Development of Erf-Mediated Craniosynostosis and Pharmacological Amelioration.

ETS2 repressor factor (ERF) insufficiency causes craniosynostosis (CRS4) in humans and mice. ERF is an ETS domain transcriptional repressor regulated by Erk1/2 phosphorylation via nucleo-cytoplasmic shuttling. Here, we analyze the onset and development of the craniosynostosis phenotype in an Erf-insufficient mouse model and evaluate the potential of the residual Erf activity augmented by pharmacological compounds to ameliorate the disease. Erf insufficiency appears to cause an initially compromised frontal bone formation and subsequent multisuture synostosis, reflecting distinct roles of Erf on the cells that give rise to skull and facial bones. We treated animals with Mek1/2 and nuclear export inhibitors, U0126 and KPT-330, respectively, to increase Erf activity by two independent pathways. We implemented both a low dosage locally over the calvaria and a systemic drug administration scheme to evaluate the possible indirect effects from other systems and minimize toxicity. The treatment of mice with either the inhibitors or the administration scheme alleviated the synostosis phenotype with minimal adverse effects. Our data suggest that the ERF level is an important regulator of cranial bone development and that pharmacological modulation of its activity may represent a valid intervention approach both in CRS4 and in other syndromic forms of craniosynostosis mediated by the FGFR-RAS-ERK-ERF pathway.

Micro-Computed Tomographic Evaluation of Canal Transportation and Centering Ability of 4 Heat-Treated Nickel-Titanium Systems.

INTRODUCTION: This study aimed to evaluate and compare canal transportation and centering ability of 4 different root canal preparation systems produced with thermal treatments by means of micro-computed tomographic imaging. METHODS: Eighty mesial canals of human extracted mandibular molars were selected based on similar morphologic parameters and were randomly assigned to 4 experimental groups (n = 20) according to the canal instrumentation technique: HyFlex CM (HCM [Coltène-Whaledent, Allstätten, Switzerland]), HyFlex EDM (HEDM [Coltène-Whaledent]), WaveOne Gold (WOG [Dentsply Sirona, Ballaigues, Switzerland]), and OneCurve (OC [Micro-Mega, Besancon, France]). The specimens were scanned before and after root canal preparation using X-ray micro-computed tomographic imaging at a resolution of 19.9 µm. Apical transportation and centering ability were then analyzed at 3 different levels: 3 mm, 5 mm, and 7 mm from the apex, representing the apical, midroot, and coronal thirds of the root, respectively. One-way analysis of variance and Kruskal-Wallis tests were used to statistically compare the groups. The significance level was set at 5%. RESULTS: HCM caused less canal transportation than WOG at the 3-mm level in both the buccal and lingual canals (P < .05). Also, HCM resulted in less canal transportation than WOG and OC at the 7-mm level regarding lingual canals. No statistically significant differences were recorded between the groups when the mean centering ratios were compared. CONCLUSIONS: The 4 evaluated systems safely prepared root canals causing minimal canal transportation and producing relatively centered preparations. In terms of canal transportation, HCM performed better than WOG at the apical level and better than WOG and OC at the coronal level.

Degloving Injury of the Lower Extremity: Report of Two Cases.

OBJECTIVE: The aim of our article is to highlight the importance of the immediate treatment of lower extremity degloving injuries, in order to prevent complications. CASES PRESENTATION: Here we present two cases of degloving injury of the lower extremity, both resulting from motorway accidents. The first one concerned a 65-year-old man suffering from multiple limb fractures and a degloving injury of the right thigh, which was immediately treated with extensive debridement and primary full-thickness skin graft re-approximation. The second case involved a 63-year-old woman who presented with cervical vertebrae fractures and a degloving injury of the left posterior leg, which, due to the severity of her condition, was treated with a delayed approach resulting in skin necrosis, which required surgical debridement, alginate dressing and foam cover. CONCLUSIONS: The optimal approach to treatment of degloving injuries is challenging and they warrant immediate surgical attention. An early diagnosis and the evaluation of tissue viability are important in order to prevent limb-threatening situations.

High resolution volumetric imaging of primary and secondary tumor spheroids using multi-angle Light Sheet Fluorescence Microscopy (LSFM).

Breast cancer and Glioblastoma brain cancer are severe malignancies with poor prognosis. In this study, primary Glioblastoma and secondary breast cancer spheroids are formed and treated with the well-known Temozolomide and Doxorubicin chemotherapeutics, respectively. High resolution imaging of both primary and secondary cancer cell spheroids is possible using a custom multi-angle Light Sheet Fluorescence Microscope. Such a technique is successful in realizing preclinical drug screening, while enables the discrimination among physiologic tumor parameters.

Demonstrating Improved Multiple Transport-Mean-Free-Path Imaging Capabilities of Light Sheet Microscopy in the Quantification of Fluorescence Dynamics.

Optical microscopy constitutes, one of the most fundamental paradigms for the understanding of complex biological mechanisms in the whole-organism and live-tissue context. Novel imaging techniques such as light sheet fluorescence microscopy (LSFM) and optical projection tomography (OPT) combined with phase-retrieval algorithms (PRT) can produce highly resolved 3D images in multiple transport-mean-free-path scales. Our study aims to exemplify the microscopic capabilities of LSFM when imaging protein dynamics in Caenorhabditis elegans and the distribution of necrotic cells in cancer cell spheroids. To this end, we apply LSFM to quantify the spatio-temporal localization of the GFP-tagged aging and stress response factor DAF-16/FOXO in transgenic C. elegans. Our analysis reveals a linear nuclear localization of DAF-16::GFP across tissues in response to heat stress, using a system that outperforms confocal scanning fluorescent microscopy in imaging speed, 3D resolution and reduced photo-toxicity. Furthermore, we present how PRT can improve the depth-to-resolution-ratio when applied to image the far-red fluorescent dye DRAQ7 which stains dead cells in a T47D cancer cell spheroid recorded with a customized OPT/LSFM system. Our studies demonstrate that LSFM combined with our novel approaches enables higher resolution and more accurate 3D quantification than previously applied technologies, proving its advance as new gold standard for fluorescence microscopy.

Fluorescence Diffusion in the Presence of Optically Clear Tissues in a Mouse Head Model.

Diffuse Optical Tomography commonly neglects or assumes as insignificant the presence of optically clear regions in biological tissues, estimating their contribution as a small perturbation to light transport. The inaccuracy introduced by this practice is examined in detail in the context of a complete, based on realistic geometry, virtual fluorescence Diffuse Optical Tomography experiment where a mouse head is imaged in the presence of cerebral spinal fluid. Despite the small thickness of such layer, we point out that an error is introduced when neglecting it from the model with possibly reduction in the accuracy of the reconstruction and localization of the fluorescence distribution within the brain. The results obtained in the extensive study presented here suggest that fluorescence diffuse neuroimaging studies can be improved in terms of quantitative and qualitative reconstruction by accurately taking into account optically transparent regions especially in the cases where the reconstruction is aided by the prior knowledge of the structural geometry of the specimen. Thus, this has only recently become an affordable choice, thanks to novel computation paradigms that allow to run Monte Carlo photon propagation on a simple graphic card, hence speeding up the process a thousand folds compared to CPU-based solutions.

A customized light sheet microscope to measure spatio-temporal protein dynamics in small model organisms.

We describe a customizable and cost-effective light sheet microscopy (LSM) platform for rapid three-dimensional imaging of protein dynamics in small model organisms. The system is designed for high acquisition speeds and enables extended time-lapse in vivo experiments when using fluorescently labeled specimens. We demonstrate the capability of the setup to monitor gene expression and protein localization during ageing and upon starvation stress in longitudinal studies in individual or small groups of adult Caenorhabditis elegans nematodes. The system is equipped to readily perform fluorescence recovery after photobleaching (FRAP), which allows monitoring protein recovery and distribution under low photobleaching conditions. Our imaging platform is designed to easily switch between light sheet microscopy and optical projection tomography (OPT) modalities. The setup permits monitoring of spatio-temporal expression and localization of ageing biomarkers of subcellular size and can be conveniently adapted to image a wide range of small model organisms and tissue samples.

Development of a three-dimensional surface imaging system for melanocytic skin lesion evaluation.

Even though surface morphology is always taken into account when assessing clinically pigmented skin lesions, it is not captured by most modern imaging systems using digital imaging. Our aim is to develop a novel three-dimensional (3D) imaging technique to record detailed information of the surface anatomy of melanocytic lesions that will enable improved classification through digital imaging. The apparatus consists of three high-resolution cameras, a light source, and accompanying software. Volume measurements of specific phantoms using volumetric tubes render slightly lower values than those obtained by our 3D imaging system (mean%± SD, 3.8%± 0.98, P<0.05). To examine the reproducibility of the method, sequential imaging of melanocytic lesions is carried out. The mean%± SD differences of area, major axis length, volume, and maximum height are 2.1%± 1.1, 0.9%± 0.8, 3.8%± 2.9, and 2.5%± 3.5, respectively. Thirty melanocytic lesions are assessed, including common and dysplastic nevi and melanomas. There is a significant difference between nevi and melanomas in terms of variance in height and boundary asymmetry (P<0.001). Moreover, dysplastic nevi have significantly higher variances in pigment density values than common nevi (P<0.001). Preliminary data suggest that our instrument has great potential in the evaluation of the melanocytic lesions. However, these findings should be confirmed in larger-scale studies.

Kinetics of T-cell receptor-dependent antigen recognition determined in vivo by multi-spectral normalized epifluorescence laser scanning.

Detection of multiple fluorophores in conditions of low signal represents a limiting factor for the application of in vivo optical imaging techniques in immunology where fluorescent labels report for different functional characteristics. A noninvasive in vivo Multi-Spectral Normalized Epifluorescence Laser scanning (M-SNELS) method was developed for the simultaneous and quantitative detection of multiple fluorophores in low signal to noise ratios and used to follow T-cell activation and clonal expansion. Colocalized DsRed- and GFP-labeled T cells were followed in tandem during the mounting of an immune response. Spectral unmixing was used to distinguish the overlapping fluorescent emissions representative of the two distinct cell populations and longitudinal data reported the discrete pattern of antigen-driven proliferation. Retrieved values were validated both in vitro and in vivo with flow cytometry and significant correlation between all methodologies was achieved. Noninvasive M-SNELS successfully quantified two colocalized fluorescent populations and provides a valid alternative imaging approach to traditional invasive methods for detecting T cell dynamics.

A matrix-free algorithm for multiple wavelength fluorescence tomography.

In the recent years, there has been an increase in applications of non-contact diffusion optical tomography. Especially when the objective is the recovery of fluorescence targets. The non-contact acquisition systems with the use of a CCD-camera produce much denser sampled boundary data sets than fibre-based systems. When model-based reconstruction methods are used, that rely on the inversion of a derivative operator, the large number of measurements poses a challenge since the explicit formulation and storage of the Jacobian matrix could be in general not feasible. This problem is aggravated further in applications, where measurements at multiple wavelengths are used. We present a matrix-free model-based reconstruction method, that addresses the problems of large data sets and reduces the computational cost and memory requirements for the reconstruction. The idea behind the matrix-free method is that information about the Jacobian matrix could be available through matrix times vector products so that the creation and storage of big matrices can be avoided. We tested the method for multiple wavelength fluorescence tomography with simulated and experimental data from phantom experiments, and we found substantial benefits in computational times and memory requirements.

3D shape based reconstruction of experimental data in Diffuse Optical Tomography.

Diffuse optical tomography (DOT) aims at recovering three-dimensional images of absorption and scattering parameters inside diffusive body based on small number of transmission measurements at the boundary of the body. This image reconstruction problem is known to be an ill-posed inverse problem, which requires use of prior information for successful reconstruction. We present a shape based method for DOT, where we assume a priori that the unknown body consist of disjoint subdomains with different optical properties. We utilize spherical harmonics expansion to parameterize the reconstruction problem with respect to the subdomain boundaries, and introduce a finite element (FEM) based algorithm that uses a novel 3D mesh subdivision technique to describe the mapping from spherical harmonics coefficients to the 3D absorption and scattering distributions inside a unstructured volumetric FEM mesh. We evaluate the shape based method by reconstructing experimental DOT data, from a cylindrical phantom with one inclusion with high absorption and one with high scattering. The reconstruction was monitored, and we found a 87% reduction in the Hausdorff measure between targets and reconstructed inclusions, 96% success in recovering the location of the centers of the inclusions and 87% success in average in the recovery for the volumes.

Diffuse photon propagation in multilayered geometries.

Diffuse optical tomography (DOT) is an emerging functional medical imaging modality which aims to recover the optical properties of biological tissue. The forward problem of the light propagation of DOT can be modelled in the frequency domain as a diffusion equation with Robin boundary conditions. In the case of multilayered geometries with piecewise constant parameters, the forward problem is equivalent to a set of coupled Helmholtz equations. In this paper, we present solutions for the multilayered diffuse light propagation for a three-layer concentric sphere model using a series expansion method and for a general layered geometry using the boundary element method (BEM). Results are presented comparing these solutions to an independent Monte Carlo model, and for an example three layered head model.