Hyperspectral Imaging and Classification for Grading Skin Erythema

International audienceErythema is an inflammatory condition of the skin that is commonly used as a feature to monitor the progression of cutaneous diseases or treatment induced side effects. In radiation therapy, skin erythema is routinely assessed visually by an expert using standardized grading criteria. However, visual assessment (VA) is subjective and commonly used grading tools are too coarse to score the onset of erythema. Therefore, an objective method capable of quantitatively grading early erythema changes may help identify patients at higher risk for developing severe radiation induced skin toxicities. The purpose of this study is to investigate the feasibility of using hyperspectral imaging (HSI) for quantitative assessment of early erythema and to characterize its performance against VA documented on conventional digital photographic red-green-blue (RGB) images. Erythema was induced artificially on 3 volunteers in a controlled pilot study; and was subsequently measured using HSI and color imaging. HSI and color imaging data was analyzed using linear discriminant analysis (LDA) to perform classification. The classification results, including accuracy, and precision, demonstrated that HSI is superior to color imaging in skin erythema assessment

Prx1 Expressing Cells Are Required for Periodontal Regeneration of the Mouse Incisor

Previous studies have shown that post-natal skeletal stem cells expressing Paired-related homeobox 1 (PRX1 or PRRX1) are present in the periosteum of long bones where they contribute to post-natal bone development and regeneration. Our group also identified post-natal PRX1 expressing cells (pnPRX1+ cells) in mouse calvarial synarthroses (sutures) and showed that these cells are required for calvarial bone regeneration. Since calvarial synarthroses are similar to dentoalveolar gomphosis (periodontium) and since there is no information available on the presence or function of pnPRX1+ cells in the periodontium, the present study aimed at identifying and characterizing pnPRX1+ cells within the mouse periodontium and assess their contribution to periodontal development and regeneration. Here we demonstrated that pnPRX1+ cells are present within the periodontal ligament (PDL) of the mouse molars and of the continuously regenerating mouse incisor. By means of diphtheria toxin (DTA)-mediated conditional ablation of pnPRX1+ cells, we show that pnPRX1+ cells contribute to post-natal periodontal development of the molars and the incisor, as ablation of pnPRX1+ cells in 3-days old mice resulted in a significant enlargement of the PDL space after 18 days. The contribution of pnPRX1+ cells to periodontal regeneration was assessed by developing a novel non-critical size periodontal defect model. Outcomes showed that DTA-mediated post-natal ablation of pnPRX1+ cells results in lack of regeneration in periodontal non-critical size defects in the regeneration competent mouse incisors. Importantly, gene expression analysis of these cells shows a profile typical of quiescent cells, while gene expression analysis of human samples of periodontal stem cells (PDLSC) confirmed that Prx1 is highly expressed in human periodontium. In conclusion, pnPRX1+ cells are present within the continuously regenerating PDL of the mouse incisor, and at such location they contribute to post-natal periodontal development and regeneration. Since this study further reports the presence of PRX1 expressing cells within human periodontal ligament, we suggest that studying the mouse periodontal pnPRX1+ cells may provide significant information for the development of novel and more effective periodontal regenerative therapies in humans

Postnatal Calvarial Skeletal Stem Cells Expressing PRX1 Reside Exclusively in the Calvarial Sutures and Are Required for Bone Regeneration

Summary Post-natal skeletal stem cells expressing PRX1 (pnPRX1+) have been identified in the calvaria and in the axial skeleton. Here we characterize the location and functional capacity of the calvarial pnPRX1+ cells. We found that pnPRX1+ reside exclusively in the calvarial suture niche and decrease in number with age. They are distinct from preosteoblasts and osteoblasts of the sutures, respond to WNT signaling in vitro and in vivo by differentiating into osteoblasts, and, upon heterotopic transplantation, are able to regenerate bone. Diphtheria toxin A (DTA)-mediated lineage ablation of pnPRX1+ cells and suturectomy perturb regeneration of calvarial bone defects and confirm that pnPRX1+ cells of the sutures are required for bone regeneration. Orthotopic transplantation of sutures with traceable pnPRX1+ cells into wild-type animals shows that pnPRX1+ cells of the suture contribute to calvarial bone defect regeneration. DTA-mediated lineage ablation of pnPRX1+ does not, however, interfere with calvarial development

Rapamycin induces glucose intolerance in mice by reducing islet mass, insulin content, and insulin sensitivity

Rapamycin, a specific inhibitor for mTOR complex 1, is an FDA-approved immunosuppressant for organ transplant. Recent developments have raised the prospect of using rapamycin to treat cancer or diabetes and to delay aging. It is therefore important to assess how rapamycin treatment affects glucose homeostasis. Here, we show that the same rapamycin treatment reported to extend mouse life span significantly impaired glucose homeostasis of aged mice. Moreover, rapamycin treatment of lean C57B/L6 mice reduced glucose-stimulated insulin secretion in vivo and ex vivo as well as the insulin content and beta cell mass of pancreatic islets. Confounding the diminished capacity for insulin release, rapamycin decreased insulin sensitivity. The multitude of rapamycin effects thus all lead to glucose intolerance. As our findings reveal that chronic rapamycin treatment could be diabetogenic, monitoring glucose homeostasis is crucial when using rapamycin as a therapeutic as well as experimental reagent

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

In Vivo 3D Histomorphometry Quantifies Bone Apposition and Skeletal Progenitor Cell Differentiation

Histomorphometry and Micro-CT are commonly used to assess bone remodeling and bone microarchitecture. These approaches typically require separate cohorts of animals to analyze 3D morphological changes and involve time-consuming immunohistochemistry preparation. Intravital Microscopy (IVM) in combination with mouse genetics may represent an attractive option to obtain bone architectural measurements while performing longitudinal monitoring of dynamic cellular processes in vivo. In this study we utilized two-photon, multicolor fluorescence IVM together with a lineage tracing reporter mouse model to image skeletal stem cells (SSCs) in their calvarial suture niche and analyze their differentiation fate after stimulation with an agonist of the canonical Wnt pathway (recombinant Wnt3a). Our in vivo histomorphometry analyses of bone formation, suture volume, and cellular dynamics showed that recombinant Wnt3a induces new bone formation, differentiation and incorporation of SSCs progeny into newly forming bone. IVM technology can therefore provide additional dynamic 3D information to the traditional static 2D histomorphometry

Intravital fluorescence microscopy with negative contrast.

Advances in intravital microscopy (IVM) have enabled the studies of cellular organization and dynamics in the native microenvironment of intact organisms with minimal perturbation. The abilities to track specific cell populations and monitor their interactions have opened up new horizons for visualizing cell biology in vivo, yet the success of standard fluorescence cell labeling approaches for IVM comes with a "dark side" in that unlabeled cells are invisible, leaving labeled cells or structures to appear isolated in space, devoid of their surroundings and lacking proper biological context. Here we describe a novel method for "filling in the void" by harnessing the ubiquity of extracellular (interstitial) fluid and its ease of fluorescence labelling by commonly used vascular and lymphatic tracers. We show that during routine labeling of the vasculature and lymphatics for IVM, commonly used fluorescent tracers readily perfuse the interstitial spaces of the bone marrow (BM) and the lymph node (LN), outlining the unlabeled cells and forming negative contrast images that complement standard (positive) cell labeling approaches. The method is simple yet powerful, offering a comprehensive view of the cellular landscape such as cell density and spatial distribution, as well as dynamic processes such as cell motility and transmigration across the vascular endothelium. The extracellular localization of the dye and the interstitial flow provide favorable conditions for prolonged Intravital time lapse imaging with minimal toxicity and photobleaching