A novel ACVR1 mutation in the glycine/serine-rich domain found in the most benign case of a fibrodysplasia ossificans progressiva variant reported to date

Fibrodysplasia Ossificans Progressiva (FOP) is a rare, autosomal dominant condition, classically characterised by heterotopic ossification beginning in childhood and congenital great toe malformations; occurring in response to a c.617 G>A ACVR1 mutation in the functionally important glycine/serine-rich domain of exon 6. Here we describe a novel c.587 T>C mutation in the glycine/serine-rich domain of ACVR1, associated with delayed onset of heterotopic ossification and an exceptionally mild clinical course. Absence of great toe malformations, the presence of early ossification of the cervical spine facets joints, plus mild bilateral camptodactyly of the 5th fingers, together with a novel ACVR1 mutation, are consistent with the 'FOP-variant' syndrome. The c.587 T>C mutation replaces a conserved leucine with proline at residue 196. Modelling of the mutant protein reveals a steric clash with the kinase domain that will weaken interactions with FKBP12 and induce exposure of the glycine/serine-rich repeat. The mutant receptor is predicted to be hypersensitive to ligand stimulation rather than being constitutively active, consistent with the mild clinical phenotype. This case extends our understanding of the 'FOP-variant' syndrome.

Dependence of light attenuation and backscattering on collagen concentration and chondrocyte density in agarose scaffolds

Optical coherence tomography (OCT) has been applied for high resolution imaging of articular cartilage. However, the contribution of individual structural elements of cartilage on OCT signal has not been thoroughly studied. We hypothesize that both collagen and chondrocytes, essential structural components of cartilage, act as important light scatterers and that variation in their concentrations can be detected by OCT through changes in backscattering and attenuation. To evaluate this hypothesis, we established a controlled model system using agarose scaffolds embedded with variable collagen concentrations and chondrocyte densities. Using OCT, we measured the backscattering coefficient (µb) and total attenuation coefficient (µt) in these scaffolds. Along our hypothesis, light backscattering and attenuation in agarose were dependent on collagen concentration and chondrocyte density. Significant correlations were found between µt and chondrocyte density (ρ = 0.853, p < 0.001) and between µt and collagen concentration (ρ = 0.694, p < 0.001). µb correlated significantly with chondrocyte density (ρ = 0.504, p < 0.001) but not with collagen concentration (ρ = 0.103, p = 0.422) of the scaffold. Thus, quantitation of light backscattering and, especially, attenuation could be valuable when evaluating the integrity of soft tissues, such as articular cartilage with OCT.

Alteration of the C-terminal ligand specificity of the Erbin PDZ domain by allosteric mutational effects

Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain. We constructed a library of PDZ domains by randomizing residues in direct contact with position 0 and in a loop that is close to but does not contact position 0. We used phage display to select for PDZ variants that bind to 19 peptide ligands differing only at position 0. To verify that each obtained PDZ domain exhibited the correct binding specificity, we selected peptide ligands for each domain. Despite intensive efforts, we were only able to evolve Erbin PDZ domain variants with selectivity for the aliphatic C-terminal side chains Val, Ile and Leu. Interestingly, many PDZ domains with these three distinct specificities contained identical amino acids at positions that directly contact position 0 but differed in the loop that does not contact position 0. Computational modeling of the selected PDZ domains shows how slight conformational changes in the loop region propagate to the binding site and result in different binding specificities. Our results demonstrate that second-sphere residues could be crucial in determining protein binding specificity.

SERS-barcoded colloidal gold NP assemblies as imaging agents for use in biodiagnostics

There is a growing need for new biodiagnostics that combine high throughput with enhanced spatial resolution and sensitivity. Gold nanoparticle (NP) assemblies with sub-10 nm particle spacing have the benefits of improving detection sensitivity via Surface enhanced Raman scattering (SERS) and being of potential use in biomedicine due to their colloidal stability. A promising and versatile approach to form solution-stable NP assemblies involves the use of multi-branched molecular linkers which allows tailoring of the assembly size, hot-spot density and interparticle distance. We have shown that linkers with multiple anchoring end-groups can be successfully employed as a linker to assemble gold NPs into dimers, linear NP chains and clustered NP assemblies. These NP assemblies with diameters of 30-120 nm are stable in solution and perform better as SERS substrates compared with single gold NPs, due to an increased hot-spot density. Thus, tailored gold NP assemblies are potential candidates for use as biomedical imaging agents. We observed that the hot-spot density and in-turn the SERS enhancement is a function of the linker polymer concentration and polymer architecture. New deep Raman techniques like Spatially Offset Raman Spectroscopy (SORS) have emerged that allow detection from beneath diffusely scattering opaque materials, including biological media such as animal tissue. We have been able to demonstrate that the gold NP assemblies could be detected from within both proteinaceous and high lipid containing animal tissue by employing a SORS technique with a backscattered geometry.

Analysis of focal adhesion turnover: A quantitative live-cell imaging example

Recent advances in optical and fluorescent protein technology have rapidly raised expectations in cell biology, allowing quantitative insights into dynamic intracellular processes like never before. However, quantitative live-cell imaging comes with many challenges including how best to translate dynamic microscopy data into numerical outputs that can be used to make meaningful comparisons rather than relying on representative data sets. Here, we use analysis of focal adhesion turnover dynamics as a straightforward specific example on how to image, measure, and analyze intracellular protein dynamics, but we believe this outlines a thought process and can provide guidance on how to understand dynamic microcopy data of other intracellular structures.

Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging

Here we describe a protocol for advanced CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis). The CUBIC protocol enables simple and efficient organ clearing, rapid imaging by light-sheet microscopy and quantitative imaging analysis of multiple samples. The organ or body is cleared by immersion for 1–14 d, with the exact time required dependent on the sample type and the experimental purposes. A single imaging set can be completed in 30–60 min. Image processing and analysis can take <1 d, but it is dependent on the number of samples in the data set. The CUBIC clearing protocol can process multiple samples simultaneously. We previously used CUBIC to image whole-brain neural activities at single-cell resolution using Arc-dVenus transgenic (Tg) mice. CUBIC informatics calculated the Venus signal subtraction, comparing different brains at a whole-organ scale. These protocols provide a platform for organism-level systems biology by comprehensively detecting cells in a whole organ or body.

Imaging tumour distribution of a polymeric drug delivery platformin vivoby PET-MRI

Background Over the past decade, molecular imaging has played a key role in the progression of drug delivery platforms from concept to commercialisation. Of the molecular imaging techniques commonly utilised, positron emission tomography (PET) can yield a breadth of information not easily accessible by other methodologies and when combined with other complementary imaging modalities, is a powerful tool for pre- and clinical development of therapeutics. However, very little research has focussed on the information available from complimentary imaging modalities. This paper reports on the data-rich methodologies of contrast enhanced PET/CT and PET/MRI for probing efficacy of polymer drug delivery platforms. Results The information available from an ExiTron nano 6000 contrast enhanced PET/CT and a gadolinium (Gd) enhanced PET/MRI image of a 64Cu labeled HBP in the same mouse was qualitatively compared. Conclusions Gd contrast enhanced PET/MRI offers a powerful methodology for investigating the distribution of polymer drug delivery platforms in vivo and throughout a tumour volume. Furthermore, information about depth of penetration away from primary blood vessels can be gleaned, potentially leading to development of more efficacious delivery vehicles for clinical use.

Synthesis of a multimodal molecular imaging probe based on a hyperbranched polymer architecture

Molecular imaging is utilised in modern medicine to aid in the diagnosis and treatment of disease by allowing its spatiotemporal state to be examined in vivo. This study focuses on the development of novel multimodal molecular imaging agents based on hyperbranched polymers that combine the complementary capabilities of optical fluorescence imaging and positron emission tomography-computed tomography (PET/CT) into one construct. RAFT-mediated polymerisation was used to prepare two hydrophilic hyperbranched polymers that were differentiated by their size and level of branching. The multiple functional end-groups facilitated covalent attachment of both near infrared fluorescent dyes for optical imaging, as well as a copper chelator allowing binding of 64Cu as a PET radio nuclei. In vivo multimodal imaging of mice using PET/CT and planar optical imaging was first used to assess the biodistribution of the polymeric materials and it was shown that the larger and more branched polymer had a significantly longer circulation time. The larger constructs were also shown to exhibit enhanced accumulation in solid tumours in a murine B16 melanoma model. Importantly, it was demonstrated that the PET modality gave rise to high sensitivity immediately after injection of the agent, while the optical modality facilitated extended longitudinal studies, thus highlighting how the complementary capabilities of the molecular imaging agents can be useful for studying various diseases, including cancer.

Molecular imaging with polymers

Polymers open up new possibilities in the field of molecular imaging, allowing sensitive and robust agents that can be imaged over long periods of time. This review highlights some recent advances in polymeric molecular imaging agents in both (pre)clinical and emerging applications.

Regional changes in choroidal thickness associated with accommodation

Purpose. To characterize the changes occurring in choroidal thickness (ChT) across the posterior pole during accommodation using enhanced-depth imaging optical coherence tomography (OCT). Methods. Forty participants (mean age 21 ± 2 years) had measures of ChT and ocular biometry taken during accommodation to 0, 3, and 6 diopter (D) stimuli, with the Spectralis OCT and Lenstar biometer. A Badal optometer and cold mirror system was mounted on both instruments, allowing measurement collection while subjects viewed an external fixation target at varying accommodative demands. Results. The choroid exhibited significant thinning during accommodation to the 6 D stimulus in both subfoveal (mean change, −5 ± 7 μm) and parafoveal regions (P < 0.001). The magnitude of these changes varied by parafoveal meridian, with the largest changes seen in the temporal (−9 ± 12 μm) and inferotemporal (−8 ± 8 μm) meridians (P < 0.001). Axial length increased with accommodation (mean change, +5 ± 11 μm at 3 D, +14 ± 13 μm at 6 D), and these changes were weakly negatively associated with the choroidal changes (r2 = 0.114, P < 0.05). Conclusions. A small, but significant thinning of the choroid was observed at the 6 D accommodation demand, which was greatest in the temporal and inferotemporal parafoveal choroid, and increased with increasing eccentricity from the fovea. The regional variation in the parafoveal thinning corresponds to the distribution of the nonvascular smooth muscle within the uvea, which may implicate these cells as the potential mechanism by which the choroid thins during accommodation.

Significant deterioration in nanomechanical quality occurs through incomplete extrafibrillar mineralization in rachitic bone: Evidence from in-situ synchrotron X-ray scattering and backscattered electron imaging

Bone diseases such as rickets and osteoporosis cause significant reduction in bone quantity and quality, which leads to mechanical abnormalities. However, the precise ultrastructural mechanism by which altered bone quality affects mechanical properties is not clearly understood. Here we demonstrate the functional link between altered bone quality (reduced mineralization) and abnormal fibrillar-level mechanics using a novel, real-time synchrotron X-ray nanomechanical imaging method to study a mouse model with rickets due to reduced extrafibrillar mineralization. A previously unreported N-ethyl-N-nitrosourea (ENU) mouse model for hypophosphatemic rickets (Hpr), as a result of missense Trp314Arg mutation of the phosphate regulating gene with homologies to endopeptidase on the X chromosome (Phex) and with features consistent with X-linked hypophosphatemic rickets (XLHR) in man, was investigated using in situ synchrotron small angle X-ray scattering to measure real-time changes in axial periodicity of the nanoscale mineralized fibrils in bone during tensile loading. These determine nanomechanical parameters including fibril elastic modulus and maximum fibril strain. Mineral content was estimated using backscattered electron imaging. A significant reduction of effective fibril modulus and enhancement of maximum fibril strain was found in Hpr mice. Effective fibril modulus and maximum fibril strain in the elastic region increased consistently with age in Hpr and wild-type mice. However, the mean mineral content was ∼21% lower in Hpr mice and was more heterogeneous in its distribution. Our results are consistent with a nanostructural mechanism in which incompletely mineralized fibrils show greater extensibility and lower stiffness, leading to macroscopic outcomes such as greater bone flexibility. Our study demonstrates the value of in situ X-ray nanomechanical imaging in linking the alterations in bone nanostructure to nanoscale mechanical deterioration in a metabolic bone disease. Copyright

Benefits of blended learning for the first year medical imaging students in preparation for clinical placement

Although there is a plethora of definitions of blended learning, the underlying distinguishing feature is the combination of traditional content delivery and the utilisation of technology. Within Medical Imaging undergraduate education there is evidence of advantages and increased student engagement when utilising a blended learning approach. Although the embedding of technology has been proven to be a useful teaching tool, “Educators should tailor their teaching media to learner’s needs rather than assume that web based learning is intrinsically superior”. This study aims to determine which clinical learning tools are perceived to be the most useful to the student in preparing them for placements.

Stabilization of exotic minority phases in a multicomponent self-assembled molecular network

Trimesic acid (TMA) and alcohols were recently shown to self-assemble into a stable, two-component linear pattern at the solution/highly oriented pyrolytic graphite (HOPG) interface. Away from equilibrium, the TMA/alcohol self-assembled molecular network (SAMN) can coexist with pure-TMA networks. Here, we report on some novel characteristics of these non-equilibrium TMA structures, investigated by scanning tunneling microscopy (STM). We observe that both the chicken-wire and flower-structure TMA phases can host 'guest' C60 molecules within their pores, whereas the TMA/alcohol SAMN does not offer any stable adsorption sites for the C60 molecules. The presence of the C60 molecules at the solution/solid interface was found to improve the STM image quality. We have taken advantage of the high-quality imaging conditions to observe unusual TMA bonding geometries at domain boundaries in the TMA/alcohol SAMN. Boundaries between aligned TMA/alcohol domains can give rise to doubled TMA dimer rows in two different configurations, as well as a tripled-TMA row. The boundaries created between non-aligned domains can create geometries that stabilize TMA bonding configurations not observed on surfaces without TMA/alcohol SAMNs, including small regions of the previously predicted 'super flower' TMA bonding geometry and a tertiary structure related to the known TMA phases. These structures are identified as part of a homologic class of TMA bonding motifs, and we explore some of the reasons for the stabilization of these phases in our multicomponent system.

Single 2×1 domain orientation on Si(001) surfaces using aperiodic Bi line arrays

Studies of Bi heteroepitaxy on Si(001) have shown that lines grow to lengths of up to 500nm if the substrate is heated to above the Bi desorption temperature (500°C) during or after Bi deposition. Unlike many other nanoline systems, the lines formed by this nonequilibrium growth process have no detectable width dispersion. Although much attention has been given to the atomic geometery of the line, in this paper, we focus on how the lines can be used to create a majority 2×1 domain orientation. It is demonstrated that the Bi lines can be used to produce a single-domain orientation on Si(001) if the lines are grown on Si(001) surfaces with a regular distribution of single height steps. This is a compelling example of how a nanoscale motif can be used to modify mesoscopic surface structure on Si(001).

From vision to actions: Towards adaptive and autonomous humanoid robots

Although robotics research has seen advances over the last decades robots are still not in widespread use outside industrial applications. Yet a range of proposed scenarios have robots working together, helping and coexisting with humans in daily life. In all these a clear need to deal with a more unstructured, changing environment arises. I herein present a system that aims to overcome the limitations of highly complex robotic systems, in terms of autonomy and adaptation. The main focus of research is to investigate the use of visual feedback for improving reaching and grasping capabilities of complex robots. To facilitate this a combined integration of computer vision and machine learning techniques is employed. From a robot vision point of view the combination of domain knowledge from both imaging processing and machine learning techniques, can expand the capabilities of robots. I present a novel framework called Cartesian Genetic Programming for Image Processing (CGP-IP). CGP-IP can be trained to detect objects in the incoming camera streams and successfully demonstrated on many different problem domains. The approach requires only a few training images (it was tested with 5 to 10 images per experiment) is fast, scalable and robust yet requires very small training sets. Additionally, it can generate human readable programs that can be further customized and tuned. While CGP-IP is a supervised-learning technique, I show an integration on the iCub, that allows for the autonomous learning of object detection and identification. Finally this dissertation includes two proof-of-concepts that integrate the motion and action sides. First, reactive reaching and grasping is shown. It allows the robot to avoid obstacles detected in the visual stream, while reaching for the intended target object. Furthermore the integration enables us to use the robot in non-static environments, i.e. the reaching is adapted on-the- fly from the visual feedback received, e.g. when an obstacle is moved into the trajectory. The second integration highlights the capabilities of these frameworks, by improving the visual detection by performing object manipulation actions.