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Over the past years, pituitary hormones and their receptors have been shown to have non-traditional actions that allow them to bypass the hypothalamus-pituitary-effector glands axis. Bone cells—osteoblasts and osteoclasts—express receptors for growth hormone, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), adrenocorticotrophic hormone (ACTH), prolactin, oxytocin, and vasopressin. Independent skeletal actions of pituitary hormones on bone have been studied using genetically modified mice with haploinsufficiency and by activating or inactivating the receptors pharmacologically, without altering systemic effector hormone levels. On another front, the discovery of a TSH variant (TSH-βv) in immune cells in the bone marrow and skeletal action of FSHβ through tumor necrosis factor α provides new insights underscoring the integrated physiology of bone-immune-endocrine axis. Here we discuss the interaction of each pituitary hormone with bone and the potential it holds in understanding bone physiology and as a therapeutic target.

Silver nanoparticles (AgNPs) were synthesized using the whole plant of Duchesnea indica (DI) which was extracted in different solvents; the antimicrobial effects of the extract were investigated in this study. The extraction of DI was performed using three different solvents: water, pure ethanol (EtOH), and pure dimethyl sulfoxide (DMSO). AgNP formation was monitored by measuring the UV–Vis spectrum of each reaction solution. After synthesis for 48 h, the AgNPs were collected and the negative surface charge and size distribution of the synthesized AgNPs were measured using dynamic light scattering (DLS). The AgNP structure was determined by high-resolution powder X-ray diffraction (XRD) and the AgNP morphology was investigated using transmission electron microscopy (TEM). AgNP antibacterial activities were evaluated against Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, and Pseudomonas aeruginosa using the disc diffusion method. Additionally, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were also determined. Biosynthesized AgNPs showed enhanced antibacterial activity against B. cereus, S. aureus, E. coli, S. enteritidis, and P. aeruginosa compared with that of pristine solvent extract. These results suggest that AgNPs synthesized from extracts of DI are promising antibacterial agents against pathogenic bacteria and can be further applied in the food industry.

Highly concentrated antibody formulations are oftentimes required for subcutaneous, self-administered biologics. Here, we report the creation of a unique formulation for our first–in– class FSH–blocking humanized antibody, MS-Hu6, which we propose to move to the clinic for osteoporosis, obesity, and Alzheimer’s disease. The studies were carried out using our Good Laboratory Practice (GLP) platform, compliant with the Code of Federal Regulations (Title 21, Part 58). We first used protein thermal shift, size exclusion chromatography, and dynamic light scattering to examine MS-Hu6 concentrations between 1 and 100 mg/mL. We found that thermal, monomeric, and colloidal stability of formulated MS-Hu6 was maintained at a concentration of 100 mg/mL. The addition of the antioxidant L–methionine and chelating agent disodium EDTA improved the formulation’s long–term colloidal and thermal stability. Thermal stability was further confirmed by Nano differential scanning calorimetry (DSC). Physiochemical properties of formulated MS-Hu6, including viscosity, turbidity, and clarity, conformed with acceptable industry standards. That the structural integrity of MS-Hu6 in formulation was maintained was proven through Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. Three rapid freeze–thaw cycles at –80°C/25°C or –80°C/37°C further revealed excellent thermal and colloidal stability. Furthermore, formulated MS-Hu6, particularly its Fab domain, displayed thermal and monomeric storage stability for more than 90 days at 4°C and 25°C. Finally, the unfolding temperature (Tm) for formulated MS-Hu6 increased by >4.80°C upon binding to recombinant FSH, indicating highly specific ligand binding. Overall, we document the feasibility of developing a stable, manufacturable and transportable MS-Hu6 formulation at a ultra–high concentration at industry standards. The study should become a resource for developing biologic formulations in academic medical centers.

The past decade has seen significant advances in our understanding of skeletal homeostasis and the mechanisms that mediate the loss of bone integrity in disease. Recent breakthroughs have arisen mainly from identifying disease-causing mutations and modeling human bone disease in rodents, in essence, highlighting the integrative nature of skeletal physiology. It has become increasingly clear that bone cells, osteoblasts, osteoclasts, and osteocytes, communicate and regulate the fate of each other through RANK/RANKL/OPG, liver X receptors (LXRs), EphirinB2-EphB4 signaling, sphingolipids, and other membrane-associated proteins, such as semaphorins. Mounting evidence also showed that critical developmental pathways, namely, bone morphogenetic protein (BMP), NOTCH, and WNT, interact each other and play an important role in postnatal bone remodeling. The skeleton communicates not only with closely situated organs, such as bone marrow, muscle, and fat, but also with remote vital organs, such as the kidney, liver, and brain. The metabolic effect of bone-derived osteocalcin highlights a possible role of skeleton in energy homeostasis. Furthermore, studies using genetically modified rodent models disrupting the reciprocal relationship with tropic pituitary hormone and effector hormone have unraveled an independent role of pituitary hormone in skeletal remodeling beyond the role of regulating target endocrine glands. The cytokine-mediated skeletal actions and the evidence of local production of certain pituitary hormones by bone marrow-derived cells displays a unique endocrine-immune-skeletal connection. Here, we discuss recently elucidated mechanisms controlling the remodeling of bone, communication of bone cells with cells of other lineages, crosstalk between bone and vital organs, as well as opportunities for treating diseases of the skeleton.

Pharmacological and genetic studies over the past decade have established the follicle-stimulating hormone (FSH) as an actionable target for diseases affecting millions, namely osteoporosis, obesity, and Alzheimer’s disease. Blocking FSH action prevents bone loss, fat gain, and neurodegeneration in mice. We recently developed a first-in-class, humanized, epitope-specific FSH-blocking antibody, MS-Hu6, with a KD of 7.52 nM. Using a Good Laboratory Practice (GLP)-compliant platform, we now report the efficacy of MS-Hu6 in preventing and treating osteoporosis in mice and parameters of acute safety in monkeys. Biodistribution studies using 89Zr-labeled, biotinylated or unconjugated MS-Hu6 in mice and monkeys showed localization to bone and bone marrow. The MS-Hu6 displayed a β phase t½ of 7.5 days (180 hr) in humanized Tg32 mice. We tested 217 variations of excipients using the protein thermal shift assay to generate a final formulation that rendered MS-Hu6 stable in solution upon freeze-thaw and at different temperatures, with minimal aggregation, and without self-, cross-, or hydrophobic interactions or appreciable binding to relevant human antigens. The MS-Hu6 showed the same level of “humanness” as human IgG1 in silico and was non-immunogenic in ELISpot assays for IL-2 and IFN-γ in human peripheral blood mononuclear cell cultures. We conclude that MS-Hu6 is efficacious, durable, and manufacturable, and is therefore poised for future human testing.

There is increasing evidence that anterior pituitary hormones, traditionally thought to have unitary functions in regulating single endocrine targets, act on multiple somatic tissues, such as bone, fat, and liver. There is also emerging evidence for anterior pituitary hormone action on brain receptors in mediating central neural and peripheral somatic functions. Here, we have created the most comprehensive neuroanatomical atlas on the expression of TSHR, LHCGR, and FSHR. We have used RNAscope, a technology that allows the detection of mRNA at single-transcript level, together with protein level validation, to document Tshr expression in 173 and Fshr expression in 353 brain regions, nuclei and subnuclei identified using the Atlas for the Mouse Brain in Stereotaxic Coordinates. We also identified Lhcgr transcripts in 401 brain regions, nuclei and subnuclei. Complementarily, we used ViewRNA, another single-transcript detection technology, to establish the expression of FSHR in human brain samples, where transcripts were co-localized in MALAT1-positive neurons. In addition, we show high expression for all three receptors in the ventricular region—with yet unknown functions. Intriguingly, Tshr and Fshr expression in the ependymal layer of the third ventricle was similar to that of the thyroid follicular cells and testicular Sertoli cells, respectively. In contrast, Fshr was localized to NeuN-positive neurons in the granular layer of the dentate gyrus in murine and human brain—both are Alzheimer’s disease-vulnerable regions. Our atlas thus provides a vital resource for scientists to explore the link between the stimulation or inactivation of brain glycoprotein hormone receptors on somatic function. New actionable pathways for human disease may be unmasked through further studies.

There is increasing evidence that anterior pituitary hormones, traditionally thought to have unitary functions in regulating single endocrine targets, act on multiple somatic tissues, such as bone, fat, and liver. There is also emerging evidence for anterior pituitary hormone action on brain receptors in mediating central neural and peripheral somatic functions. Here, we have created the most comprehensive neuroanatomical atlas on the expression of TSHRs, LHCGRs and FSHRs. We have used RNAscope, a technology that allows the detection of mRNA at single-transcript level, together with protein level validation, to document Tshr expression in 173 and Fshr expression in 353 brain regions, nuclei and sub–nuclei identified using the Atlas for the Mouse Brain in Stereotaxic Coordinates. We also identified Lhcgr transcripts in 401 brain regions, nuclei and sub–nuclei. Complementarily, we used ViewRNA, another single-transcript detection technology, to establish the expression of FSHRs in human brain samples, where transcripts were co–localized in MALAT1–positive neurons. In addition, we show high expression for all three receptors in the ventricular region—with yet unknown functions. Intriguingly, Tshr and Fshr expression in the ependymal layer of the third ventricle was similar to that of the thyroid follicular cells and testicular Sertoli cells, respectively. TSHRs were expressed specifically in tanycytes. In contrast, Fshrs were localized to NeuN–positive neurons in the granular layer of the dentate gyrus in murine and human brain—both are Alzheimer’s disease vulnerable regions. Our atlas thus provides a vital resource for scientists to explore the link between the stimulation or inactivation of brain glycoprotein hormone receptors on somatic function. New actionable pathways for human disease may be unmasked through further studies.

Pharmacological and genetic studies over the past decade suggest that FSH is an actionable target for diseases affecting millions, notably osteoporosis, obesity and Alzheimer's disease (AD). Blocking FSH action prevents bone loss (1, 2), fat and energy metabolism (3) and AD–like features in mice (4). We recently developed a first–in–class, humanized, epitope–specific FSH blocking antibody that binds to a 13–amino–acid–long sequence of FSHβ—"MS-Hu6"—with a KD of 7.52 nM (5). We showed that MS-Hu6 bound specifically to FSHβ and its different glycosylated forms, namely FSHβ21/18 and FSHβ24, without binding to LH and TSH. Here, using a GLP–compliant platform, we report the efficacy of MS-Hu6 in preventing obesity, osteoporosis and AD in mice. Notably, MS-Hu6-treated mice showed lower body weight and fat mass, increased lean mass (qNMR) and evidence of beiging in ThermoMice (IVIS imaging) compared with IgG–treated mice. Consistent with this, the thermogenic genes Ucp1 and Cidea were upregulated, whereas Pparg expression was attenuated in fat depots. Treatment of ThermoMice for 8 weeks also increased bone mineral density (BMD), improved microstructure (micro-CT), elevated bone formation (dynamic histomorphometry), and upregulated the osteoblastic genes Alp and Col1a1. The increase in bone mass and improved microstructure were replicated in C.J.R's lab using female mice 24 weeks post–ovariectomy. Preliminary testing using AD mice, namely APP/PS1 mice, showed that MS-Hu6 prevented the impairment in recognition and contextual memory. Biodistribution studies using 89Zr–labelled, biotinylated or unconjugated MS-Hu6 in mice and monkeys showed localization to bone, bone marrow and fat depots. MS-Hu6 displayed a β phase t½ of 13 days (316 hours) in humanized Tg32 mice, and bound endogenous FSH. In monkeys, an acute single injection of MS-Hu6 did not affect vitals, and biochemical parameters remained within the normative range. We tested 215 variations of excipients using the protein thermal shift assay to generate a final formulation that rendered MS-Hu6 stable in solution upon freeze–thaw and at different temperatures, with minimal aggregation, and without self–, cross–, or hydrophobic interactions or appreciable binding to relevant human antigens. MS-Hu6 showed the same level of "humanness" as human IgG1 in silico, and was non–immunogenic in ELISPOT assays for IL-2 and IFNγ in human peripheral blood mononuclear cell cultures. In conclusion, MS-Hu6 is efficacious, durable and manufacturable, and is therefore poised for future human testing as a multipurpose therapeutic for obesity, osteoporosis, and perhaps for AD.References: 1Sun et al., Cell, 2006, PMID: 16630814; Ji et al, PNAS, 2018, PMID: 29440419; 3Liu et al., Nature, 2017, PMID: 28538730; 4Xiong et al., Nature (In press); 5Gera et al., PNAS, 2020, PMID: 33127753 Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Pharmacological and genetic studies over the past decade have established FSH as an actionable target for diseases affecting millions, notably osteoporosis, obesity and Alzheimer’s disease (AD). Blocking FSH action prevents bone loss, fat gain and AD–like features in mice. We recently developed a first–in–class, humanized, epitope–specific FSH blocking antibody, MS-Hu6, with a KD of 7.52 nM. Using a GLP–compliant platform, we now report the efficacy of MS-Hu6 in preventing obesity and osteoporosis in mice, and parameters of acute safety in monkeys. Biodistribution studies using 89Zr–labelled, biotinylated or unconjugated MS-Hu6 in mice and monkeys showed localization to bone, bone marrow and fat depots. MS-Hu6 displayed a β phase t½ of 13 days (316 hours) in humanized Tg32 mice, and bound endogenous FSH. We tested 215 variations of excipients using the protein thermal shift assay to generate a final formulation that rendered MS-Hu6 stable in solution upon freeze–thaw and at different temperatures, with minimal aggregation, and without self–, cross–, or hydrophobic interactions or appreciable binding to relevant human antigens. MS-Hu6 showed the same level of “humanness” as human IgG1 in silico, and was non–immunogenic in ELISPOT assays for IL-2 and IFNγ in human peripheral blood mononuclear cell cultures. We conclude that MS-Hu6 is efficacious, durable and manufacturable, and is therefore poised for future human testing as a multipurpose therapeutic.

The nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway plays a critical role in skeletal homeostasis. Preclinical data using NO and its donors and genetically modified mice demonstrated that NO was required in bone remodeling and partly mediated the anabolic effects of mechanical stimuli and estrogen. However, the off-target effects and tachyphylaxis of NO limit its long-term use, and previous clinical trials using organic nitrates for osteoporosis have been disappointing. Among the other components in the downstream pathway, targeting cGMP-specific phosphodiesterase to promote the NO-cGMP-PKG signal is a viable option. There are growing in vitro and in vivo data that, among many other PDE families, PDE5A is highly expressed in skeletal tissue, and inhibiting PDE5A using currently available PDE5A inhibitors might increase the osteoanabolic signal and protect the skeleton. These preclinical data open the possibility of repurposing PDE5A inhibitors for treating osteoporosis. Further research is needed to address the primary target bone cell of PDE5A inhibition, the contribution of direct and indirect effects of PDE5A inhibition, and the pathophysiological changes in skeletal PDE5A expression in aging and hypogonadal animal models.

Today, research in biomedicine often requires the knowledge and technologies in diverse fields. Therefore, there is an increasing need for collaborative team science that crosses traditional disciplines. Here, we discuss our own lessons from both interdisciplinary and transdisciplinary teams, which ultimately ushered us to expand our research realm beyond bone biology.

The data of head-to-head comparisons of the anti-fracture efficacy of bone modifying agents (BMAs) in patients with hormone receptor-positive breast cancer receiving aromatase inhibitor (AI) are not available. Therefore, we conducted a network meta-analysis to compare the efficacy of different BMAs in patients with breast cancer receiving adjuvant AI. We performed a network meta-analysis to compare the change of bone mineral densities (BMDs) and the risk of fracture in the selected studies using a random effect model. The primary outcomes are the change of BMD of lumbar spine (LS) and total hip (TH) from the baseline (ΔBMD, %) at 1 and 2 years and the risk of fracture. We identified and included a total of 16 randomized controlled trials for this analysis. All BMAs included (risedronate, zoledronate, and denosumab) were associated with a significant increase in BMD of LS and TH at 1 and 2 years compared with no upfront treatment group. Among BMAs, zoledronate and denosumab use resulted in significantly higher BMD of LS and TH at 1 and 2 years compared with risedronate. The risk of fracture was significantly lower in the patients who received denosumab or risedronate compared with the patients without upfront treatment (Relative risk (RR) [95% CI] 0.51 [0.38–0.67] and 0.54 [0.35–0.83], respectively). Among the bisphosphonates, zoledronate increased BMD the most, but risedronate, not zoledronate, use was associated with lower risk of fracture. Denosumab increased BMD not only of LS but also of the cortical-bone-rich hip, and showed a significant reduction of fracture risk.

Core–shell particles are very well known for their unique features. Their distinctive inner core and outer shell structure allowed promising biomedical applications at both nanometer and micrometer scales. The primary role of core–shell particles is to deliver the loaded drugs as they are capable of sequence-controlled release and provide protection of drugs. Among other biomedical polymers, poly (lactic-co-glycolic acid) (PLGA), a food and drug administration (FDA)-approved polymer, has been recognized for the vehicle material. This review introduces PLGA core–shell nano/microparticles and summarizes various drug-delivery systems based on these particles for cancer therapy and tissue regeneration. Tissue regeneration mainly includes bone, cartilage, and periodontal regeneration.

Thyrotropin (TSH), traditionally seen as a pituitary hormone that regulates thyroid glands, has additional roles in physiology including skeletal remodeling. Population-based observations in people with euthyroidism or subclinical hyperthyroidism indicated a negative association between bone mass and low-normal TSH. The findings of correlative studies were supported by small intervention trials using recombinant human TSH (rhTSH) injection, and genetic and case-based evidence. Genetically modified mouse models, which disrupt the reciprocal relationship between TSH and thyroid hormone, have allowed us to examine an independent role of TSH. Since the first description of osteoporotic phenotype in haploinsufficient Tshr+/– mice with normal thyroid hormone levels, the antiosteoclastic effect of TSH has been documented in both in vitro and in vivo studies. Further studies showed that increased osteoclastogenesis in Tshr-deficient mice was mediated by tumor necrosis factor α. Low TSH not only increased osteoclastogenesis, but also decreased osteoblastogenesis in bone marrow–derived primary osteoblast cultures. However, later in vivo studies using small and intermittent doses of rhTSH showed a proanabolic effect, which suggests that its action might be dose and frequency dependent. TSHR was shown to interact with insulin-like growth factor 1 receptor, and vascular endothelial growth factor and Wnt pathway might play a role in TSH’s effect on osteoblasts. The expression and direct skeletal effect of a biologically active splice variant of the TSHβ subunit (TSHβv) in bone marrow–derived macrophage and other immune cells suggest a local skeletal effect of TSHR. Further studies of how locally secreted TSHβv and systemic TSHβ interact in skeletal remodeling through the endocrine, immune, and skeletal systems will help us better understand the hyperthyroidism-induced bone disease.

Objective: Glaucoma is a well-recognized side effect of corticosteroids. However, steroid-induced glaucoma typically refers to that caused by exogenous corticosteroid administration. Glaucoma secondary to endogenous overproduction of corticosteroids has only been reported in a few case reports. We aim to bring attention to glaucoma as a rare but important manifestation of endogenous hypercortisolism. Methods: Patient history, physical exam, laboratory results, and imaging studies were reviewed. Results: We report a case of glaucoma as the initial presentation of Cushing disease (CD). The patient was diagnosed with glaucoma 16 months prior to his endocrinology evaluation. At our initial encounter, the patient had a cushingoid appearance. Levels of 24-hour urinary cortisol and late-night salivary cortisol were elevated. Serum cortisol was not suppressed by 1 mg of dexamethasone overnight, but it was suppressed by 8 mg of dexamethasone. Adrenocorticotropic hormone was also elevated. All other pituitary hormone axes were unremarkable (thyroid-stimulating hormone, free thyroxine, follicle-stimulating hormone, luteinizing hormone, growth hormone, prolactin, and insulin-like growth factor). Pituitary magnetic resonance imaging suggested a small adenoma (2 to 3 mm); therefore, the patient underwent inferior petrosal sinus sampling. The results were consistent with CD. Transsphenoidal resection was performed and final pathology confirmed an adrenocorticotropic hormone-positive adenoma. Hypercortisolism and intraocular pressures improved after the surgery. Conclusion: Glaucoma can lead to irreversible blindness if left untreated or uncontrolled. However, endogenous hypercortisolism-induced glaucoma can be reversed with treatment of the underlying CD. Thus, heightened awareness of extraocular manifestations of secondary causes of glaucoma such as endogenous hypercortisolism is necessary in order to promote prompt evaluation and treatment.

An ideal root canal sealer creates a bacteria-resistant seal and exhibits antimicrobial activity, biocompatibility, and osteoconductivity. The aim of this study was to assess the effects of 3 root canal sealers on cell viability, inflammatory response, and osteogenic potential in MC3T3-E1 cells.AH Plus (Dentsply Caulk, Milford, DE), MTA Fillapex (Angelus Solucxoes Odontologicas, Londrina, Brazil), and EndoSequence BC (Brasseler, Savannah, GA) were mixed according to the manufacturer's instructions, and samples were prepared as extraction media (final dilution: 1/10). Lipopolysaccharide (LPS) (100 ng/mL) treatment was used to induce an inflammatory response in this study. Cell viability was evaluated using the Water soluble tetrazolium-1 (WST-1) assay. The levels of inflammatory mediators (interleukin 6 and tumor necrosis factor alpha) and osteogenic marker genes (ALP and OCN) were measured by reverse-transcription polymerase chain reaction and real-time polymerase chain reaction. Osteogenic potential was evaluated by alkaline phosphatase staining and alizarin red staining.Calcium silicate-based sealers such as MTA Fillapex and EndoSequence BC showed strong cell viability compared with AH Plus. AH Plus, MTA Fillapex, and EndoSequence BC decreased the levels of LPS-induced inflammatory mediators (P .05). The expression of osteogenic marker genes, alkaline phosphatase activity, and mineralized nodule formation decreased with LPS treatment. However, AH Plus and calcium silicate-based sealers increased the osteogenic potential reduced by LPS treatment (P .05).Calcium silicate-based sealers exhibit anti-inflammatory effects and induce osteogenic differentiation in MC3T3-E1 cells.

Background:Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis.Methods:To address whether ERFE functions also in bone and whether the mechanism of ERFE action in bone involves BMPs, we utilize the Erfe-/- mouse model as well as β–thalassemic (Hbbth3/+) mice with systemic loss of ERFE expression. In additional, we employ comprehensive skeletal phenotyping analyses as well as functional assays in vitro to address mechanistically the function of ERFE in bone.Results:We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low–bone–mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP–mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in Hbbth3/+mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss.Conclusions:Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in β–thalassemia.Funding:YZG acknowledges the support of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK107670 to YZG and DK095112 to RF, SR, and YZG). MZ acknowledges the support of the National Institute on Aging (U19 AG60917) and NIDDK (R01 DK113627). TY acknowledges the support of the National Institute on Aging (R01 AG71870). SR acknowledges the support of NIDDK (R01 DK090554) and Commonwealth Universal Research Enhancement (CURE) Program Pennsylvania.

Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis. We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low–bone–mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP–mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in β–thalassemic (Hbbth3/+) mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss. Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in β–thalassemia.

The data of head-to-head comparisons of the effect of bone-modifying agents (BMAs) in patients with androgen deprivation therapy (ADT) for prostate cancer without skeletal metastasis is limited. Thus, we conducted a network meta-analysis to compare each BMA for the efficacy of bone mineral densities (BMDs) and the risk of fracture. We performed a network meta-analysis to compare the change of BMDs and the risk of vertebral fracture in the studies included using a random-effect model. The primary outcomes are the change of BMD of the lumbar spine (LS) and the total hip (TH) from the baseline at 1 year from the initiation of the BMA and the risk of vertebral fracture. We identified and included 15 studies in this analysis. All BMAs except risedronate showed a significant increase of BMD of the LS compared with groups without BMA, among which zoledronate showed the most BMD gain. At TH, bisphosphonates (alendronate, pamidronate, and zoledronate) and denosumab showed significant elevation compared with the no-BMA group. Denosumab was associated with the most BMD gain at the TH. Only denosumab reduced the risk of vertebral fracture (relative risk [95% confidence interval]: 0.40 [0.20–0.81]). Although zoledronate showed the highest BMD gain at the LS, it did not reduce the risk of vertebral fracture in this analysis. Most bisphosphonates and denosumab significantly increased BMD at the LS and the TH in patients receiving ADT for prostate cancer without skeletal metastasis. In particular, zoledronate and denosumab were the most potent BMAs in terms of BMD increment at the LS and the TH, respectively. However, denosumab, not zoledronate, was the only BMA that showed a significant risk reduction of vertebral fracture. We need further studies to examine the change of bone quality and the effect on the risk of non-vertebral and hip fractures.

We report that two widely-used drugs for erectile dysfunction, tadalafil and vardenafil, trigger bone gain in mice through a combination of anabolic and antiresorptive actions on the skeleton. Both drugs were found to enhance osteoblastic bone formation in vivo using a unique gene footprint and to inhibit osteoclast formation. The target enzyme, phosphodiesterase 5A (PDE5A), was found to be expressed in mouse and human bone as well as in specific brain regions, namely the locus coeruleus, raphe pallidus, and paraventricular nucleus of the hypothalamus. Localization of PDE5A in sympathetic neurons was confirmed by coimmunolabeling with dopamine β-hydroxylase, as well as by retrograde bone-brain tracing using a sympathetic nerve-specific pseudorabies virus, PRV152. Both drugs elicited an antianabolic sympathetic imprint in osteoblasts, but with net bone gain. Unlike in humans, in whom vardenafil is more potent than tadalafil, the relative potencies were reversed with respect to their osteoprotective actions in mice. Structural modeling revealed a higher binding energy of tadalafil to mouse PDE5A compared with vardenafil, due to steric clashes of vardenafil with a single methionine residue at position 806 in mouse PDE5A. Collectively, our findings suggest that a balance between peripheral and central actions of PDE5A inhibitors on bone formation together with their antiresorptive actions specify the osteoprotective action of PDE5A blockade.

An imbalance in bone formation and bone resorption results in the bone loss that characterizes osteoporosis. An absolute or relative increase in bone resorption by osteoclasts can significantly contribute to this bone loss. Cathepsin K is a lysosomal protease responsible for bone degradation by osteoclasts. Pharmacological use of cathepsin K inhibitors such as odanacatib for osteoporosis has shown benefit, though the clinical development of these agents had to be discontinued due to potential adverse events. Preclinical and clinical studies on cathepsin K and its inhibitors have shown the potential for an adjuvant antiresorptive agent, and continue to guide future research.

Bone loss in women accelerates during perimenopause, and continues into old age. To-date, there has been little progress made in stratifying for fracture risk in premenopausal and early postmenopausal women. Epidemiologic data suggests that changes in serum FSH could predict decrements in bone mass during peri- and postmenopause. In bone, FSH stimulates osteoclast formation by releasing osteoclastogenic cytokines. Here, we address the evidence for bone loss across the menopausal transition, discuss strategies for detection and treatment of early postmenopausal osteoporosis, and describe the role FSH plays in physiology and likely in pathophysiology of early postmenopausal bone loss.

Objective: Ectopic Cushing syndrome secondary to thymic carcinoid is a rare disorder that may be difficult to diagnose and manage.Methods: We describe a case of severe Cushing syndrome secondary to a large, adrenocorticotropic hormone (ACTH)-producing thymic carcinoid in a patient with a history of primary hyperaldosteronism.Results: A 43-year-old female with a 20-year history of an aldosterone-secreting adrenocortical adenoma following a right adrenalectomy presented with acute onset of proximal muscle weakness, swelling, facial hirsutism, and severe hypokalemia. Ectopic Cushing syndrome was suspected based on the sudden symptom onset and markedly elevated 24-hour urine cortisol and ACTH levels. Magnetic resonance imaging revealed an empty pituitary sella and a large (7.3 cm) mediastinal mass visible on chest computed tomography. The mass was resected by video-assisted thoracoscopic surgery, resulting in resolution of symptoms and cortisol levels. Pathology assessment confirmed well-differentiated thymic carcinoid with positive ACTH staining.Conclusion: This case highlights the clinical features, challenges in diagnostic work up, treatment modalities, and associated endocrine findings in a thymic carcinoid abutting the heart in a patient presenting with ectopic ACTH secretion.Abbreviations: ACTH = adrenocorticotropic hormone;EAS = ectopic adrenocorticotropic hormone syndrome;MRI = magnetic resonance imaging;VATS = video-assisted thoracoscopic surgery

The primitive neurohypophyseal nonapeptide oxytocin (OXT) has established functions in parturition, lactation, appetite, and social behavior. We have shown that OXT has direct actions on the mammalian skeleton, stimulating bone formation by osteoblasts and modulating the genesis and function of bone-resorbing osteoclasts. We deleted OXT receptors (OXTRs) selectively in osteoblasts and osteoclasts using Col2.3Cre and Acp5Cre mice, respectively. Both male and female Col2.3Cre + : Oxtr fl/fl mice recapitulate the low-bone mass phenotype of Oxtr +/− mice, suggesting that OXT has a prominent osteoblastic action in vivo. Furthermore, abolishment of the anabolic effect of estrogen in Col2.3Cre + : Oxtr fl/fl mice suggests that osteoblastic OXTRs are necessary for estrogen action. In addition, the high bone mass in Acp5Cre + : Oxtr fl/fl mice indicates a prominent action of OXT in stimulating osteoclastogenesis. In contrast, we found that in pregnant and lactating Col2.3Cre + : Oxtr fl/fl mice, elevated OXT inhibits bone resorption and rescues the bone loss otherwise noted during pregnancy and lactation. However, OXT does not contribute to ovariectomy-induced bone loss. Finally, we show that OXT acts directly on OXTRs on adipocytes to suppress the white-to-beige transition gene program. Despite this direct antibeiging action, injected OXT reduces total body fat, likely through an action on OXT-ergic neurons. Consistent with an antiobesity action of OXT, Oxt −/− and Oxtr −/− mice display increased total body fat. Overall, the actions of OXT on bone mass and body composition provide the framework for future therapies for osteoporosis and obesity.

In this study, mixture flow of sand and water within a hydrocyclone was investigated using the Eulerian-Eulerian model based on a Computational Fluid Dynamics (CFD) technique. Experiments were also performed to validate the numerical analyses results. In Eulerian-Eulerian analyses, both solid and liquid phases are regarded as continua. Accordingly, conservation equations, such as continuity and momentum equations, are applied to each phase of sand and water. In this study, the RNG k-e model was used as a turbulence model. The results of the computational analysis were compared with those obtained experimentally. The CFD and experimental results were largely consistent with each other. The accuracy of the analysis model was compared with the experiment, and the effects of inlet volume fraction of solid and inlet velocity of the mixture on separation efficiency were examined. Separation efficiency decreased as inlet velocity increased. In addition, separation efficiency was high when solid concentration was low (3%), but there was no relationship between concentration and separation efficiency when the solid concentration was above 6%.

Pituitary hormones have long been thought solely to regulate single targets. Challenging this paradigm, we discovered that both anterior and posterior pituitary hormones, including FSH, had other functions in physiology. We have shown that FSH regulates skeletal integrity, and, more recently, find that FSH inhibition reduces body fat and induces thermogenic adipose tissue. A polyclonal antibody raised against a short, receptor-binding epitope of FSHβ was found not only to rescue bone loss postovariectomy, but also to display marked antiobesity and probeiging actions. Questioning whether a single agent could be used to treat two medical conditions of public health importance--osteoporosis and obesity--we developed two further monoclonal antibodies, Hf2 and Mf4, against computationally defined receptor-binding epitopes of FSHβ. Hf2 has already been shown to reduce body weight and fat mass and cause beiging in mice on a high-fat diet. Here, we show that Hf2, which binds mouse Fsh in immunoprecipitation assays, also increases cortical thickness and trabecular bone volume, and microstructural parameters, in sham-operated and ovariectomized mice, noted on microcomputed tomography. This effect was largely recapitulated with Mf4, which inhibited bone resorption by osteoclasts and stimulated new bone formation by osteoblasts. These effects were exerted in the absence of alterations in serum estrogen in wild-type mice. We also reconfirm the existence of Fshrs in bone by documenting the specific binding of fluorescently labeled FSH, FSH-CH, in vivo. Our study provides the framework for the future development of an FSH-based therapeutic that could potentially target both bone and fat.

Background Chlormadinone acetate (CMA) is a derivative of progesterone and is used as an oral contraceptive. The aim of this study was to investigate the effects of CMA on odontogenic differentiation and mineralization of human dental pulp cells (hDPCs) and related signaling pathways. Methods Cell viability was determined by the water-soluble tetrazolium (WST)-1 assay. Odontogenic differentiation of hDPCs was evaluated by real-time polymerase chain reaction using odontogenic marker genes, such as alkaline phosphatase (ALP), osteocalcin (OCN), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1). Mineralization of hDPCs was evaluated by ALP staining and alizarin red staining. The extracellular signal-regulated kinase (ERK) pathway was examined by Western blot analysis. Results There was no statistically significant difference in cell viability between the control and CMA-treated groups. Our analysis of odontogenic marker genes indicated that CMA enhanced the expression of those genes. CMA-treated hDPCs showed increased ALP activity and formation of mineralized nodules, compared with control-treated cells. In addition, CMA stimulation resulted in phosphorylation of ERK and resulted in inhibition of downstream molecules by the ERK inhibitor U0126. Conclusions These findings suggest that CMA improves odontogenic differentiation and mineralization of hDPCs through the ERK signaling pathway.

Bone formation and resorption are tightly coupled, and dysfunction of either process leads to bone diseases, such as osteoporosis. Bone-forming agents have been explored clinically to increase bone density; however, long-term efficacy of these strategies is limited due to the accompanying increase in resorption in response to increased bone formation. Axonal guidance molecules have recently been shown to regulate formation-resorption coupling and thus have the potential for osteoporosis therapy. In this issue of the JCI, Kim et al. demonstrate that osteoclast-secreted SLIT3 influences bone formation and resorption by promoting osteoblast migration and suppressing osteoclast differentiation. Activation of SLIT3/ROBO signaling in ovariectomized mice increased bone mass, suggesting that SLIT3 should be further explored as a therapeutic target.

The traditional view of follicle-stimulating hormone (FSH) as a reproductive hormone is changing. It has been shown that FSH receptors (FSHRs) are expressed in various extra-gonadal tissues and mediate the biological effects of FSH at those sites. Molecular, animal, epidemiologic, and clinical data suggest that elevated serum FSH may play a significant role in the evolution of bone loss and obesity, as well as contributing to cardiovascular and cancer risk. This review summarizes recent data on FSH action beyond reproduction.

Here, we will describe a combinatorial approach for drug discovery using two technologies—genomic connectivity mapping (C-MAPping) and structural modeling—to repurpose drugs, using bisphosphonates as a paradigm. Bisphosphonates are the mainstay of therapy for the skeletal fragility associated with aging, menopause, cancer, and chronic inflammatory diseases. Using mRNA microarrays, we generated a gene signature of bisphosphonate action on human osteoclasts. Interrogation of the Connectivity Map (C-MAP) identified potential bisphosphonate mimetics, which included 1,5-isoquinolinediol, a PARP inhibitor, and tyrphostin AG1478, an inhibitor of human EGFR (HER). In silico modeling suggested that bisphosphonates, due to a similarity of chemical structure to pyrophosphate, could bind to the kinase domain of the EGFR. Confirmatory in vitro testing showed a potent inhibition of osteoclast formation in vitro with the two agents, respectively. This was accompanied by reduced Akt phosphorylation, also suggesting increased apoptosis. Additionally, consistent with previous results, bisphosphonates attenuated EGF-induced cell proliferation of EGFR-overexpressing non-small cell lung cancer cells (H1666), and reduced cell viability of those driven by a known activating mutation, namely EGFR△ E746-A750 (HCC827). Thus, C-MAP together with computational modeling not only allowed us to discover bisphosphonate-like, osteoprotective actions of two anticancer drugs, but more importantly prompted studies on the use of bisphosphonates for EGFR-driven cancers.

Fibrous sheath interacting protein 1 (FSIP1) is a cancer antigen expressed in the majority of breast cancer tissues and is associated with poor prognosis. However, the role of FSIP1 in the progression and drug sensitivity of triple-negative breast cancer (TNBC) has not been explored. Here, we show that FSIP1 deficiency by shRNA-mediated knockdown or CRISPR-Cas9–mediated knockout significantly inhibits the proliferation and invasion of TNBC cells and impairs chemotherapy-induced growth inhibition in vivo. Computational modeling predicted that FSIP1 binds to ULK1, and this was established by coimmunoprecipitation. FSIP1 deficiency promoted autophagy, enhanced AMP-activated protein kinase (AMPK) signaling, and decreased mechanistic target of rapamycin (mTOR) and Wnt/β-catenin activity. In contrast, knockdown of AMPK or inhibition of autophagy restored the sensitivity to chemotherapy drugs in TNBC cells. Our findings uncover a role of FSIP1 as well as mechanisms underlying FSIP1 action in drug sensitivity and may, therefore, aid in design of TNBC therapies.

In this study, the Enneagram test was done for the technical high school students, and the personality types of those students were categorized as the following three types: head, breast, and belly, which are three main types of the Enneagram test. Through the discussion between expert course tutors and an Enneagram professional, respective effective teaching-learning methods were chosen for each type of student groups, and the three groups began to study the robot programming course. Students were advised to study with VPL and TPL, and the results of their learning were examined using robots. The results showed that learners of respective personality types preferred VPL and TPL differently. Learners' achievement was found remarkably different according to their preference of VPL and TPL (programming tool). When not considering the Enneagram personality type factor, it could happen that part of personality type learners could not get desirable results.

For effective utilization of downtown area, many studies about underground have been performed around the world, and double-deck tunnel have being operated in USA, Europe and China, etc. (A86 East Duplex in France, M30 tunnel project in Spain, SR-99 in seattle, USA, Yangtze river tunnel in China) In Korea, the research about network type double-deck tunnel in deep underground space is in progress to solve the traffic jam and secure the ground space. In this study, a number of factors required for double-deck tunnel in deep underground are analyzed through the existing ventilation design outline and unique ventilation design factors for network type double-deck tunnel are established by reviewing design cases of overseas double-deck tunnel.

The Study of Women’s Health Across the Nation has taught us that impending ovarian failure during late perimenopause is associated with a sharp rise in serum FSH, which coincides with the most rapid rate of bone loss and the onset of visceral adiposity. At this time in a woman’s life, serum estrogen levels are largely unaltered, so the hypothesis that hypoestrogenemia is the sole cause of bone loss and visceral obesity does not offer a full explanation. An alternative explanation, arising from animal models and human data, is that both physiologic aberrations, obesity and osteoporosis, arise at least in part from rising FSH levels. Here, we discuss recent findings on the mechanism through which FSH exerts biological actions on bone and fat and review clinical data that support a role for FSH in causing osteoporosis and obesity. We will also provide a conceptual framework for using a single anti-FSH agent to prevent and treat both osteoporosis and obesity in women across the menopausal transition.