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Hypophosphatasia (HPP) is a clinically heterogeneous rare, inherited disorder of bone and mineral metabolism with extensive allelic heterogeneity in the ALPL gene. In this report, we present a family with heterozygous parents (maternal p.(Glu191Lys), paternal p.(Gly334Asp) mutations in the ALPL gene) and four children (one genotypically normal, one heterozygous carrier and two compound heterozygous) showing an unexpected high phenotypic variability. One of the compound heterozygous showed clinical symptoms of the mild childhood form mainly affecting the teeth. The other one was more seriously affected with severe failure to thrive, delayed motor development, need for oxygen supply and profound mineralization deficit compatible with an infantile form of HPP. Functional in vitro studies identified p.(Glu191Lys) as mild (68%, no dominant-negative effect) and p.(Gly334Asp) as severely affected allele (1.2%, dominant-negative effect). In vitro simulation of the children's genetic status showed a residual AP activity of 29%, while the biochemical AP activity in the serum was comparably reduced in both children (22 and 36 U/l). This family report indicates that mapping ALPL mutations within the gene does not necessarily help to predict the clinical severity of the phenotype. Therefore, results of prenatal diagnostics have to be interpreted with caution and prenatal genetic diagnosis and counseling for HPP should be provided within an experienced multidisciplinary team. Research about other confounding factors is urgently needed.

Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements – it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types.

Enhanced osteoclast formation and function is a fundamental cause of alterations to bone structure and plays an important role in several diseases impairing bone quality. Recent work revealed that TRP calcium channels 3 and 6 might play a special role in this context. By analyzing the bone phenotype of TRPC6-deficient mice we detected a regulatory effect of TRPC3 on osteoclast function. These mice exhibit a significant decrease in bone volume per tissue volume, trabecular thickness and -number together with an increased number of osteoclasts found on the surface of trabecular bone. Primary bone marrow mononuclear cells from TRPC6-deficient mice showed enhanced osteoclastic differentiation and resorptive activity. This was confirmed in vitro by using TRPC6-deficient RAW 264.7 cells. TRPC6 deficiency led to an increase of TRPC3 in osteoclasts, suggesting that TRPC3 overcompensates for the loss of TRPC6. Raised intracellular calcium levels led to enhanced NFAT-luciferase reporter gene activity in the absence of TRPC6. In line with these findings inhibition of TRPC3 using the specific inhibitor Pyr3 significantly reduced intracellular calcium concentrations and normalized osteoclastic differentiation and resorptive activity of TRPC6-deficient cells. Interestingly, an up-regulation of TRPC3 could be detected in a cohort of patients with low bone mineral density by comparing micro array data sets of circulating human osteoclast precursor cells to those from patients with high bone mineral density, suggesting a noticeable contribution of TRP calcium channels on bone quality. These observations demonstrate a novel regulatory function of TRPC channels in the process of osteoclastic differentiation and bone loss.

1,25-dihydroxyvitamin D3 (1,25D3) was reported to induce premature organismal aging in fibroblast growth factor-23 (Fgf23) and klotho deficient mice, which is of main interest as 1,25D3 supplementation of its precursor cholecalciferol is used in basic osteoporosis treatment. We wanted to know if 1,25D3 is able to modulate aging processes on a cellular level in human mesenchymal stem cells (hMSC). Effects of 100 nM 1,25D3 on hMSC were analyzed by cell proliferation and apoptosis assay, β-galactosidase staining, VDR and surface marker immunocytochemistry, RT-PCR of 1,25D3-responsive, quiescence- and replicative senescence-associated genes. 1,25D3 treatment significantly inhibited hMSC proliferation and apoptosis after 72 h and delayed the development of replicative senescence in long-term cultures according to β-galactosidase staining and P16 expression. Cell morphology changed from a fibroblast like appearance to broad and rounded shapes. Long term treatment did not induce lineage commitment in terms of osteogenic pathways but maintained their clonogenic capacity, their surface marker characteristics (expression of CD73, CD90, CD105) and their multipotency to develop towards the chondrogenic, adipogenic and osteogenic pathways. In conclusion, 1,25D3 delays replicative senescence in primary hMSC while the pro-aging effects seen in mouse models might mainly be due to elevated systemic phosphate levels, which propagate organismal aging.

Background Anti-resorptive bisphosphonates (BP) are used for the treatment of osteoporosis and bone metastases. Clinical studies indicated a benefit in survival and tumor relapse in subpopulations of breast cancer patients receiving zoledronic acid, thus stimulating the debate about its anti-tumor activity. Amino-bisphosphonates in nM concentrations inhibit farnesyl pyrophosphate synthase leading to accumulation of isopentenyl pyrophosphate (IPP) and the ATP/pyrophosphate adduct ApppI, which induces apoptosis in osteoclasts. For anti-tumor effects μM concentrations are needed and a sensitizer for bisphosphonate effects would be beneficial in clinical anti-tumor applications. We hypothesized that enhancing intracellular pyrophosphate accumulation via inhibition of probenecid-sensitive channels and transporters would sensitize tumor cells for bisphosphonates anti-tumor efficacy. Method MDA-MB-231, T47D and MCF-7 breast cancer cells were treated with BP (zoledronic acid, risedronate, ibandronate, alendronate) and the pyrophosphate channel inhibitors probenecid and novobiocin. We determined cell viability and caspase 3/7 activity (apoptosis), accumulation of IPP and ApppI, expression of ANKH, PANX1, ABCC1, SLC22A11, and the zoledronic acid target gene and tumor-suppressor KLF2. Results Treatment of MDA-MB-231 with BP induced caspase 3/7 activity, with zoledronic acid being the most effective. In MCF-7 and T47D either BP markedly suppressed cell viability with only minor effects on apoptosis. Co-treatment with probenecid enhanced BP effects on cell viability, IPP/ApppI accumulation as measurable in MCF-7 and T47D cells, caspase 3/7 activity and target gene expression. Novobiocin co-treatment of MDA-MB-231 yielded identical results on viability and apoptosis compared to probenecid, rendering SLC22A family members as candidate modulators of BP effects, whereas no such evidence was found for ANKH, ABCC1 and PANX1. Conclusions In summary, we demonstrate effects of various bisphosphonates on caspase 3/7 activity, cell viability and expression of tumor suppressor genes in breast cancer cells. Blocking probenecid and novobiocin-sensitive channels and transporters enhances BP anti-tumor effects and renders SLC22A family members as good candidates as BP modulators. Further studies will have to unravel if treatment with such BP-sensitizers translates into preclinical and clinical efficacy.

Consumption of soya-based nutrients is increasing in modern society because of their potentially protective effects against chronic diseases. Soya products are also heavily advertised as alternative drugs for relief from symptoms of the menopause and for hormone replacement therapy.However, because of their oestrogenic activity, negative effects of isoflavones have been postulated. Therefore, we analysed influences of soya isoflavones, major soya constituents with endocrine activity, on thyroxine (T4) binding to its distribution proteins. Serum binding of 125I-labelled l-T4 was analysed in the absence or presence of increasing concentrations of soya isoflavones using non-denaturing PAGE for analysis. Complete displacement of [125I]T4 binding to transthyretin (TTR) was observed in human serum incubated with genistein at concentrations >10μm; interference started at >0·1μm. Glycitein showed decreased and daidzein the lowest displacement potency. [125I]T4 was displaced to albumin in rat and to T4-binding globulin in human serum. Soya isoflavones also obstruct [125I]T4 binding to TTR in human cerebrospinal fluid (CSF). The inhibitory effect was confirmed in direct binding assays using purified TTR with 50% inhibitory concentration values of 0·07μm for genistein, 0·2μm for glycitein and 1·8μm for daidzein. The present study underlined a potent competition of soya isoflavones for T4 binding to TTR in serum and CSF. Isoflavones might alter free thyroid hormone concentrations resulting in altered tissue availability and metabolism. As a consequence of this interference, one could expect a disturbance in the feedback regulation of hormonal networks, including the pituitary–thyroid–periphery axis during development and in adult organisms.

Epidemiological data, animal studies and interventional studies provide evidence for a potential chemopreventive effect of selenium during development of colorectal cancer. The human glycoprotein Selenoprotein P (SeP) contains up to 50% of plasma selenium content. SeP is expressed in the gastrointestinal tract and the liver, where its expression is downregulated by various proinflammatory cytokines (Il1β, TGFβ, IFNγ). Previously, we have demonstrated dramatically reduced SeP expression in human colon adenomas. Here, we have identified a complex (A) 4 -C-(A) 4 -GG-(A) 8 -GCT-(TC) 5 -(T) 17 (bp −429 to bp − 477) repeat structure within the SeP promoter and we have analysed this regulatory DNA sequence with respect to polymorphisms, genomic instability and functional relevance to promoter activity. As opposed to the (TC) 5 variant we identified a novel (TC) 3 polymorphism within this repeat in the general population, which conferred significantly reduced basal promoter activity to reporter gene constructs in HepG2 cells. Allelic distribution of this (TC) n element was similar in colon carcinoma patients and healthy controls. Additionally, we observed genetic instability within the (T) 17 repeat motif in colon cancers of the mutator phenotype. This instability of the (T) 17 repeat had no effect on basal promoter activity in reporter gene assays. In conclusion, we characterised a complex repeat structure within the SeP promoter that may be of functional relevance to SeP gene expression. Further studies on the effect of different SeP promoter genotypes on SeP protein expression and disease susceptibility are needed.

There is growing evidence that, in addition to the reproductive system, the hypothalamic-pituitary-thyroid axis is a target of endocrine-disrupting compounds (EDCs). However, this is not reflected adequately in current screening and assessment procedures for endocrine activity that to date determine only general parameters of thyroid function. We used several in vitro and ex vivo assays in an attempt to identify suitable biomarkers for antithyroid action testing a selected panel of putative EDCs. In vitro we detected stimulation or inhibition of iodide uptake into FRTL-5 rat thyroid cells, inhibition of thyroid hormone binding to transthyretin, agonistic or antagonistic effects in a thyroid hormone receptor-dependent reporter assay, and inhibition of thyroid peroxidase using a novel assay system based on human recombinant thyroperoxidase that might be suitable for routine screening for potential EDCs. In rats, chronic application of several EDCs led to changes in thyroid morphology, alterations of thyrotropin and thyroid hormone serum levels as well as alterations in peripheral thyroid hormone-regulated end points such as malic enzyme and type I 5'-deiodinase activity. As the effects of EDCs do not reflect classic mechanisms of hormone-dependent regulation and feedback, we believe multitarget and multimodal actions of EDCs affect the hypothalamic-pituitary-thyroid axis. These complex effects require a diverse approach for screening, evaluation, and risk assessment of potential antithyroid compounds. This approach involves novel in vitro or cell-based screening assays in order to assess thyroid hormone synthesis, transport, metabolism, and action as well as in vivo assays to measure thyroid hormone-regulated tissue-specific and developmental end points in animals.

We performed exome sequencing for mutation discovery of an ENU (N-ethyl-N-nitrosourea)-derived mouse model characterized by significant elevated plasma alkaline phosphatase (ALP) activities in female and male mutant mice, originally named BAP014 (bone screen alkaline phosphatase #14). We identified a novel loss-of-function mutation within the Fam46a (family with sequence similarity 46, member A) gene (NM_001160378.1:c.469G>T, NP_001153850.1:p.Glu157*). Heterozygous mice of this mouse line (renamed Fam46a ᴱ¹⁵⁷*ᴹʰᵈᵃ) had significantly high ALP activities and apparently no other differences in morphology compared to wild-type mice. In contrast, homozygous Fam46a ᴱ¹⁵⁷*ᴹʰᵈᵃ mice showed severe morphological and skeletal abnormalities including short stature along with limb, rib, pelvis, and skull deformities with minimal trabecular bone and reduced cortical bone thickness in long bones. ALP activities of homozygous mutants were almost two-fold higher than in heterozygous mice. Fam46a is weakly expressed in most adult and embryonic tissues with a strong expression in mineralized tissues as calvaria and femur. The FAM46A protein is computationally predicted as a new member of the superfamily of nucleotidyltransferase fold proteins, but little is known about its function. Fam46a ᴱ¹⁵⁷*ᴹʰᵈᵃ mice are the first mouse model for a mutation within the Fam46a gene.

Covering all state-of-the-art experimental research methods in orthopedic surgery and trauma From bioinformatics to nanotechnology, advances in basic research ultimately drive advances in clinical care. This book provides a comprehensive summary of all current research methodologies for translational and pre-clinical studies in biomechanics and orthopedic trauma surgery. With this \"roadmap\" at hand, specialists and trainees will have the tools to conduct high-quality experimental research in any area of musculoskeletal science, with a solid understanding of how the findings can be applied in patient care. Special Features: * Utilizes the principles and methodology of modern, evidence-based medicine in pre-clinical musculoskeletal research * Offers a comprehensive analysis of in vivo models for studying different components of the musculoskeletal system * Demonstrates how principles of structural, functional, and numerical biomechanics can be utilized in well-defined experimental research studies – spanning topics from fracture fixation to gait analysis to bone remodeling * Covers the role of new macroscopic CT and ultrasound imaging techniques for assessing bone and cartilage function * Explores cutting-edge developments in cell culture research, molecular testing, and tissue engineering * Provides practical advice, a glossary of key terminology, and hundreds of illustrations to familiarize clinicians with every aspect of designing and interpreting an effective research study With 54 state-of-the-art chapters by orthopedic surgeons, musculoskeletal physicians, biologists, engineers, physicists, and mathematicians, Experimental Research Methods in Orthopedics and Trauma is the authoritative reference on the topic. It is essential for clinicians, basic researchers, and orthopedic surgical trainees who need to understand experimental research methodology, apply its findings, and participate fully in research activities.