Biomarkers of in vivo limbal stem cell function

PurposeTo evaluate in vivo parameters as biomarkers of limbal stem cell function and to establish an objective system that detects and stage limbal stem cell deficiency (LSCD).MethodsA total of 126 patients (172 eyes) with LSCD and 67 normal subjects (99 eyes) were included in this observational cross-sectional comparative study. Slit-lamp biomicroscopy, in vivo laser scanning confocal microscopy (IVCM), and anterior segment optical coherence tomography (AS-OCT) were performed to obtain the following: clinical score, cell morphology score, basal cell density (BCD), central corneal epithelial thickness (CET), limbal epithelial thickness (LET), total corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), and tortuosity coefficient. Their potential correlations with the severity of LSCD were investigated, and cutoff values were determined.ResultsAn increase clinical score correlated with a decrease in central cornea BCD, limbal BCD, CET, mean LET, maximum LET, CNFL, CNFD, CNBD, and tortuosity coefficient. Regression analyses showed that central cornea BCD, CET and CNFL were the best parameters to differentiate LSCD from normal eyes (Coef = 3.123, 3.379, and 2.223; all p < 0.05). The rank correlation analysis showed a similar outcome between the clinical scores and the central cornea BCD (ρ = 0.79), CET (ρ = 0.82), and CNFL (ρ = 0.71). A comprehensive LSCD grading formula based on a combination of these parameters was established.ConclusionsA comprehensive staging system combining clinical presentation, central cornea BCD, CET, and CNFL is established to accurately and objectively diagnose LSCD and stage its severity.     

Assessment of dental implant surface stability at the nanoscale level

ObjectivesTo study the oxide layer stability of certified dental implants of system "P", made based on TiO₂ alloy with carbon coating. To perform a comparative statistical analysis of the obtained data with the available data for the dental implants of systems "A" and "B".MethodsX-ray microtomography and X-ray fluorescence analysis were used to study soft tissue biopsy specimens. Supernatants were studied by dynamic light scattering and transmission electron microscopy when simulating free emission of nanoscale metal oxide particles from the surface of dental implants as well as when simulating physical loading. A comparative analysis of three parameters of nanoscale particles was performed by statistical data analysis. The surface of the "P" system dental implant with surface treatment was analyzed by scanning electron microscopy.ResultsBoth free emission of nanoscale oxide layer particles and yield of nano- and microscale particles during simulation of physical load were confirmed. Statistically significant differences were noted in a comparative analysis of the size and frequency of occurrence of these particles in the supernatants obtained from the surfaces of three dental implant systems. The elemental composition of the particles and the composition and structure of the "P" system dental implants themselves were analyzed.SignificanceThe developed method of dynamic light scattering can be used to compare the stability of the oxide layer of standardized medical products manufactured on the basis of the TiO₂ alloy.     

Three-year efficacy and patency follow-up of decellularized human internal mammary artery as a novel vascular graft in animal models

Background

Various investigations have reported that the internal mammary artery (IMA) is an efficient and functional choice of conduit for vascular graft surgeries, especially for coronary artery bypass grafts; however, the quest to find an ideal vascular substitute remains. We hypothesized that acellular IMA could be an appropriate graft for small-diameter vascular bypasses that could be used in various surgeries including coronary artery bypass grafting.

Reflectance confocal microscopy may enhance the accuracy of histopathologic diagnosis: A case series

Although histopathology is the time‐honored gold standard diagnostic measure in dermatology, several factors may detract from an accurate microscopic diagnosis. Limiting factors include: human error, suboptimal biopsy‐site selection or biopsy technique, and inherent restrictions of vertical tissue sectioning that lead to incomplete microscopic evaluation of the lesion. Reflectance confocal microscopy (RCM) is a non‐invasive imaging tool that allows for the cellular‐level examination of the lesion, at a horizontal plane, which may complement the subsequent vertical histopathological tissue examination. Herein, we report a case series whereby prebiopsy RCM examination enhanced the accuracy of histopathological diagnosis or allowed for a critical appraisal of initial histopathological misdiagnosis.     

Structural insights into membrane remodeling by SNX1

The sorting nexin (SNX) family of proteins deform the membrane to generate transport carriers in endosomal pathways. Here, we elucidate how a prototypic member, SNX1, acts in this process. Performing cryoelectron microscopy, we find that SNX1 assembles into a protein lattice that consists of helical rows of SNX1 dimers wrapped around tubular membranes in a crosslinked fashion. We also visualize the details of this structure, which provides a molecular understanding of how various parts of SNX1 contribute to its ability to deform the membrane. Moreover, we have compared the SNX1 structure with a previously elucidated structure of an endosomal coat complex formed by retromer coupled to a SNX, which reveals how the molecular organization of the SNX in this coat complex is affected by retromer. The comparison also suggests insight into intermediary stages of assembly that results in the formation of the retromer-SNX coat complex on the membrane.

Sorting nexins (SNXs) exist as a large family of proteins defined by the presence of a PX (phox homology) domain (1, 2). Members of this family have been found to act as coat proteins in endosomal pathways that include recycling from endosomes to the plasma membrane and retrieval from endosomes to the Golgi complex (3, 4). Defects in these transport processes is associated with various neurologic disorders including Alzheimer’s disease, Parkinson’s disease, and Down’s syndrome (5, 6).Coat proteins assemble into complexes on the membrane to initiate intracellular transport pathways by coupling two main functions: bending the membrane to generate transport carriers and binding to cargoes for their sorting into these carriers (7). Retromer, a trimeric complex consisting of Vps26, Vps29, and Vps35, has been found to couple with different SNXs to form multiple endosomal coat complexes, in which select members of the SNX family act in membrane deformation while retromer acts in cargo recognition (8–17). Recently, a detailed molecular view of this functional cooperation has been achieved by elucidating the structure of a retromer-SNX complex on the membrane (18).Notably, it has been further discovered recently that an endosomal coat complex can be formed with only SNX members. SNX1/2 have been found to heterodimerize with SNX5/6 to form the endosomal SNX–BAR sorting complex for promoting exit 1 (ESCPE-1) complex, in which SNX1/2 are proposed to act in membrane deformation while SNX5/6 act in cargo recognition (19). As such, a key question has become whether SNX that acts in membrane deformation in this type of coat complex would be organized similarly on the membrane, as previously elucidated for SNX in the context of a retromer-SNX complex (18).One of the best characterized mechanisms of membrane deformation involves proteins that possess the BAR (Bin/Amphiphysin/Rvs) domain. This domain has been shown to undergo homodimerization to form a banana-shaped structure, which can impart membrane curvature through a scaffolding mechanism that involves electrostatic interactions between the positive charges lining the concave side of the curved BAR dimer and the negative charges that line the surface of the membrane bilayer. In some cases, the BAR domain can deform the membrane through a second mechanism, which involves the formation of an amphipathic helix that inserts into one leaflet of the membrane bilayer to generate bilayer asymmetry in driving membrane curvature (20, 21).Besides the PX domain, SNX1 also possesses a BAR domain. However, studies have found that its BAR domain is not sufficient in driving membrane deformation. Instead, the PX domain as well as the linker region between the BAR and PX domains are also needed (22, 23). As such, a key goal has been to achieve a better understanding of how the various parts of SNX1 contribute to its ability to deform the membrane.Structural studies, such as those involving crystallography and single-particle electron microscopy (EM), have been advancing a molecular understanding of coat proteins (24), including components of endosomal coats (17, 19, 22, 25–27). Notably, however, these approaches solve protein structures in solution, but the functional form of coat proteins involves their association with the membrane. In this study, we have pursued cryo-EM to reveal how SNX1 is organized on the membrane to explain its ability to deform the membrane. The result advances a molecular understanding of how an endosomal coat that contains only SNXs generates transport carriers. Moreover, by comparing our SNX1 structure to the previously solved retromer-SNX structure (18), we delineate the extent to which the molecular organization of SNX on the membrane is affected by the presence of retromer. This comparison also suggests insight into intermediary stages of coat assembly that form the retromer-SNX complex on the membrane.     

Multiphoton microscopy for pre-clinical evaluation of flow-diverter stents for treating aneurysms

BackgroundConventional histological analyses are the gold standard for the study of aneurysms and vascular pathologies in pre-clinical research. Over the past decade, in vivo and ex vivo imaging using multiphoton microscopy have emerged as powerful pre-clinical tools for detailed tissue analyses that can assess morphology, the extracellular matrix (ECM), cell density and vascularisation. Multiphoton microscopy allows for deeper tissue penetration with minor phototoxicity.ObjectiveThe present study aimed to demonstrate the current status of multimodality imaging, including multiphoton microscopy, for detailed analyses of neo-endothelialisation and ECM evolution after flow-diverter stent (FDS) treatment in an experimental rabbit model of aneurysms.MethodsMultiphoton microscopy tools for assessing autofluorescence and second harmonic generation (SHG) signals from biological tissues were used to evaluate the endovascular treatment of intracranial aneurysms in an animal model of aneurysms (pig, rabbit). Results from multiphoton microscopy were compared to those from standard histology, electronic and bright field microscopy.ConclusionsThe present study describes novel evaluation modes based on multiphoton microscopy for visualising tissue morphology (e.g., collagen, elastin, and cells) to qualify and quantify the extent of neo-intimal formation of covered arteries and device integration into the arterial wall using a rabbit model of intracranial aneurysms treated with FDS.