Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published “–omics” data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in ‘skewing’ of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these “control” proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation using total protein analysis on samples run in parallel with stains such as Coomassie blue provides a more robust approach.

Recent developments in endocrinology, made possible by the combination of mouse genetics, integrative physiology and clinical observations have resulted in rapid and unanticipated advances in the field of skeletal biology. Indeed, the skeleton, classically viewed as a structural scaffold necessary for mobility, and regulator of calcium-phosphorus homoeostasis and maintenance of the haematopoietic niche has now been identified as an important regulator of male fertility and whole-body glucose metabolism, in addition to the classical insulin target tissues. These seminal findings confirm bone to be a true endocrine organ. This review is intended to detail the key events commencing from the elucidation of osteocalcin (OC) in bone metabolism to identification of new and emerging candidates that may regulate energy metabolism independently of OC.Key WordsJournal of Endocrinology (2015) 225, R1-R19

PHOSPHO1 is one of principal proteins involved in initiating bone matrix mineralisation. Recent studies have found that Phospho1 KO mice (Phospho1-R74X) display multiple skeletal abnormalities with spontaneous fractures, bowed long bones, osteomalacia and scoliosis. These analyses have however been limited to young mice and it remains unclear whether the role of PHOSPHO1 is conserved in the mature murine skeleton where bone turnover is limited. In this study, we have used ex-vivo computerised tomography to examine the effect of Phospho1 deletion on tibial bone architecture in mice at a range of ages (5, 7, 16 and 34 weeks of age) to establish whether its role is conserved during skeletal growth and maturation. Matrix mineralisation has also been reported to influence terminal osteoblast differentiation into osteocytes and we have also explored whether hypomineralised bones in Phospho1 KO mice exhibit modified osteocyte lacunar and vascular porosity. Our data reveal that Phospho1 deficiency generates age-related defects in trabecular architecture and compromised cortical microarchitecture with greater porosity accompanied by marked alterations in osteocyte shape, significant increases in osteocytic lacuna and vessel number. Our in vitro studies examining the behaviour of osteoblast derived from Phospho1 KO and wild-type mice reveal reduced levels of matrix mineralisation and modified osteocytogenic programming in cells deficient in PHOSPHO1. Together our data suggest that deficiency in PHOSPHO1 exerts modifications in bone architecture that are transient and depend upon age, yet produces consistent modification in lacunar and vascular porosity. It is possible that the inhibitory role of PHOSPHO1 on osteocyte differentiation leads to these age-related changes in bone architecture. It is also intriguing to note that this apparent acceleration in osteocyte differentiation evident in the hypomineralised bones of Phospho1 KO mice suggests an uncoupling of the interplay between osteocytogenesis and biomineralisation. Further studies are required to dissect the molecular processes underlying the regulatory influences exerted by PHOSPHO1 on the skeleton with ageing.

Bone is a strong and tough material composed of apatite mineral, organic matter and water. Changes in composition and organization of these building blocks affect bone’s mechanical integrity. Skeletal disorders often affect bone’s mineral phase, either by variations in the collagen or directly altering mineralization. The aim of the current study was to explore the differences in the mineral of brittle and ductile cortical bone at the mineral (nm) and tissue (µm) levels using two mouse phenotypes. Osteogenesis imperfecta murine (oim−/−) mice were used to model brittle bone; PHOSPHO1 mutants (Phospho1−/−) had ductile bone. They were compared to their respective wild-type controls. Femora were defatted and ground to powder to measure average mineral crystal size using X-ray diffraction (XRD), and to monitor the bulk mineral to matrix ratio via thermogravimetric analysis (TGA). XRD scans were run after TGA for phase identification, to assess the fractions of hydroxyapatite and β-tricalcium phosphate. Tibiae were embedded to measure elastic properties with nanoindentation and the extent of mineralization with backscattered electron microscopy (qbSEM). Interestingly, the mineral of brittle oim−/− and ductile Phospho1−/− bones had many similar characteristics. Both pathology models had smaller apatite crystals, lower mineral to matrix ratio, and showed more thermal conversion to β-tricalcium phosphate than their wild-types, indicating deviations from stoichiometric hydroxyapatite in the original mineral. The degree of mineralization of the bone matrix was different for each strain: oim−/− were hypermineralized, while Phospho1−/− were hypomineralized. However, alterations in the mineral were associated with reduced tissue elastic moduli in both pathologies. Results revealed that despite having extremely different whole bone mechanics, the mineral of oim−/− and Phospho1−/− has several similar trends at smaller length scales. This indicates that alterations from normal crystal size, composition, and structure will reduce the mechanical integrity of bone.

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term "western blot" suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many "optimized" western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

Bone fragility is a concern for aged and diseased bone. Measuring bone toughness and understanding fracture properties of the bone are critical for predicting fracture risk associated with age and disease and for preclinical testing of therapies. A reference point indentation technique (BioDent) has recently been developed to determine bone's resistance to fracture in a minimally invasive way by measuring the indentation distance increase (IDI) between the first and last indentations over cyclic indentations in the same position. In this study, we investigate the relationship between fracture toughness KC and reference point indentation parameters (i.e. IDI, total indentation distance (TID) and creep indentation distance (CID)) in bones from 38 mice from six types (C57Bl/6, Balb, oim/oim, oim/+, Phospho1−/− and Phospho1 wild type counterpart). These mice bone are models of healthy and diseased bone spanning a range of fracture toughness from very brittle (oim/oim) to ductile (Phospho1−/−). Left femora were dissected, notched and tested in 3-point bending until complete failure. Contralateral femora were dissected and indented in 10 sites of their anterior and posterior shaft surface over 10 indentation cycles. IDI, TID and CID were measured. Results from this study suggest that reference point indentation parameters are not indicative of stress intensity fracture toughness in mouse bone. In particular, the IDI values at the anterior mid-diaphysis across mouse types overlapped, making it difficult to discern differences between mouse types, despite having extreme differences in stress intensity based toughness measures. When more locations of indentation were considered, the normalised IDIs could distinguish between mouse types. Future studies should investigate the relationship of the reference point indentation parameters for mouse bone in other material properties of the bone tissue in order to determine their use for measuring bone quality.

Follistatin-like 3 (FSTL3) is a glycoprotein that binds and inhibits the action of TGFβ ligands such as activin. The roles played by FSTL3 and activin signaling in organ development and homeostasis are not fully understood. The authors show mice deficient in FSTL3 develop markedly enlarged testes that are also delayed in their age-related regression. These FSTL3 knockout mice exhibit increased Sertoli cell numbers, allowing for increased spermatogenesis but otherwise showing normal testicular function. The data show that FSTL3 deletion leads to increased AKT signaling and SIRT1 expression in the testis. This demonstrates a cross-talk between TGFβ ligand and AKT signaling and leads to a potential mechanism for increased cellular survival and antiaging. The findings identify crucial roles for FSTL3 in limiting testis organ size and promoting age-related testicular regression.

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