established. The glyco(sphingo)lipids are separable by normal-phase HPTLC (high-performance thin-layer chromatography) into groups of increasing sugar-chain length (lipid/ceramide mono-, di-, tri-, tetra- and > tetrasaccharides), with those corresponding to three and four hexoses being the main immunoreactive components (HPTLC immunostaining). In ELISA (enzyme-linked immunosorbent assay), reverse-phase HPTLC-isolated T. crassiceps metacestode glyco(sphingo)lipids equivalent to tri- and tetrahexoside allowed a discrimination between non-infected and infected calves (at least 80 metacestodes recovered). The formation of IgG antibodies was correlated with the infection, not with other non-specific inducing factors, as seen by the differential humoral response detected in experimentally infected (T. saginata) calves before and after Praziquantel treatment (HPTLC immunostaining and ELISA).