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For a better understanding of living tissues and materials, it is essential to study the intricate spatial relationship between cells and their surrounding tissue on the nanoscale, with a need for 3D, high‐resolution imaging techniques. In the case of bone, focused ion beam‐scanning electron microscopy (FIB‐SEM) operated in the backscattered electron (BSE) mode proves to be a suitable method to image mineralized areas with a nominal resolution of 5 nm. However, as clinically relevant samples are often resin‐embedded, the lack of atomic number (Z) contrast makes it difficult to distinguish the embedding material from unmineralized parts of the tissue, such as osteoid, in BSE images. Staining embedded samples with iodine vapor has been shown to be effective in revealing osteoid microstructure by 2D BSE imaging. Based on this idea, an iodine (Z = 53) staining protocol is developed for 3D imaging with FIB‐SEM, investigating how the amount of iodine and exposure time influences the imaging outcome. Bone samples stained with this protocol also remain compatible with confocal laser scanning microscopy to visualize the lacunocanalicular network. The proposed protocol can be applied for 3D imaging of tissues exhibiting mineralized and nonmineralized regions to study physiological and pathological biomineralization. [ABSTRACT FROM AUTHOR]

Side‐chain‐to‐side‐chain cyclization is frequently used to stabilize the α‐helical conformation of short peptides. In a previous study, we incorporated a lactam bridge between the side chains of Lys‐i and Asp‐i+4 in the nonapeptide 1Y, cyclo‐(2,6)‐(Ac‐VKRLQDLQY‐NH2), an artificial ligand of the inhibitor of DNA binding and cell differentiation (ID) protein with antiproliferative activity on cancer cells. Herein, we show that only the cyclized five‐residue segment adopts a helical turn whereas the C‐terminal residues remain flexible. Moreover, we present nine 1Y analogs arising from different combinations of hydrophobic residues (leucine, isoleucine, norleucine, valine, and tyrosine) at positions 1, 4, 7, and 9. All cyclopeptides except one build a lactam‐bridged helical turn; however, residue‐4 reveals less helix character than the neighboring Arg‐3 and Gln‐5, especially with residue‐4 being isoleucine, valine, and tyrosine. Surprisingly, only two cyclopeptides exhibit helix propagation until the C‐terminus, whereas the others share a remarkable outward tilting of the backbone carbonyl of the lactam‐bridged Asp‐6 (>40° deviation from the orientation parallel to the helix axis), which prevents the formation of the H‐bond between Arg‐3 CO and residue‐7 NH: As a result, the propagation of the helix beyond the lactam‐bridged sequence becomes unfavorable. We conclude that, depending on the amino‐acid sequence, the lactam bridge between Lys‐i and Asp‐i+4 can stabilize a helical turn but deviations from the ideal helix geometry are possible: Indeed, besides the outward tilting of the backbone carbonyls, the residues per turn increased from 3.6 (typical of a regular α‐helix) to 4.2, suggesting a partial helix unwinding. [ABSTRACT FROM AUTHOR]

Abnormal tissue levels of certain trace elements such as zinc (Zn) were reported in various types of cancer. Little is known about the role of Zn in osteosarcoma. Using confocal synchrotron radiation micro X-ray fluorescence analysis, we characterized the spatial distribution of Zn in high-grade sclerosing osteosarcoma of nine patients (four women/five men; seven knee/one humerus/one femur) following chemotherapy and wide surgical resection. Levels were compared with adjacent normal tissue. Quantitative backscattered electron imaging as well as histological examinations was also performed. On average, the ratio of medians of Zn count rates (normalized to calcium) in mineralized tumor tissue was about six times higher than in normal tissue. There was no difference in Zn levels between tumor fraction areas with a low fraction and a high fraction of mineralized tissue, which were clearly depicted using quantitative backscattered electron imaging. Moreover, we found no correlation between the Zn values and the type of tumor regression according to the Salzer-Kuntschik grading. The underlying mechanism of Zn accumulation remains unclear. Given the emerging data on the role of trace elements in other types of cancer, our novel results warrant further studies on the role of trace elements in bone cancer. Copyright © 2016 The Authors. X-Ray Spectrometry published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]

In the quest for finding the ideal synchrotron-radiation-induced imaging method for the investigation of trace element distributions in human bone samples, experiments were performed using both a scanning confocal synchrotron radiation micro X-ray fluorescence (SR-µXRF) (FLUO beamline at ANKA) setup and a full-field color X-ray camera (BAMline at BESSY-II) setup. As zinc is a trace element of special interest in bone, the setups were optimized for its detection. The setups were compared with respect to count rate, required measurement time and spatial resolution. It was demonstrated that the ideal method depends on the element of interest. Although for Ca (a major constituent of the bone with a low energy of 3.69 keV for its Kα XRF line) the color X-ray camera provided a higher resolution in the plane, for Zn (a trace element in bone) only the confocal SR-µXRF setup was able to sufficiently image the distribution. [ABSTRACT FROM AUTHOR]

Background: Idiopathic Juvenile Osteoporosis (IJO) refers to significantly lower than expected bone mass manifesting in childhood with no identifiable aetiology. IJO classically presents in early pubertal period with multiple fractures including metaphyseal and vertebral crush fractures, and low bone‐mass. Methods: Here we describe two patients and provide information on their clinical phenotype, genotype and bone material analysis in one of the patients. Results: Patient 1: 40‐year old adult male diagnosed with IJO in childhood who re‐presented with a hip fracture as an adult. Genetic analysis identified a pathogenic PLS3 hemizygous variant, c.1765del in exon 16. Patient 2: 15‐year old boy with multiple vertebral fractures and bone biopsy findings suggestive of IJO who also has a diagnosis of autism spectrum disorder. Genetic analysis identified a maternally inherited PLS3 pathogenic c.1295T>A variant in exon 12. Analyses of the transiliac bone sample revealed severe reduction of trabecular volume and bone turnover indices and elevated bone matrix mineralisation. Discussion: We propose that genetic testing for PLS3 should be undertaken in patients presenting with a current or previous history of IJO as this has implications for genetic counselling and cascade screening. The extensive evaluation of the transiliac biopsy sample of Patient 2 revealed a novel bone phenotype. Conclusion: This report includes a review of IJO and genetic causes of osteoporosis, and suggests that existing cases of IJO should be screened for PLS3. Through analysis of bone material properties in Patient 2, we can conclude that PLS3 does have a role in bone mineralisation. [ABSTRACT FROM AUTHOR]

Synthetic helical peptides are valuable scaffolds for the development of modulators of protein-protein interactions involving helical motifs. Backbone-to-side chain or side chain-to-side chain constraints have been and still are intensively exploited to stabilize short α-helices. Very often, these constraints have been combined with backbone modifications induced by C α-tetrasubstituted, β-, or γ-amino acids, which facilitate the α-peptide or α/ β/ γ-peptide adopting an α-helical conformation. In this work, we investigated the helical character of octapeptides that were cyclized by a Lys-Asp-( i, i + 4)-lactam bridge. We started with two sequences extracted from the helix-loop-helix region of the Id proteins, which are inhibitors of cell differentiation during development and in cancer. Nineteen analogs containing the lactam bridge at different positions and displaying different amino acid core triads ( i + 1,2,3) as well as outer residues were prepared by solid-phase methodology. Their conformation in water and water/2,2,2-trifluoroethanol mixtures was investigated by circular dichroism (CD) spectroscopy. The cyclopeptides could be grouped in helix-prone and non-helix-prone structures. Both the amino acid core triad ( i + 1,2,3) and the pendant residues positively or negatively affected the formation of a helical structure. Computational studies based on the NMR-derived helical structure of a cyclopeptide containing Aib at position ( i + 2) of the triad were generally in agreement with the secondary structure propensity of the cyclopeptides observed by CD spectroscopy. In conclusion, the Lys-Asp-( i, i + 4)-lactam bridge may succeed or fail in the stabilization of short helices, depending on the primary structure. Moreover, computational methods may be valuable tools to discriminate helix-prone from non-helix-prone peptide-based macrolactams. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Mice lacking the renal epithelial Ca2+ channel TRPV5 (TRPV5−/−) display impaired renal Ca2+ reabsorption, hypercalciuria, and intestinal Ca2+ hyperabsorption, due to secondary hypervitaminosis D. Using these mice, we previously demonstrated that ZK191784 acts as an intestine-specific 1,25(OH)2D3 antagonist without affecting serum calcium levels. On the other hand, it acted as an agonist in the kidney and the effects of ZK191784 on bone were ambiguous. The present study was undertaken to further evaluate the effect of the vitamin D receptor antagonist on murine bone in mice lacking TRPV5. Eight-week-old female Trpv5 +/+ and Trpv5 −/− mice were treated for 4 weeks with or without 50 µg/kg/day ZK191784. Quantitative backscattered electron imaging showed that the reduced bone matrix mineralization found in femoral bones of Trpv5 −/− mice was partially but significantly restored upon ZK191784 treatment, just as we observed for trabecular bone thickness. This supports the significance of 1,25(OH)2D3 and optimal control of Ca2+ homeostasis for bone formation and matrix mineralization. Restoration also took place at the bone gene expression level, where 1α-hydroxylase ( Cyp27b1) mRNA in femurs from ZK-treated Trpv5 −/− mice was upregulated compared to control levels. The downregulated 24-hydroxylase ( Cyp24a1) gene expression in femoral bone indicated local vitamin D resistance in the mice treated with ZK191784. Phosphate homeostasis was unaffected between the groups as shown by unaltered serum $PO_{4}^{3- } $ and fibroblast growth factor (FGF) 23 as well as Fgf23 mRNA expression in bone. In conclusion, circulating 1,25(OH)2D3 is important for optimal control of Ca2+ homeostasis but also for controlled bone formation and matrix mineralization. J. Cell. Physiol. 228: 402-407, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Little is known whether trabecular bone matrix mineralization is altered at the site of osteoporotic vertebral fractures. Bone mineralization density distribution (BMDD) was assessed in trabecular bone of acute, single-level compression fractures of the spine at various stages of fracture repair using quantitative backscattered electron imaging (qBEI). The grading of the repair stage was performed by histological methods. From 20 patients, who underwent either kyphoplasty ( n = 18) or vertebroplasty ( n = 2), a vertebral bone biopsy was taken prior to cement augmentation. Six patients took bisphosphonates (BP) prior to fracture. Three study groups were formed: N1 = early-, N2 = late-healing and B = BP treatment at late healing stage. In general, all groups had an altered BMDD when compared to historical normative reference data. Mean matrix mineralization (CaMean) was significantly ( p < 0.001) lower in all groups (N1: −5%, N2: −16%, and B2: −16%). In N2, CaMean was −13.1% ( p < 0.001) lower than N1. At this stage, deposition of new bone matrix and/or formation of woven bone are seen, which also explains the more heterogeneous matrix mineralization (CaWidth). Moreover, BP treatment (B2) led to a significant reduction in CaWidth (−28.5%, p < 0.001), when compared to N2. Bone tissue from vertebrae with acute compression fractures reveals a large variation in matrix mineralization depending on the stage of repair. Bisphosphonate treatment does affect the mineralization pattern of tissue repair. The low mineralization values found in early stage of repair suggest that altered bone material properties may play a role in the occurrence of fragility fractures of the spine. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1089-1094, 2012 [ABSTRACT FROM AUTHOR]

Bone is the major store for Ca2+ in the body and plays an important role in Ca2+ homeostasis. During bone formation and resorption Ca2+ must be transported to and from bone by osteoblasts and osteoclasts, respectively. However, little is known about the Ca2+ transport machinery in these bone cells. In this study, we examined the epithelial Ca2+ channel TRPV6 in bone. TRPV6 mRNA is expressed in human and mouse osteoblast-like cells as well as in peripheral blood mononuclear cell-derived human osteoclasts and murine tibial bone marrow-derived osteoclasts. Also other transcellular Ca2+ transport genes, calbindin-D9k and/or -D28K, Na+/Ca2+ exchanger 1, and plasma membrane Ca2+ ATPase (PMCA1b) were expressed in these bone cell types. Immunofluorescence and confocal microscopy on human osteoblasts and osteoclasts and mouse osteoclasts revealed TRPV6 protein at the apical domain and PMCA1b at the osteoidal domain of osteoblasts, whereas in osteoclasts TRPV6 was predominantly found at the bone-facing site. TRPV6 was dynamically expressed in human osteoblasts, showing maximal expression during mineralization of the extracellular matrix. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) did not change TRPV6 expression in both mineralizing and non-mineralizing SV-HFO cultures. Lentiviral transduction-mediated overexpression of TRPV6 in these cells did not alter mineralization. Bone microarchitecture and mineralization were unaffected in Trpv6D541A/D541A mice in which aspartate 541 in the pore region was replaced with alanine to render TRPV6 channels non-functional. In summary, TRPV6 and other proteins involved in transcellular Ca2+ transport are dynamically expressed in bone cells, while TRPV6 appears not crucial for bone metabolism and matrix mineralization in mice. J. Cell. Physiol. 227: 1951-1959, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Based on clinical trials showing the efficacy to reduce vertebral and non-vertebral fractures, strontium ranelate (SrR) has been approved in several countries for the treatment of postmenopausal osteoporosis. Hence, it is of special clinical interest to elucidate how the Sr uptake is influenced by dietary Ca deficiency as well as by the formula of Sr administration, SrR versus strontium chloride (SrCl2). Three-month-old ovariectomized rats were treated for 90 days with doses of 25 mg kg−1 d−1 and 150 mg kg−1 d−1 of SrR or SrCl2 at low (0.1% Ca) or normal (1.19% Ca) Ca diet. Vertebral bone tissue was analysed by confocal synchrotron-radiation-induced micro X-ray fluorescence and by backscattered electron imaging. Principal component analysis and k-means clustering of the acquired elemental maps of Ca and Sr revealed that the newly formed bone exhibited the highest Sr fractions and that low Ca diet increased the Sr uptake by a factor of three to four. Furthermore, Sr uptake in bone of the SrCl2-treated animals was generally lower compared with SrR. The study clearly shows that inadequate nutritional calcium intake significantly increases uptake of Sr in serum as well as in trabecular bone matrix. This indicates that nutritional calcium intake as well as serum Ca levels are important regulators of any Sr treatment. [ABSTRACT FROM AUTHOR]

The article investigates the measurement of chemical species of lead (Pb) in tidemarks of human articular cartilage using X-ray absorption near edge structure (XANES) analysis. It discusses the application of spatially resolved X-ray absorption spectroscopy (XAS) to determine the species of Pb in the tidemark and human bones. It also explores the information contains in XANES spectra about the chemical state of the absorbing element.

The article details the use of a stack of cards model, which describes the size and arrangement of mineral particles (MP) in bone tissues, for the analysis of the results of a scanning small-angle X-ray scattering experiment on a pathological bone biopsy. The similar description of the model and the tissue's ultrastructure is discussed. In addition, the parameters to characterize the fluctuations in positional correlation of the MP are derived.

Texture analysis with microbeam scanning diffraction enables the local mapping of three-dimensional crystallite orientation in heterogeneous natural and synthetic materials. Cortical (compact) bone is an example of a hierarchically structured biocomposite, which is built mainly of cylindrical osteons, having a lamellar texture at the micrometre level. In this work, a combination of microbeam synchrotron X-ray texture analysis with thin sections of osteonal bone is used to measure the three-dimensional distribution of the c-axis orientation of the mineral apatite in bone with positional resolution of 1 µm. The data reduction procedure needed to go from the stereographic projection of X-ray intensity to the determination of the local orientation of mineralized collagen fibrils is described. The procedure can be applied to other mineralized tissues (such as trabecular bone and chitin) with micrometre scale and biologically controlled fibrillar texture. [ABSTRACT FROM AUTHOR]

Analyzes the texture of the human vertebral bone material, a collagen-mineral nanocomposite, within single trabecula by collecting two-dimensional small-angle X-ray scattering (SAXS) and X-ray diffraction (XRD) data. Reconstruction of three-dimensional information by combining measurements with different specimen orientations; Pole figure analysis of the SAXS and XRD data.

Focuses on the development of small-angle X-ray scattering and wide angle X-ray diffraction instrument to analyze bone and cartilage structure. Details on the structure of biological materials; Evaluation of the approach to bone and cartilage investigation; Information on the properties of the nanoparticles.

Monoclonal antibodies, fusion proteins including the immunoglobulin fragment c (Ig Fc) CH2‐CH3 domains, and engineered antibodies are prominent representatives of an important class of drugs and drug candidates, which are referred to as biotherapeutics or biopharmaceuticals. These recombinant proteins are highly heterogeneous due to their glycosylation pattern. In addition, enzyme‐independent reactions, like deamidation, dehydration, and oxidation of sensitive side chains, may contribute to their heterogeneity in a minor amount. To investigate the biological impact of a spontaneous chemical modification, especially if found to be recurrent in a biotherapeutic, it would be necessary to reproduce it in a homogeneous manner. Herein, we undertook an explorative study towards the chemical synthesis of the IgG1 Fc CH3 domain, which has been shown to undergo spontaneous changes like succinimide formation and methionine oxidation. We used Fmoc‐solid‐phase peptide synthesis (SPPS) and native chemical ligation (NCL) to test the accessibility of large fragments of the IgG1 Fc CH3 domain. In general, the incorporation of pseudoproline dipeptides improved the quality of the crude peptide precursors; however, sequences larger than 44 residues could not be achieved by standard stepwise elongation with Fmoc‐SPPS. In contrast, the application of NCL with cysteine residues, which were either native or introduced ad hoc, allowed the assembly of the C‐terminal IgG1 Fc CH3 sequence 371 to 450. The syntheses reported here show advantages and limitations of the chemical approaches chosen for the preparation of the synthetic IgG1 Fc CH3 domain and will guide future plans towards the synthesis of both the native and selectively modified full‐length domain. Different fragments of the lgG1 Fc CH3 domain have been synthesized by Fmoc‐SPPS and evaluated for their suitability as precursors for native chemical ligation [ABSTRACT FROM AUTHOR]