Long-term in vivo degradation of Mg-Zn-Ca elastic stable intramedullary nails and their influence on the physis of juvenile sheep.

The use of bioresorbable magnesium (Mg)-based elastic stable intramedullary nails (ESIN) is highly promising for the treatment of pediatric long-bone fractures. Being fully resorbable, a removal surgery is not required, preventing repeated physical and psychological stress for the child. Further, the osteoconductive properties of the material support fracture healing. Nowadays, ESIN are exclusively implanted in a non-transphyseal manner to prevent growth discrepancies, although transphyseal implantation would often be required to guarantee optimized fracture stabilization. Here, we investigated the influence of trans-epiphyseally implanted Mg-Zinc (Zn)-Calcium (Ca) ESIN on the proximal tibial physis of juvenile sheep over a period of three years, until skeletal maturity was reached. We used the two alloying systems ZX10 (Mg-1Zn-0.3Ca, in wt%) and ZX00 (Mg-0.3Zn-0.4Ca, in wt%) for this study. To elaborate potential growth disturbances such as leg-length differences and axis deviations we used a combination of in vivo clinical computed tomography (cCT) and ex vivo micro CT (µCT), and also performed histology studies on the extracted bones to obtain information on the related tissue. Because there is a lack of long-term data regarding the degradation performance of magnesium-based implants, we used cCT and µCT data to evaluate the implant volume, gas volume and degradation rate of both alloying systems over a period of 148 weeks. We show that transepiphyseal implantation of Mg-Zn-Ca ESIN has no negative influence on the longitudinal bone growth in juvenile sheep, and that there is no axis deviation observed in all cases. We also illustrate that 95 % of the ESIN degraded over nearly three years, converging the time point of full resorption. We thus conclude that both, ZX10 and ZX00, constitute promising implant materials for the ESIN technique.

Comparison of a resorbable magnesium implant in small and large growing-animal models.

Fracture treatment in children needs new implant materials to overcome disadvantages associated with removal surgery. Magnesium-based implants constitute a biocompatible and bioresorbable alternative. In adults and especially in children, implant safety needs to be evaluated. In children the bone turnover rate is higher and implant material might influence growth capacity, and the long-term effect of accumulated particles or ions is more critical due to the host's prolonged post-surgery lifespan. In this study we aimed to investigate the degradation behavior of ZX00 (Mg-0.45Zn-0.45Ca; in wt.%) in a small and a large animal model to find out whether there is a difference between the two models (i) in degradation rate and (ii) in bone formation and in-growth. Our results 6, 12 and 24 weeks after ZX00 implantation showed no negative effects on bone formation and in-growth, and no adverse effects such as fibrotic or sclerotic encapsulation. The degradation rate did not significantly differ between the two growing-animal models, and both showed slow and homogeneous degradation performance. Our conclusion is that small animal models may be sufficient to investigate degradation rates and provide preliminary evidence on bone formation and in-growth of implant materials in a growing-animal model. STATEMENT OF SIGNIFICANCE: The safety of implant material is of the utmost importance, especially in children, who have enhanced bone turnover, more growth capacity and longer postoperative lifespans. Magnesium (Mg)-based implants have long been of great interest in pediatric orthopedic and trauma surgery, due to their good biocompatibility, biodegradability and biomechanics. In the study documented in this manuscript we investigated Mg-Zn-Ca implant material without rare-earth elements, and compared its outcome in a small and a large growing-animal model. In both models we observed bone formation and in-growth which featured no adverse effects such as fibrotic or sclerotic encapsulation, and slow homogeneous degradation performance of the Mg-based implant material.

Modelling the elastic scattering in diagnostic radiology: the importance of structure form factors.

The importance of structure form factors in describing elastic scattering in diagnostic radiology was studied through a Monte Carlo code built to reproduce scattering in large water samples. The code, developed by us, considers all relevant interactions, including multiple scattering and interference due to scattering by the liquid structure. Geometrical conditions and energies similar to those found in radiology were used. The secondary to primary radiation ratio using the usual free atom approximation and the structure form factor was obtained and both approaches were compared. Calculations of radiological parameters such as the angular distribution of photons incident on the detector and the fraction of scattered photons stopped by anti-scattering grids were also performed considering mammography, thorax and abdomen radiography conditions. The results have shown that S(beta)/P depends on the experimental set-up, being more important for low momentum transfers and sample sizes for which the multiple scattering is not expected to be significant, as in the case of mammography. It was also verified that large samples increase the probability of multiple scattering, masking the structure peak in S(beta) and making the sample structure important just for relatively thin samples. Considering mammography-like geometry, the maximum of the S(beta)/P distribution considering structure form factors occurs around 15 degrees while the correspondent maximum without considering the structure factors occurs around 10 degrees for any sample thickness. S(beta)/P is almost independent of the irradiation field, with the maximum remaining at 15 degrees and 10 degrees for the SFF and FAFF, respectively. The cases studied in this paper stress some conditions in which it is mandatory to use SFF, but since it requires no further significant efforts, the SFF approach is recommended as a standard procedure when describing the elastic scattering process in radiology.

Characterization of inverted membrane vesicles from the halophilic archaeon Haloferax volcanii.

Membrane-related processes in archaea, the third and most-recently described domain of life, are in general only poorly understood. One obstacle to a functional understanding of archaeal membrane-associated activities corresponds to a lack of archaeal model membrane systems. In the following, characterization of inverted archaeal membrane vesicles, prepared from the halophilic archaeon Haloferax volcanii, is presented. The inverted topology of the vesicles was revealed by defining the orientation of membrane-bound enzymes that in intact cells normally face the cytoplasm or of other protein markers, known to face the exterior medium in intact cells. Electron microscopy, protease protection assays and lectin-binding experiments confirmed the sealed nature of the vesicles. Upon alkalinization of the external medium, the vesicles were able to generate ATP, reflecting the functional nature of the membrane preparation. The availability of preparative scale amounts of inverted archaeal membrane vesicles provides a platform for the study of various membrane-related phenomena in archaea.

[Introduction to tissue optics and optical dosimetry]. / Einführung in die Gewebeoptik und optische Dosimetrie.

The spatial distribution of radiation during medical laser application is determined by the characteristics of the beam (power, time, beam geometry) and the optical properties of the tissue. The irradiance E (in W/m2) describes the primary laser beam. Scattered radiation, in turn, is taken into account by the fluence rate phi (also in W/m2). The basic parameters of tissue optics are the absorption coefficient mu a, the scattering coefficient mu s and the anisotropy factor g. In addition, derived parameters are also used, i.e., total attenuation coefficient mu t, reduced scattering coefficient mu s', effective attenuation coefficient mu eff, mean free path of a photon d and penetration depth delta. Further tissue properties are the diffuse reflectance Rd and the back-scattering factor k. In an one-dimensional model the fluence rate phi in tissue is a nearly exponential function characterized by the penetration depth delta. At the tissue surface, the relationship exists phi = kE. This model is compared with the results of a computer program based on the finite element method.

Influence of pulse duration on ultrashort laser pulse ablation of biological tissues.

Ablation characteristics of ultrashort laser pulses were investigated for pulse durations in the range of 130 fs-10 ps. Tissue samples used in the study were dental hard tissue (dentin) and water. We observed differences in ablation crater morphology for craters generated with pulse durations in the 130 fs-1 ps and the 5 ps-10 ps range. For the water experiment, the surface ablation and subsequent propagation of stress waves were monitored using Mach-Zehnder interferometry. For 130 fs-1 ps, energy is deposited on the surface while for longer pulses the beam penetrates into the sample. Both studies indicate that a transition occurs between 1 and 5 ps.

The signal recognition particle of Archaea.

It is becoming increasingly clear that similarities exist in the manner in which extracytoplasmic proteins are targeted to complexes responsible for translocating these proteins across membranes in each of the three domains of life. In Eukarya and Bacteria, the signal recognition particle (SRP) directs nascent polypeptides to membrane-embedded translocation sites. In Archaea, the SRP protein targeting pathway apparently represents an intermediate between the bacterial and eukaryal systems. Understanding the archaeal SRP pathway could therefore reveal universal aspects of targeting not detected in current comparisons of the eukaryal and bacterial systems while possibly identifying aspects of the process either not previously reported or unique to Archaea.

Collagen structure and nonlinear susceptibility: effects of heat, glycation, and enzymatic cleavage on second harmonic signal intensity.

BACKGROUND AND OBJECTIVE: Helical macromolecules such as collagen and DNA are characterized by nonlinear optical properties, including nonlinear susceptibility. Because collagen is the predominant component of most biological tissues, as well as the major source of second harmonic generation (SHG), it is reasonable to assume that changes in harmonic signal can be attributed to structural changes in collagen. The purpose of this study is to determine whether various modifications of collagen structure affect second harmonic intensity. STUDY DESIGN/MATERIALS AND METHODS: SHG was measured in tissues from cows, humans, and chickens. The effects of beam polarization, thermal denaturation, glyco-oxidative damage, and enzymatic cleavage of tissues on second harmonic intensity was studied. RESULTS: The second harmonic intensity differed considerably among different tissues, as did the effect of the incident beam polarization. In structurally modified collagen, SHG was significantly degraded from SHG in intact collagen. CONCLUSION: These structural modifications are representative of changes that occur in pathophysiologic conditions such as thermal injury, diabetes, tumor invasion, and abnormal wound healing. The ability to assess these changes rapidly and noninvasively has considerable clinical applicability. SHG analysis might provide a unique tool for monitoring these structural changes of collagen.

Temperature distribution for combined laser hyperthermia-photodynamic therapy in the esophagus.

In recent years photodynamic laser therapy (PDT) has been tested in animal and clinical studies for treatment of esophageal cancer. In several animal experiments a synergistic effect was found by simultaneously applying PDT and hyperthermia (HT). In this paper an optical fibre system is described which can be used in the esophagus for combined PDT with a 1 W dye laser and HT with a 15 W Nd:YAG laser. A phantom was built simulating the geometry of the esophagus using cow muscle. The spatial temperature field during HT was measured versus irradiation time. The results were compared with calculations using a coupled Monte Carlo laser transport/finite difference heat transport model using the LATIS computer program. Measurements and calculations yield a realistic description of the temperature distribution during HT under various experimental conditions. The LATIS program allows the prediction of the effects of blood perfusion for in vivo situations. The results show that perfusion has considerable influence on the temperature field, reducing the effective depth in tissue for HT.

[In vitro experiments with Nd:YAG laser surgery on the turbinates]. / In-vitro-Experimente zur Nd:YAG-Laserchirurgie an den Nasenmuscheln.

BACKGROUND: Turbinate surgery is a therapeutic method for the treatment of the obstruction of nasal respiration. In this paper the dimensions of the laser lesions are described. In addition macro- and microscopical findings after laser surgery are given. METHODS: 10 human lower and 4 middle turbinates in vitro were treated with the Nd:YAG-laser in the non-contact mode (1064 nm, 2.5-25 W, cw). Stripe-like lesion with 3 cm length were produced. In addition the posterior end of the lower turbinates and the head of the middle turbinates were vaporized. RESULTS: Width, depth and volume of the lesions are given in dependence of laser power and irradiation time. The histological changes immediately after laser treatment are described. CONCLUSIONS: The energy doses for a clinical relevant stripe-like laser lesion of 3-4 cm in length of the lower turbinate is about 1500 Ws using Nd:YAG-laser. For evaporation of a posterior end of the lower turbinate 360 Ws are required using Nd:YAG-laser. For evaporation of the head of the middle turbinate a doses of about 1500 Ws are required using Nd:YAG-laser.

The SecA subunit of Escherichia coli preprotein translocase is exposed to the periplasm.

SecA undergoes conformational changes during translocation, inserting domains into and across the membrane or enhancing the protease resistance of these domains. We now show that some SecA bound at SecYEG is accessible from the periplasm to a membrane-impermeant probe in cells with a permeabilized outer membrane but an intact plasma membrane.

125I-labeled fasciculin 2: a new tool for quantitation of acetylcholinesterase densities at synaptic sites by EM-autoradiography.

Radio-iodinated fasciculin 2 (Fas2), a polypeptide anticholinesterase toxin from Mamba venom, was used as a new probe for localizing and quantifying acetylcholinesterase (AChE) at mouse neuromuscular junctions (NMJs) by quantitative electron microscope autoradiography. We demonstrate that 125I-Fas2 binds very specifically to the NMJs of mouse sternomastoid muscles, with very little binding to other regions in the muscles. Junctional AChE-site densities obtained from the autoradiograms were similar to those previously obtained for the same muscles using 3H-DFP. The use of 125I-Fas2 with EM-autoradiography is simpler and provides higher resolution and sensitivity, as well as considerably lower non-specific binding than previously attainable with 3H-DFP. The advantages and limitations of this procedure are discussed.

Endogenous SecA catalyzes preprotein translocation at SecYEG.

SecA is found in the cytosol and bound to the plasma membrane of Escherichia coli. Binding occurs either with high affinity at SecYEG or with low affinity to lipid. Domains of 65 and 30 kDa of SecYEG-bound SecA insert into the membrane upon interaction with preprotein and ATP. Azide blocks preprotein translocation, in vivo and in vitro, through interacting with SecA and preventing SecA deinsertion. This provides a measure of the translocation relevance of each form of SecA membrane association. We now report that azide acts exclusively on SecA that is cycling at SecYEG and has no effect on SecA lipid associations. SecA molecules recovered with sucrose gradient-purified inner membrane vesicles ("endogenous" SecA) support translocation at the same rate as "added" SecA molecules bound at SecYEG. Both endogenous and added SecA yield the same proteolytic fragments, which are distinct from those obtained from SecA once it has inserted into membranes at SecYEG or from SecA at lipidic sites. Endogenous and added SecA differ, however, in their resistance to urea extraction. The translocation supported by either endogenous or added SecA is blocked by azide or by antibody to SecY. We conclude that SecA functions in preprotein translocation only through cycling at SecYEG.

The protease-protected 30 kDa domain of SecA is largely inaccessible to the membrane lipid phase.

SecA binds to the inner membrane of Escherichia coli through low affinity lipid interactions or with high affinity at SecYEG, the integral domain of preprotein translocase. Upon addition of preprotein and nucleotide, a 30 kDa domain of SecYEG-bound SecA is protected from proteolysis via membrane insertion. Such protection could result from some combination of insertion into the lipid phase, into a proteinaceous environment or across the membrane. To assess the exposure of SecYEG-bound SecA to membrane lipids, a radiolabeled, photoactivatable and lipid-partitioning crosslinker, 3-trifluoromethyl-3-(m[125I]iodophenyl) diazirine benzoic acid ester, was incorporated into inner membrane vesicles. The 30 kDa domain of SecYEG-bound SecA, inserted into the membrane in response to translocation ligands, is 18-fold less labeled than SecY, which is labeled effectively. In contrast, incorporation of the purified 30 kDa SecA fragment into crosslinker-containing detergent micelles or addition of detergent to crosslinker-containing membranes bearing the protease-protected SecA domain readily allows for labeling of this domain. We propose that the protease-inaccessible 30 kDa SecA domain is shielded from the fatty acyl membrane phase by membrane-spanning SecYEG helices and/or is largely exposed to the periplasm.

Both an N-terminal 65-kDa domain and a C-terminal 30-kDa domain of SecA cycle into the membrane at SecYEG during translocation.

SecA, a 102-kDa hydrophilic protein, couples the energy of ATP binding to the translocation of preprotein across the bacterial inner membrane. SecA function and topology were studied with metabolically labeled [35S]SecA and with inner membrane vesicles from cells that overexpressed SecYEGDFyajC, the integral domain of preprotein translocase. During translocation in the presence of ATP and preprotein, a 65-kDa N-terminal domain of SecA is protected from proteolytic digestion through insertion into the membrane, as previously reported for a 30-kDa C-terminal domain [Economou, A. & Wickner, W. (1994) Cell 78, 835-843]. Insertion of both domains occurs at saturable SecYEGDFyajC sites and is rapidly followed by deinsertion. SecA also associates nonsaturably and unproductively with lipid. In the presence of ATP, yet without involvement of preprotein or SecYEG, lipid-bound SecA forms domains that are protease-resistant and that remain so even upon subsequent membrane disruption. Unlike the [35S]SecA that inserts into the membrane at SecYEGDFyajC as it promotes preprotein translocation, lipid-associated [35S]SecA does not chase from its protease-resistant state upon the addition of excess SecA. The finding that two domains of SecA (which together represent most regions of the polypeptide chain) cycle into the membrane during preprotein translocation, as well as the distinction between the membrane association of SecA at translocation sites of SecYEGDFyajC and at nonproductive lipid sites, are fundamental to the study of the role of SecA in preprotein movement.

Inclusion volume solid-phase synthesis.

Solid-phase synthesis of peptides was carried out using only the volume of the solvent included in the swollen solid-phase resin heads (inclusion volume synthesis). This approach enables (i) the use of higher concentrations of activated amino acids, resulting in increased coupling rates, (ii) drastically decreased consumption of solvents, and (iii) the construction of multiple peptide synthesizers having virtually no reaction vessels.

Placas radiográficas amplificadoras: estudo da contribuiçäo dos raios-x secundários / Amplifier radiographic plaques: contribuition study of secundary x-rays

Placas amplificadoras fluorescentes para filmes de raio-X são usadas em radiologia a fim de reduzir a dose de radiação. Estas placas produzem luz visível que aumenta a eficiência do filme. Adicionalmente, raios-X secundários são originados devido à efeito fotoelétrico, espalhamento elástico (Rayleigh( e inelástico (Compton). A taxa de contagem e a distribuição angular destes raios-X foram medidos, mostrando que a razão da radiação secundária pela primária incidente no filme de raio-X é cerca de 20 por cento.

To reduce the radiation dose in radiology, fluorescent intensifying screens for X-ray films are used. They produce visible light which increases the efficiency of the film. ln addition, secondary X-rays arise due to the photoelectric effect, elastic (Rayleigh) and inelastic (Compton) scattering. The counting rate and angular distribution of these X-rays were measured, showing that the ratio of secondary-to-primary radiation incident on the X-ray film is about 20 %

SecYEG and SecA are the stoichiometric components of preprotein translocase.

The transport of large preproteins across the Escherichia coli plasma membrane is catalyzed by preprotein translocase, comprised of the peripherally bound SecA subunit and an integrally bound heterotrimeric domain consisting of the SecY, SecE, and SecG subunits. We have now placed the secY, secE, and secG genes under the control of an arabinose-inducible promoter on a multicopy plasmid. Upon induction, all three of the proteins are strongly overexpressed and recovered in the plasma membrane fraction. These membranes show a strong enhancement of 1) translocation ATPase activity, 2) preprotein translocation, 3) capacity for SecA binding, and 4) formation of the membrane-inserted form of SecA. These data establish that SecY, SecE, and SecG constitute the integral membrane domain of preprotein translocase.

Generation and utilization of synthetic combinatorial libraries.

The use of combinatorial chemistry is fundamentally changing the pace and scope of basic research and drug discovery. Since the introduction of synthetic peptide libraries several years ago, combinatorial chemistry has proven to be a powerful tool for the generation of immense molecular diversities of peptides, peptidomimetics and new organic compounds. This article briefly reviews methods for the generation and application of combinatorial libraries, with particular emphasis on soluble synthetic combinatorial libraries. The utility of these molecular diversities for basic research and drug discovery has been demonstrated through the identification of numerous highly active compounds such as antigenic peptides, receptor ligands, antimicrobial compounds and enzyme inhibitors.