Unilateral Condylar Hyperplasia: A 3-Dimensional Quantification of Asymmetry

Purpose

Objective quantifications of facial asymmetry in patients with Unilateral Condylar Hyperplasia (UCH) have not yet been described in literature. The aim of this study was to objectively quantify soft-tissue asymmetry in patients with UCH and to compare the findings with a control group using a new method.

Material and Methods

Thirty 3D photographs of patients diagnosed with UCH were compared with 30 3D photographs of healthy controls. As UCH presents particularly in the mandible, a new method was used to isolate the lower part of the face to evaluate asymmetry of this part separately. The new method was validated by two observers using 3D photographs of five patients and five controls.

Results

A significant difference (0.79 mm) between patients and controls whole face asymmetry was found. Intra- and inter-observer differences of 0.011 mm (−0.034–0.011) and 0.017 mm (−0.007–0.042) respectively were found. These differences are irrelevant in clinical practice.

Conclusion

After objective quantification, a significant difference was identified in soft-tissue asymmetry between patients with UCH and controls. The method used to isolate mandibular asymmetry was found to be valid and a suitable tool to evaluate facial asymmetry.     

Cellular entry pathway and gene transfer capacity of TAT-modified lipoplexes

Several reports have shown a fast and efficient translocation of TAT-modified lipoplexes and particles into the cell cytoplasm. However, neither the uptake mechanism nor the biological effect of TAT-modified lipoplexes has been studied in detail. In this report we show that the increase in gene transfer of TAT-modified lipoplexes depends on the amount of cationic lipid in the lipoplexes and on the way TAT was coupled to the lipoplexes. We demonstrate that the cellular uptake of both TAT-modified and unmodified lipoplexes is very fast and, in contrast to previous publications, temperature-dependent. Additionally, after internalization TAT-modified as well as unmodified lipoplexes end up in lysosomal vesicles, indicating the involvement of clathrin-mediated endocytosis. Furthermore, chlorpromazine, a specific inhibitor of clathrin-dependent endocytosis, strongly inhibits the cellular uptake and biological activity of both the TAT-modified and unmodified lipoplexes. We also found that the uptake and biological activity of these lipoplexes are diminished when cholesterol in the cell membrane was bound by filipin, an inhibitor of the lipid-raft mediated pathway. Considering these data, we conclude that TAT-modified and unmodified lipoplexes are mainly internalized via a cholesterol-dependent clathrin-mediated pathway.     

Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples

We present a truly quantitative fluorescence recovery after photobleaching (FRAP) model for use with the confocal laser scanning microscope based on the photobleaching of a long line segment. The line FRAP method is developed to complement the disk FRAP method reported before. Although being more subject to the influence of noise, the line FRAP model has the advantage of a smaller bleach region, thus allowing for faster and more localized measurements of the diffusion coefficient and mobile fraction. The line FRAP model is also very well suited to examine directly the influence of the bleaching power on the effective bleaching resolution. We present the outline of the mathematical derivation, leading to a final analytical expression to calculate the fluorescence recovery. We examine the influence of the confocal aperture and the bleaching power on the measured diffusion coefficient to find the optimal experimental conditions for the line FRAP method. This will be done for R-phycoerythrin and FITC-dextrans of various molecular weights. The ability of the line FRAP method to measure correctly absolute diffusion coefficients in three-dimensional samples will be evaluated as well. Finally we show the application of the method to the simultaneous measurement of free green fluorescent protein diffusion in the cytoplasm and nucleus of living A549 cells.     

Targeted molecular engineering of a family 11 endoxylanase to decrease its sensitivity towards Triticum aestivum endoxylanase inhibitor types

The Bacillus subtilis endoxylanase XynA (BSXY) is frequently used to improve the functionality of arabinoxylan-containing material in cereal based industries. The presence of endogenous Triticum aestivum xylanase inhibitors (TAXI-I and TAXI-II) in wheat is a real concern as they have a direct negative impact on the efficiency of this enzyme. Here, we used the recently determined structure of the complex between TAXI-I and an endoxylanase of Aspergillus niger to develop inhibitor-insensitive BSXY variants by site-directed mutagenesis of strategically chosen amino acids. We either induced steric hindrance to reject the inhibitors or interrupted key interactions with the inhibitors in the endoxylanase substrate-binding groove. The first strategy was successfully applied to position G12 where G12W combined inhibition insensitivity with unharmed catalytic performance. Variants from the second strategy showed altered inhibitor sensitivities concomitant with changes in enzyme activities and allowed to gain insight in the binding-mode of both TAXI-I and TAXI-II with BSXY.     

Nestling telomere shortening,but not telomere length,reflects developmental stress and predicts survival in wild birds

Developmental stressors often have long-term fitness consequences, but linking offspring traits to fitness prospects has remained a challenge. Telomere length predicts mortality in adult birds, and may provide a link between developmental conditions and fitness prospects. Here, we examine the effects of manipulated brood size on growth, telomere dynamics and post-fledging survival in free-living jackdaws. Nestlings in enlarged broods achieved lower mass and lost 21% more telomere repeats relative to nestlings in reduced broods, showing that developmental stress accelerates telomere shortening. Adult telomere length was positively correlated with their telomere length as nestling (r = 0.83). Thus, an advantage of long telomeres in nestlings is carried through to adulthood. Nestling telomere shortening predicted post-fledging survival and recruitment independent of manipulation and fledgling mass. This effect was strong, with a threefold difference in recruitment probability over the telomere shortening range. By contrast, absolute telomere length was neither affected by brood size manipulation nor related to survival. We conclude that telomere loss, but not absolute telomere length, links developmental conditions to subsequent survival and suggest that telomere shortening may provide a key to unravelling the physiological causes of developmental effects on fitness.     

Recipient leukocyte infusion enhances the local and systemic graft-versus-neuroblastoma effect of allogeneic bone marrow transplantation in mice

Allogeneic hematopoietic stem cell transplantation and donor leukocyte infusion (DLI) may hold potential as a novel form of immunotherapy for high-risk neuroblastoma. DLI, however, carries the risk of graft-versus-host disease (GvHD). Recipient leukocyte infusion (RLI) induces graft-versus-leukemia responses without GvHD in mice and is currently being explored clinically. Here, we demonstrate that both DLI and RLI, when given to mixed C57BL/6 → A/J radiation chimeras carrying subcutaneous Neuro2A neuroblastoma implants, can slow the local growth of such tumors. DLI provoked full donor chimerism and GvHD; RLI produced graft rejection but left mice healthy. Flow cytometric studies showed that the chimerism of intratumoral leukocytes paralleled the systemic chimerism. This was associated with increased CD8/CD4 ratios, CD8⁺ T-cell IFN-γ expression and NK-cell Granzyme B expression within the tumor, following both DLI and RLI. The clinically safe anti-tumor effect of RLI was further enhanced by adoptively transferred naïve recipient-type NK cells. In models of intravenous Neuro2A tumor challenge, allogeneic chimeras showed superior overall survival over syngeneic chimeras. Bioluminescence imaging in allogeneic chimeras challenged with luciferase-transduced Neuro2A cells showed both DLI and RLI to prolong metastasis-free survival. This is the first experimental evidence that RLI can safely produce a local and systemic anti-tumor effect against a solid tumor. Our data indicate that RLI may provide combined T-cell and NK-cell reactivity effectively targeting Neuro2A neuroblastoma.     

Fluid flow in the osteocyte mechanical environment: a fluid–structure interaction approach

Osteocytes are believed to be the primary sensor of mechanical stimuli in bone, which orchestrate osteoblasts and osteoclasts to adapt bone structure and composition to meet physiological loading demands. Experimental studies to quantify the mechanical environment surrounding bone cells are challenging, and as such, computational and theoretical approaches have modelled either the solid or fluid environment of osteocytes to predict how these cells are stimulated in vivo. Osteocytes are an elastic cellular structure that deforms in response to the external fluid flow imposed by mechanical loading. This represents a most challenging multi-physics problem in which fluid and solid domains interact, and as such, no previous study has accounted for this complex behaviour. The objective of this study is to employ fluid–structure interaction (FSI) modelling to investigate the complex mechanical environment of osteocytes in vivo. Fluorescent staining of osteocytes was performed in order to visualise their native environment and develop geometrically accurate models of the osteocyte in vivo. By simulating loading levels representative of vigorous physiological activity ( $3,000\,\upmu \upvarepsilon $ compression and 300 Pa pressure gradient), we predict average interstitial fluid velocities $(\sim 60.5\,\upmu \text{ m/s })$ and average maximum shear stresses $(\sim 11\, \text{ Pa })$ surrounding osteocytes in vivo. Interestingly, these values occur in the canaliculi around the osteocyte cell processes and are within the range of stimuli known to stimulate osteogenic responses by osteoblastic cells in vitro. Significantly our results suggest that the greatest mechanical stimulation of the osteocyte occurs in the cell processes, which, cell culture studies have indicated, is the most mechanosensitive area of the cell. These are the first computational FSI models to simulate the complex multi-physics mechanical environment of osteocyte in vivo and provide a deeper understanding of bone mechanobiology.     

Effect of enzyme concentrations on protoplast isolation and protoplast culture of <Emphasis Type="Italic">Spathiphyllum</Emphasis> and <Emphasis Type="Italic">Anthurium</Emphasis>

Vital protoplasts from Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were isolated from both somatic embryos and leaves. The highest yields were obtained when 1.5% cellulase, 0.5% macerase and 0.5% driselase were used for Spathiphyllum wallisii leaves and 0.5% cellulase, 0.3% macerase and 0.5% driselase for Anthurium scherzerianum embryos. About 1 × 10⁶ protoplasts g⁻¹ and 1 × 10⁵ protoplasts g⁻¹ could be isolated from leaves and embryos, respectively. For protoplast fusion Spathiphyllum wallisii ‘Alain’ and Anthurium scherzerianum ‘238’ were mixed in a 1:1 ratio in a fusion solution containing 1 mM CaCl₂·2H₂O, 1 mM MES and 0.5 M mannitol. Fusion was performed by protoplast alignment under 500 V cm⁻¹ alternating current for 60 s and subsequent generation of two pulses of 4500 V cm⁻¹ direct current during 50 μs. Development until colony stage was achieved using agarose beads for protoplast culture.