Diese Arbeit wurde von der National Science Foundation (DBI 0227541) unterstützt.

<ol>
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High-throughput und genaue Auswertung der Pflanze Orgel Morphologie und Farbe ist wegen seiner quantitativer Natur und oft subjektiven Quantifizierung schwierig. TA bietet objektive und genaue Messungen von mehreren Früchten morphologischen und kolorimetrischen Eigenschaften in einem High-Throughput-und semi-automatische Weise. Der Nutzen der TA hat in Studien, die zur Identifizierung von Tomatenfrucht Form QTL 2,4,5 führte nachgewiesen. Darüber hinaus hat die Einstufung von cultivars6 und die Taxonomie der eine Gruppe von Arten wurden anhand der Ausgabe von TA 15,16. Hohe Ausdruck der Tomatenfrucht Form Gen so ergibt sich längliche Früchte 17. Mit TA-, Blatt-und cotyledon Form wurde in den Zeilen auszudrücken Sonne zu einem hohen Niveau 7 bestimmt, dass der Einsatz effektiv genutzt werden kann, um Morphologie der anderen Pflanzenorgane sowie zu messen. Darüber hinaus hat TA wurde von entscheidender Bedeutung bei der Identifizierung der genetischen Grundlagen der Größe der Samen in Tomaten und Sonnenblumen 8,9. Schließlich ist die Color Test-Modul in TA implementiert genauer und präziser und günstiger als andere Methoden, um Obst Farbe in Tomaten zu analysieren. Zusammenfassend hat TA zu einem wichtigen Werkzeug für die objektive und zuverlässige Beurteilung der Morphologie und Farbvariationen der Pflanzenorgane.

tomato analyzer: a useful software application to collect accurate and detailed morphological and colorimetric data from two-dimensional objects

Mess-Obst Morphologie und Farbe Züge Gemüse-und Obstkulturen in eine objektive und reproduzierbare Weise ist für detaillierte phänotypische Analysen dieser Merkmale wichtig. Tomato Analyzer (TA) ist ein Software-Programm, dass 37 Attribute im Zusammenhang mit zweidimensionalen Form in eine semi-automatische und reproduzierbare Weise 1,2 Maßnahmen. Viele dieser Attribute, wie Winkel am distalen und proximalen Enden der Frucht und Bereichen Einzug, sind schwer zu manuell zu quantifizieren. Grundlegende Mess-, Obst-Shape-Index, Blockiness, Homogenität, Proximale Fruit End Shape, Distal Fruit End Shape, Asymmetrie, Internal Exzentrizität, Breiten-Section und Morphometrie: Die Attribute werden in zehn Kategorien innerhalb der Software organisiert. Die letzte Kategorie erfordert weder Vorkenntnisse noch vorgegebenen Vorstellungen von der Gestalt Attribute, bietet so morphometrische Analyse eine unvoreingenommene Option, die besser an High-Throughput-Analysen können als Attribut Analyse angepasst. TA bietet auch die Color Test-Anwendung, die entworfen, um Farbmessungen aus gescannten Bildern zu sammeln und erlauben das Scannen Geräte kalibriert werden mit Farbstandards 3 war. TA bietet verschiedene Optionen für den Export und analysieren Formattribut, morphometrische und Farbdaten. Die Daten können in eine Excel-Datei im Batch-Modus (mehr als 100 Bilder auf einmal) exportiert werden oder exportiert als einzelne Bilder. Der Anwender kann zwischen Ausgang, dass die durchschnittliche zeigt für jedes Attribut für die Objekte in jedem Bild (einschließlich Standardabweichung), oder eine Ausgabe, die die Werte des Attributs zeigt für jedes Objekt auf dem Bild zu wählen. TA hat sich als wertvolles und effektives Werkzeug für indentifying und Bestätigung Tomatenfrucht Form Quantitative Trait Loci (QTL), sowie die Durchführung von eingehenden Analysen der Wirkung der wichtigsten Obst-Form Gene auf Pflanzenmorphologie worden. Außerdem können TA verwendet werden, um objektiv zu klassifizieren Frucht in verschiedener Form Kategorien. Schließlich können Früchte in Form und Farbe Züge in anderen Pflanzenarten sowie andere Pflanzenorgane wie Blätter und Samen mit TA ausgewertet werden.

Watch this Scientific Journal Video about Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects at JoVE.com

Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects

Tomato Analyzer (TA) quantifiziert Attribute zweidimensionale Formen und Farben in eine reproduzierbare und genaue Weise. Eine Schritt-für-Schritt-Verfahren zur Erlangung hoher Qualität digitalisierten Bilder von Tomatenfrucht, morphologische und Farbe analysiert diese Bilder und mehrere Anwendungen unter Verwendung der Daten durch die Software erzeugt werden beschrieben.

Tomato Analyzer: A Useful Software-Anwendung, um genaue und detaillierte morphologische und farbmetrischen Daten von zweidimensionalen Objekten sammeln

Measuring fruit morphology and color traits of vegetable and fruit crops in an objective and reproducible way is important for detailed phenotypic ...

tomato-analyzer-useful-software-application-to-collect-accurate

Research

JoVE Journal

Biology

26.1K Aufrufe.  The Ohio State University. Tomato Analyzer (TA) quantifiziert Attribute zweidimensionale Formen und Farben in eine reproduzierbare und genaue Weise. Eine Schritt-für-Schritt-Verfahren zur Erlangung hoher Qualität digitalisierten Bilder von Tomatenfrucht, morphologische und Farbe analysiert diese Bilder und mehrere Anwendungen unter Verwendung der Daten durch die Software erzeugt werden beschrieben.

Serie: Tomato Analyzer: A Useful Software Application to Collect Accurate and Detailed Morphological and Colorimetric Data from Two-dimensional Objects

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

Die Beurteilung Zweidimensionale Kristallisation Trials of Small Membranproteinen für Strukturbiologie Studien Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Forschung

Biologie

Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living cells. The proposed instrument extends the sensitivity of label-free optical microscopy to nanoscale changes in organelle size and shape and can be used to accelerate the study of the structure-function relationship pertaining to organelle dynamics underlying fundamental biological processes, such as programmed cell death or cellular differentiation. The microscope can be easily implemented on existing microscopy platforms, and can therefore be disseminated to individual laboratories, where scientists can implement and use the proposed methods with unrestricted access.
The proposed technique is able to characterize subcellular structure by observing the cell through two-dimensional optical Gabor filters. These filters can be tuned to sense with nanoscale (10's of nm) sensitivity, specific morphological attributes pertaining to the size and orientation of non-spherical subcellular organelles. While based on contrast generated by elastic scattering, the technique does not rely on a detailed inverse scattering model or on Mie theory to extract morphometric measurements. This technique is therefore applicable to non-spherical organelles for which a precise theoretical scatter description is not easily given, and provides distinctive morphometric parameters that can be obtained within unstained living cells to assess their function. The technique is advantageous compared with digital image processing in that it operates directly on the object's field transform rather than the discretized object's intensity. It does not rely on high image sampling rates and can therefore be used to rapidly screen morphological activity within hundreds of cells at a time, thus greatly facilitating the study of organelle structure beyond individual organelle segmentation and reconstruction by fluorescence confocal microscopy of highly magnified digital images of limited fields of view.
In this demonstration we show data from a marine diatom to illustrate the methodology. We also show preliminary data collected from living cells to give an idea of how the method may be applied in a relevant biological context.

We demonstrate a dark-field microscopy method based on Gabor-like filtering to measure subcellular dynamics within single living cells. The technique is sensitive to alterations in the structure of organelles, such as mitochondrial fragmentation.

Optical Microscopy Scatter auf zweidimensionalen Gabor-Filtern

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living ...

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.

Sorting of Streptomyces Cell Pellets Using a Complex Object Parametric Analyzer and Sorter

Liquid-grown Streptomyces cultures are characterized by mycelial pellets that are heterogeneous in size. We here describe a method to analyze and sort such pellets in a high-throughput manner. These pellets can be used for further analyses, which will provide leads to understand and control growth heterogeneity.

Sortierung der<em&gt; Streptomyces</em&gt; Zell Pellets mit einem komplexen Objekt Parametric Analyzer und Sorter

Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

Hochdurchsatz-Analyse von Bild Tumorsphäroide: Eine benutzerfreundliche Software-Anwendung, um die Größe der Sphäroide automatisch und präzise messen

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

Workflow für Hoch Inhalt, einzelne Zell Quantifizierung der Fluoreszenzmarker von Universal Mikroskop Daten, Unterstützung von Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Ein Verfahren zur Auswahl Struktur-Schalt Aptamere zu einem Farbmetrik Gold-Nanopartikeln Assay angewandt

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, Rhizoctonia bataticola. In the literature, comprehensive and detailed step-by-step protocols on disease assays are sparse. This article provides complete details on the steps involved in setting up a blotting paper technique for quickly screening genotypes for resistance to DRR. The blotting paper technique is easy and less expensive. Another method, based on the sick pot approach, is a mimic of natural infection and can be applied to study the interacting components&#8212;plant, pathogen, and environment&#8212;involved in the disease triangle.
Moreover, in nature, DRR occurs mostly in rainfed chickpea cultivation areas, where soil moisture recedes as crop growth advances. Drought stress is known to predispose chickpea plants to DRR disease. Pathomorphological and molecular understanding of plant-pathogen interaction under drought stress can pave the way for the identification of elite DRR-resistant varieties from the chickpea germplasm pool. This article provides a stepwise methodology for the preparation of a sick pot and subsequent disease assay. Overall, the information presented herein will help researchers prepare R. bataticola fungal inoculum, maintain this pathogen, set up the blotting paper technique, prepare sick culture and sick pot, and assess pathogen infection in chickpea plants.

This study presents methodologies to study the pathomorphological and molecular mechanisms underlying chickpea&#8211;Rhizoctonia bataticola interaction. The blotting paper method is useful to rapidly study chickpea genotype responses, while the sick pot-based method can be used to simultaneously impose drought and R. bataticola infection and screen for tolerant genotypes.

Dry Root Rot Disease Assays in Chickpea: eine detaillierte Methodik

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolierung von hochwertigen atrialen und ventrikulären Myozyten der Maus für die simultane Messung von Ca2+ Transienten und L-Typ Calciumstrom

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron. 
It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled.

This protocol demonstrates how to image biological cryo-preserved samples using cryo-structured illumination microscopy. We demonstrate the methodology by imaging the cytoskeleton of U2OS cells.

Kryostrukturierte Beleuchtung Mikroskopische Datenerfassung von kryogen konservierten Zellen

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Verwendung eines Echtzeit-Zellstoffwechselflussanalysators zur Überwachung der Bioenergetik des Osteoblast

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...