Mouse Skeleton Research Articles

The hinge-1 domain of Flna is not necessary for diverse physiological functions in mice.

The filamins are cytoskeletal binding proteins that dynamically crosslink actin into orthogonal networks or bundle it into stress fibres. The domain structure of filamin proteins is very well characterised, with an N-terminal actin-binding region, followed by 24 immunoglobulin-like repeat units. The repeat domains are separated into distinct segments by two regions of low-complexity known as hinge-1 and hinge-2. The role of hinge-1 especially has been proposed to be essential for protein function as it provides flexibility to the otherwise rigid protein, and is a target for cleavage by calpain. Hinge-1 protects cells from otherwise destructive forces, and the products of calpain cleavage are involved in critical cellular signalling processes, such as survival during hypoxia. Pathogenic variants in FLNA encoding Filamin A, including those that remove the hinge-1 domain, cause a wide range of survivable developmental disorders. In contrast, complete loss of function of this gene is embryonic lethal in human and mouse. In this study, we show that removing filamin A hinge-1 from mouse (FlnaΔH1), while preserving its expression level leads to no obvious developmental phenotype. Detailed characterisation of the skeletons of FlnaΔH1 mice showed no skeletal phenotype reminiscent of that found in the FLNA-causing skeletal dysplasia. Furthermore, nuclear functions of FLNA are maintained with loss of Filamin A hinge-1. We conclude that hinge-1 is dispensable for filamin A protein function during development over the murine lifespan.

Members of an array of zinc-finger proteins specify distinct Hox chromatin boundaries

Partitioning of repressive from actively transcribed chromatin in mammalian cells fosters cell-type-specific gene expression patterns. While this partitioning is reconstructed during differentiation, the chromatin occupancy of the key insulator, CCCTC-binding factor (CTCF), is unchanged at the developmentally important Hox clusters. Thus, dynamic changes in chromatin boundaries must entail other activities. Given its requirement for chromatin loop formation, we examined cohesin-based chromatin occupancy without known insulators, CTCF and Myc-associated zinc-finger protein (MAZ), and identified a family of zinc-finger proteins (ZNFs), some of which exhibit tissue-specific expression. Two such ZNFs foster chromatin boundaries at the Hox clusters that are distinct from each other and from MAZ. PATZ1 was critical to the thoracolumbar boundary in differentiating motor neurons and mouse skeleton, while ZNF263 contributed to cervicothoracic boundaries. We propose that these insulating activities act with cohesin, alone or combinatorially, with or without CTCF, to implement precise positional identity and cell fate during development.

Increased β2-adrenergic signaling promotes fracture healing through callus neovascularization in mice.

Traumatic brain injury (TBI) leads to skeletal changes, including bone loss in the unfractured skeleton, and paradoxically accelerates healing of bone fractures; however, the mechanisms remain unclear. TBI is associated with a hyperadrenergic state characterized by increased norepinephrine release. Here, we identified the β2-adrenergic receptor (ADRB2) as a mediator of skeletal changes in response to increased norepinephrine. In a murine model of femoral osteotomy combined with cortical impact brain injury, TBI was associated with ADRB2-dependent enhanced fracture healing compared with osteotomy alone. In the unfractured 12-week-old mouse skeleton, ADRB2 was required for TBI-induced decrease in bone formation and increased bone resorption. Adult 30-week-old mice had higher bone concentrations of norepinephrine, and ADRB2 expression was associated with decreased bone volume in the unfractured skeleton and better fracture healing in the injured skeleton. Norepinephrine stimulated expression of vascular endothelial growth factor A and calcitonin gene-related peptide-α (αCGRP) in periosteal cells through ADRB2, promoting formation of osteogenic type-H vessels in the fracture callus. Both ADRB2 and αCGRP were required for the beneficial effect of TBI on bone repair. Adult mice deficient in ADRB2 without TBI developed fracture nonunion despite high bone formation in uninjured bone. Blocking ADRB2 with propranolol impaired fracture healing in mice, whereas the ADRB2 agonist formoterol promoted fracture healing by regulating callus neovascularization. A retrospective cohort analysis of 72 patients with long bone fractures indicated improved callus formation in 36 patients treated with intravenous norepinephrine. These findings suggest that ADRB2 is a potential therapeutic target for promoting bone healing.

Long-term oral meclozine administration improves survival rate and spinal canal stenosis during postnatal growth in a mouse model of achondroplasia in both sexes.

Achondroplasia (ACH) is a skeletal dysplasia characterized by short-limbed short stature caused by the gain-of-function mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. Activated FGFR3, which is a negative regulator of bone elongation, impairs the growth of long bones and the spinal arch by inhibiting chondrocyte proliferation and differentiation. Most patients with ACH have spinal canal stenosis in addition to short stature. Meclozine has been found to inhibit FGFR3 via drug repurposing. A 10-d treatment with meclozine promoted long-bone growth in a mouse model of ACH (Fgfr3ach mice). This study aimed to evaluate the effects of long-term meclozine administration on promoting bone growth and the spinal canal in Fgfr3ach mice. Meclozine (2mg/kg/d) was orally administered to Fgfr3ach mice for 5 d per wk from the age of 7d to 56d. Meclozine (2mg/kg/d) significantly reduced the rate of death or paralysis and improved the length of the body, cranium, and long bones in male and female Fgfr3ach mice. Micro-computed tomography analysis revealed that meclozine ameliorated kyphotic deformities and trabecular parameters, including BMD, bone volume/tissue volume, trabecular thickness, and trabecular number at distal femur of Fgfr3ach mice in both sexes. Histological analyses revealed that the hypertrophic zone in the growth plate was restored in Fgfr3ach mice following meclozine treatment, suggesting upregulation of endochondral ossification. Skeletal preparations demonstrated that meclozine restored the spinal canal diameter in Fgfr3ach mice in addition to improving the length of each bone. The 2mg/kg/d dose of meclozine reduced the rate of spinal paralysis caused by spinal canal stenosis, maintained the growth plate structure, and recovered the bone quality and growth of axial and appendicular skeletons of Fgfr3ach mice in both sexes. Long-term meclozine administration has the potential to ameliorate spinal paralysis and bone growth in patients with ACH.

Rapamycin does not alter bone microarchitecture or material properties quality in young-adult and aged female C57BL/6 mice.

Advancing age is the strongest risk factor for osteoporosis and skeletal fragility. Rapamycin is an FDA-approved immunosuppressant that inhibits the mechanistic target of rapamycin (mTOR) complex, extends lifespan, and protects against aging-related diseases in multiple species; however, the impact of rapamycin on skeletal tissue is incompletely understood. We evaluated the effects of a short-term, low-dosage, interval rapamycin treatment on bone microarchitecture and strength in young-adult (3 mo old) and aged female (20 mo old) C57BL/6 mice. Rapamycin (2mg/kg body mass) was administered via intraperitoneal injection 1×/5d for a duration of 8wk; this treatment regimen has been shown to induce geroprotective effects while minimizing the side effects associated with higher rapamycin dosages and/or more frequent or prolonged delivery schedules. Aged femurs exhibited lower cancellous bone mineral density, volume, trabecular connectivity density and number, higher trabecular thickness and spacing, and lower cortical thickness compared to young-adult mice. Rapamycin had no impact on assessed microCT parameters. Flexural testing of the femur revealed that both yield strength and ultimate strength were lower in aged mice compared to young-adult mice. There were no effects of rapamycin on these or other measures of bone biomechanics. Age, but not rapamycin, altered local and global measures of bone turnover. These data demonstrate that short-term, low-dosage interval rapamycin treatment does not negatively or positively impact the skeleton of young-adult and aged mice.

Bone-Derived IGF-I Regulates Radial Bone Growth in Adult Male Mice.

Insulin-like growth factor-I (IGF-I) levels, which are reduced by age, and cortical bone dimensions are major determinants of fracture risk in elderly subjects. Inactivation of liver-derived circulating IGF-I results in reduced periosteal bone expansion in young and older mice. In mice with lifelong depletion of IGF-I in osteoblast lineage cells, the long bones display reduced cortical bone width. However, it has not previously been investigated whether inducible inactivation of IGF-I locally in bone in adult/old mice affects the bone phenotype. Adult tamoxifen-inducible inactivation of IGF-I using a CAGG-CreER mouse model (inducible IGF-IKO mice) substantially reduced IGF-I expression in bone (-55%) but not in liver. Serum IGF-I and body weight were unchanged. We used this inducible mouse model to assess the effect of local IGF-I on the skeleton in adult male mice, avoiding confounding developmental effects. After tamoxifen-induced inactivation of the IGF-I gene at 9 months of age, the skeletal phenotype was determined at 14 months of age. Computed tomography analyses of tibia revealed that the mid-diaphyseal cortical periosteal and endosteal circumferences and calculated bone strength parameters were decreased in inducible IGF-IKO mice compared with controls. Furthermore, 3-point bending showed reduced tibia cortical bone stiffness in inducible IGF-IKO mice. In contrast, the tibia and vertebral trabecular bone volume fraction was unchanged. In conclusion, inactivation of IGF-I in cortical bone with unchanged liver-derived IGF-I in older male mice resulted in reduced radial growth of cortical bone. This suggests that not only circulating IGF-I but also locally derived IGF-I regulates the cortical bone phenotype in older mice.

Study on the mechanism of NLRP3 effect on the skeleton of de-ovalized mice

Postmenopausal osteoporosis caused by estrogen deficiency affects millions of women worldwide. By influencing both osteoblast and osteoclast development, NOD-like receptor thermoprotein structural domain-associated protein 3 (NLRP3) is a key player in the etiology of osteoporosis (OP). The purpose of this research was to look into the mechanism of action of NLRP3 in osteoporosis caused by a lack of estrogen, highlighting that NLRP3 induces osteoblast pyroptosis and thus inflammatory responses in de-ovulated mice, thereby inhibiting osteogenic differentiation and participating in the development of osteoporosis. In de-ovulated mice, we found an enhanced inflammatory response and suppression of osteogenic activity. In vitro experiments, we found a significant increase in markers of cell pyroptosis and inflammatory responses and a significant decrease in markers of osteogenic differentiation in osteoblasts from de-ovulated mice. However, knockdown of the NLRP3 gene inhibited this cell pyroptosis and improved osteogenic differentiation of osteoblasts. Our findings indicate a potential therapeutic potential for the treatment of estrogen deficiency-induced osteoporosis by demonstrating the critical role that NLRP3 inflammatory vesicles and their downstream-mediated cellular pyroptosis play in bone differentiation.

Experimental design and power calculation in omics circadian rhythmicity detection using the cosinor model.

Circadian clocks are 24-h endogenous oscillators in physiological and behavioral processes. Though recent transcriptomic studies have been successful in revealing the circadian rhythmicity in gene expression, the power calculation for omics circadian analysis have not been fully explored. In this paper, we develop a statistical method, namely CircaPower, to perform power calculation for circadian pattern detection. Our theoretical framework is determined by three key factors in circadian gene detection: sample size, intrinsic effect size and sampling design. Via simulations, we systematically investigate the impact of these key factors on circadian power calculation. We not only demonstrate that CircaPower is fast and accurate, but also show its underlying cosinor model is robust against variety of violations of model assumptions. In real applications, we demonstrate the performance of CircaPower using mouse pan-tissue data and human post-mortem brain data, and illustrate how to perform circadian power calculation using mouse skeleton muscle RNA-Seq pilot as case study. Our method CircaPower has been implemented in an R package, which is made publicly available on GitHub ( https://github.com/circaPower/circaPower).

Long-term effects of canagliflozin treatment on the skeleton of aged UM-HET3 mice.

Sodium glucose cotransporter-2 inhibitors (SGLT2is) promote urinary glucose excretion and decrease plasma glucose levels independent of insulin. Canagliflozin (CANA) is an SGLT2i, which is widely prescribed, to reduce cardiovascular complications, and as a second-line therapy after metformin in the treatment of type 2 diabetes mellitus. Despite the robust metabolic benefits, reductions in bone mineral density (BMD) and cortical fractures were reported for CANA-treated subjects. In collaboration with the National Institute on Aging (NIA)-sponsored Interventions Testing Program (ITP), we tested skeletal integrity of UM-HET3 mice fed control (137 mice) or CANA-containing diet (180ppm, 156 mice) from 7 to 22months of age. Micro-computed tomography (micro-CT) revealed that CANA treatment caused significant thinning of the femur mid-diaphyseal cortex in both male and female mice, did not affect trabecular bone architecture in the distal femur or the lumbar vertebra-5 in male mice, but was associated with thinning of the trabeculae at the distal femur in CANA-treated female mice. In male mice, CANA treatment is associated with significant reductions in cortical bone volumetric BMD by micro-CT, and by quantitative backscattered scanning electron microscopy. Raman microspectroscopy, taken at the femur mid-diaphyseal posterior cortex, showed significant reductions in the mineral/matrix ratio and an increased carbonate/phosphate ratio in CANA-treated male mice. These data were supported by thermogravimetric assay (TGA) showing significantly decreased mineral and increased carbonate content in CANA-treated male mice. Finally, the sintered remains of TGA were subjected to X-ray diffraction and showed significantly higher fraction of whitlockite, a calcium orthophosphate mineral, which has higher resorbability than hydroxyapatite. Overall, long-term CANA treatment compromised bone morphology and mineral composition of bones, which likely contribute to increased fracture risk seen with this drug.

Osteoblast-Specific Overexpression of Nucleolar Protein NO66/RIOX1 in Mouse Embryos Leads to Osteoporosis in Adult Mice.

In previous study, we showed that nucleolar protein 66 (NO66) is a chromatin modifier and negatively regulates Osterix activity as well as mesenchymal progenitor differentiation. Genetic ablation of the NO66 (RIOX1) gene in cells of the Prx1-expressing mesenchymal lineage leads to acceleration of osteochondrogenic differentiation and a larger skeleton in adult mice, whereas mesenchyme-specific overexpression of NO66 inhibits osteochondrogenesis resulting in dwarfism and osteopenia. However, the impact of NO66 overexpression in cells of the osteoblast lineage in vivo remains largely undefined. Here, we generated osteoblast-specific transgenic mice overexpressing a FLAG-tagged NO66 transgene driven by the 2.3 kB alpha-1type I collagen (Col1a1) promoter. We found that overexpression of NO66 in cells of the osteoblast lineage did not cause overt defects in developmental bones but led to osteoporosis in the long bones of adult mice. This includes decreased bone volume (BV), bone volume density (bone volume/total volume, BV/TV), and bone mineral density (BMD) in cancellous compartment of long bones, along with the accumulation of fatty droplets in bone marrow. Ex vivo culture of the bone marrow mesenchymal stem/stromal cells (BMSCs) from adult Col1a1-NO66 transgenic mice showed an increase in adipogenesis and a decrease in osteogenesis. Taken together, these data demonstrate a crucial role for NO66 in adult bone formation and homeostasis. Our Col1a1-NO66 transgenic mice provide a novel animal model for the mechanistic and therapeutic study of NO66 in osteoporosis.

Cortex Specific Overexpression of BAF170 and Growth of the Cranial Base, Cranial Vault, and Facial Skeleton in Mice

The fossil record and craniofacial disease reveal the tight correspondence between brain and skull shape, yet it is not understood how changes in brain growth and consequent changes in brain morphology impact growth of the skull. We tested whether variation in brain size impacted the growth of the cranial base, cranial vault, and facial skeleton in postnatal mice using a transgenic murine model of cortex specific overexpression of Chromatin Remodeling Complex BAF170 Subunit (BAF170cOE) that produces a mouse with a distinctively smaller cerebral cortex. Due to the unique growth mechanisms of the cranial base, cranial vault, and facial skeleton, and their unique anatomic relationships to the cerebral cortex, areas of bone growth may be dissimilarly affected by a reduction in cortical size. Since the cranial vault is anatomically proximate to the cerebral cortex, and because we hypothesized that brain growth impacts skull growth, we anticipated growth of the cranial vault would be more strongly affected relative to growth of the cranial base and facial skeleton. Three dimensional coordinates of landmarks describing the cranial base, cranial vault, and facial skeleton were collected from microcomputed tomography (microCT) images of BAF170cOE mice and unaffected littermates at birth (postnatal day 0 or P0), P14, P28, and P42. Sample sizes ranged between 5 and 10. Growth difference matrix analysis (GDMA) was used to statistically compare growth between genotypes. We found statistically significant differences in cranial vault growth from P0 to P14 between genotypes. There were no significant differences in growth between genotypes for any of the skull modules from P14 to P28 or from P28 to P42. Reduced cortical size of BAF170cOE mice is associated with changes in skull growth during the earliest interval studied here, with significant impacts noted on the growth of the cranial vault. Our results support the hypothesis that brain growth has an effect on skull growth, regardless of the brain decreasing in size. The specificity of the BAF170 Subunit to cause reduced cortical size and lack of direct effect on bone formation allows for future investigation of the mechanisms by which brain and skull growth are integrated.

Hypermethylation of mitochondrial DNA facilitates bone metastasis of renal cell carcinoma.

Kidney cancers including clear cell carcinoma (RCC) are identified with very vulnerable mitochondria DNA (mtDNA) and frequent epigenetic aberrations. Bone metastasis from RCC is prevalent and destructive. Bone marrow contains a quite hypoxic microenvironment that usually insitigate 50% of hypermethylation events in conferring a selective advantage for tumor growth. We hypothesized that hypermethylation of mtDNA in RCC cells would significantly contribute to bone metastatic tumor progression. Methylation-specific polymerase chain reaction assay (MSP) was adopted to measure the methylation status of D-loop region of mtDNA in 15 pairs of bone metastatic and primary RCC as well as tumor adjescent normal kidney tissues. mtDNA copy number was examined by the real-time quantitative polymerase chain reaction (qPCR). Western blotting analysis was used to measure the accumulation of several DNA methyltransferases (DNMTs) in the mitochondria and nucleus fractions of bone metastatic RCC cells. mRNA expression of mitochondria encoded genes was examined by RT-PCR. Reactive oxygen species (ROS), mitochondrial membrane potential and ATP content were measured using in vitro cells treated with de-methylation drug 5-Azacytidine (5-Aza). Non-invasive bioluminescent imaging was performed to monitor tumor occurrence in skeleton in mice. Our results showed that the D-loop region in bone metastatic tumor cells was markedly hypermethylated than those in primary RCC tumor cells, that is associated with a decreased mtDNA copy number and accumulation of DNMT1 in the mitochondria. The bone-tropism tumor colonization and progression of RCC cells was significantly suppressed by demethylating the D-loop region of mtDNA and reducing the intracellular level of ROS and ATP by 5-Aza treatment. In conclusion, our study provided a direct association between hypermethylation of mtDNA in RCC with bone metastastic tumor growth.

A Novel Single-Step-Labeled 212Pb-CaCO3 Microparticle for Internal Alpha Therapy: Preparation, Stability, and Preclinical Data from Mice.

Lead-212 is recognized as a promising radionuclide for targeted alpha therapy for tumors. Many studies of 212Pb-labeling of various biomolecules through bifunctional chelators have been conducted. Another approach to exploiting the cytotoxic effect is coupling the radionuclide to a microparticle acting as a carrier vehicle, which could be used for treating disseminated cancers in body cavities. Calcium carbonate may represent a suitable material, as it is biocompatible, biodegradable, and easy to synthesize. In this work, we explored 212Pb-labeling of various CaCO3 microparticles and developed a protocol that can be straightforwardly implemented by clinicians. Vaterite microparticles stabilized by pamidronate were effective as 212Pb carriers; labeling yields of ≥98% were achieved, and 212Pb was strongly retained by the particles in an in vitro stability assessment. Moreover, the amounts of 212Pb reaching the kidneys, liver, spleen, and skeleton of mice following intraperitoneal (i.p.) administration were very low compared to i.p. injection of unbound 212Pb2+, indicating that CaCO3-bound 212Pb exhibited stability when administered intraperitoneally. Therapeutic efficacy was observed in a model of i.p. ovarian cancer for all the tested doses, ranging from 63 to 430 kBq per mouse. Lead-212-labeled CaCO3 microparticles represent a promising candidate for treating intracavitary cancers.

Rapamycin impairs bone accrual in young adult mice independent of Nrf2.

Advanced age is the strongest risk factor for osteoporosis. The immunomodulator drug rapamycin extends lifespan in numerous experimental model organisms and is being investigated as a potential therapeutic to slow human aging, but little is known about the effects of rapamycin on bone. We evaluated the impact of rapamycin treatment on bone mass, architecture, and indices of bone turnover in healthy adult (16-20weeks old at treatment initiation) female wild-type (ICR) and Nrf2-/- mice, a mouse model of oxidative damage and aging-related disease vulnerability. Rapamycin (4mg/kg bodyweight) was administered by intraperitoneal injection every other day for 12weeks. Mice treated with rapamycin exhibited lower femur bone mineral content, bone mineral density, and bone volume compared to vehicle-treated mice. In midshaft femur diaphysis (cortical bone), rapamycin-treated mice had lower cortical volume and thickness, and in the distal femur metaphysis (cancellous bone), rapamycin-treated mice had higher trabecular spacing and lower connectivity density. Mice treated with rapamycin exhibited lower bone volume, bone volume fraction, and trabecular thickness in the 5th lumbar vertebra. Rapamycin-treated mice had lower levels of bone formation in the distal femur metaphysis compared to vehicle-treated mice which occurred co-incidentally with increased serum CTX-1, a marker of global bone resorption. Rapamycin had no impact on tibia inflammatory cytokine gene expression, and we found no independent effects of Nrf2 knockout on bone, nor did we find any interactions between genotype and treatment. These data show that rapamycin may have a negative impact on the skeleton of adult mice that should not be overlooked in the clinical context of its usage as a therapy to retard aging and reduce the incidence of age-related pathologies.

Mitochondrial Sirt3 contributes to the bone loss caused by aging or estrogen deficiency.

Altered mitochondria activity in osteoblasts and osteoclasts has been implicated in the loss of bone mass associated with aging and estrogen deficiency — the 2 most common causes of osteoporosis. However, the mechanisms that control mitochondrial metabolism in bone cells during health or disease remain unknown. The mitochondrial deacetylase sirtuin-3 (Sirt3) has been earlier implicated in age-related diseases. Here, we show that deletion of Sirt3 had no effect on the skeleton of young mice but attenuated the age-related loss of bone mass in both sexes. This effect was associated with impaired bone resorption. Osteoclast progenitors from aged Sirt3-null mice were able to differentiate into osteoclasts, though the differentiated cells exhibited impaired polykaryon formation and resorptive activity, as well as decreased oxidative phosphorylation and mitophagy. The Sirt3 inhibitor LC-0296 recapitulated the effects of Sirt3 deletion in osteoclast formation and mitochondrial function, and its administration to aging mice increased bone mass. Deletion of Sirt3 also attenuated the increase in bone resorption and loss of bone mass caused by estrogen deficiency. These findings suggest that Sirt3 inhibition and the resulting impairment of osteoclast mitochondrial function could be a novel therapeutic intervention for the 2 most important causes of osteoporosis.

Effect of high phosphate diet on the formation of dentin in Fam20c-deficient mice.

FAM20C (family with sequence similarity 20-member C), a kinase that phosphorylates secretory proteins, plays essential roles in various biological processes. In humans, mutations in FAM20C gene cause Raine syndrome, an autosomal recessive hereditary disease manifesting a broad spectrum of developmental defects including skeletal and craniofacial deformities. Our previous studies revealed that inactivation of Fam20c in mice led to hypophosphatemic rickets and that high phosphate (hPi) diet significantly improved the development of the skeleton in Fam20c-deficient mice. In this study, we evaluated the effects of hPi diet on the formation of dentin in Fam20c-deficient mice, using plain x-ray radiography, micro-computed tomography (µCT), histology, and immunohistochemistry. Plain x-ray radiography and µCT analyses showed that the hPi diet improved the dentin volume fraction and dentin mineral density of the Fam20c-deficient mice. Histology analyses further demonstrated that the hPi diet dramatically improved the integrity of the mandibular first molars and prevented pulp infection and dental abscesses in Fam20c-deficient mice. Our results support that the hPi diet significantly increased the formation and mineralization of dentin in Fam20c-deficient mice, implying that hypophosphatemia is a significant contributor to the dentin defects in Fam20c-deficient subjects.

Amelioration of bone fragility by pulsed electromagnetic fields in type 2 diabetic KK-Ay mice involving Wnt/β-catenin signaling.

Type 2 diabetes mellitus (T2DM) results in compromised bone microstructure and quality, and subsequently increased risks of fractures. However, it still lacks safe and effective approaches resisting T2DM bone fragility. Pulsed electromagnetic fields (PEMFs) exposure has proven to be effective in accelerating fracture healing and attenuating osteopenia/osteoporosis induced by estrogen deficiency. Nevertheless, whether and how PEMFs resist T2DM-associated bone deterioration remain not fully identified. The KK-Ay mouse was used as the T2DM model. We found that PEMF stimulation with 2 h/day for 8 wk remarkably improved trabecular bone microarchitecture, decreased cortical bone porosity, and promoted trabecular and cortical bone material properties in KK-Ay mice. PEMF stimulated bone formation in KK-Ay mice, as evidenced by increased serum levels of bone formation (osteocalcin and P1NP), enhanced bone formation rate, and increased osteoblast number. PEMF significantly suppressed osteocytic apoptosis and sclerostin expression in KK-Ay mice. PEMF exerted beneficial effects on osteoblast- and osteocyte-related gene expression in the skeleton of KK-Ay mice. Nevertheless, PEMF exerted no effect on serum biomarkers of bone resorption (TRAcP5b and CTX-1), osteoclast number, or osteoclast-specific gene expression (TRAP and cathepsin K). PEMF upregulated gene expression of canonical Wnt ligands (including Wnt1, Wnt3a, and Wnt10b), but not noncanonical Wnt5a. PEMF also upregulated skeletal protein expression of downstream p-GSK-3β and β-catenin in KK-Ay mice. Moreover, PEMF-induced improvement in bone microstructure, mechanical strength, and bone formation in KK-Ay mice was abolished after intragastric administration with the Wnt antagonist ETC-159. Together, our results suggest that PEMF can improve bone microarchitecture and quality by enhancing the biological activities of osteoblasts and osteocytes, which are associated with the activation of the Wnt/β-catenin signaling pathway. PEMF might become an effective countermeasure against T2DM-induced bone deterioration.NEW & NOTEWORTHY PEMF improved trabecular bone microarchitecture and suppressed cortical bone porosity in T2DM KK-Ay mice. It attenuated T2DM-induced detrimental consequence on trabecular and cortical bone material properties. PEMF resisted bone deterioration in KK-Ay mice by enhancing osteoblast-mediated bone formation. PEMF also significantly suppressed osteocytic apoptosis and sclerostin expression in KK-Ay mice. The therapeutic potential of PEMF on T2DM-induced bone deterioration was associated with the activation of Wnt/ß-catenin signaling.

Teratogenic Toxicity Evaluation of Bladder Cancer-Specific Oncolytic Adenovirus on Mice.

In our previous studies, we had demonstrated the efficiency and specificity of constructed bladder tissue-specific adenovirus Ad-PSCAE-UPII-E1A-AR (APU-EIA-AR) on bladder cancer. The virus biodistribution and body toxicity in nude mice have also been investigated. However, the safety of the bladder cancer-specific oncolytic adenovirus on fetal mice and F1 mice should be under intense investigation. In order to evaluate the teratogenic toxicity of bladder cancer-specific oncolytic adenovirus APU-EIA-AR on mice, in this study, we investigated the fetal mice weight, fetal body length and tail length, fetal skeleton development, as well as the F1 mice weight, growth curve, and major organ pathology. These teratogenic toxicity data of bladder tissue-specific adenovirus Ad-PSCAE- UPII-E1A-AR (AD) would provide safe information prior to embarking on clinical trials. On the sixth day of being fertilized, the pregnant mice began to be intramuscularly administrated with AD (1×107VP, 1×108VP, 1×109VP) every other day for ten days. The pregnant mice were then divided into two groups. One group was euthanized on the seventeenth day; the fetal mice were taken out, and the bone structure of the infants was observed. The other group was observed until natural childbirth. The Filial Generation (F1) is fed for 30 days; the variations in the growth progress and development were assessed. The mice were then euthanized; The tissues from major organs were harvested and observed under the microscope. In the process of teratogenic toxicity test, the Placenta weight, fetal mice weight, body length, and a tail length of mice fetal in adenovirus treated group did not reveal any alteration. Meanwhile, comparing with the PBS group, there is no obvious change in the skeleton of fetal mice treated with adenovirus. During the development process of F1 mice treated with adenovirus, the changes in mice weight show statistical significance. However, in the progress of the growth curve, this difference is not very obvious. Furthermore, the pathological section showed no obvious alteration in major organs. Our study demonstrated that bladder cancer-specific adenovirus Ad-PSCAE-UPII- E1A-AR appears safe in pregnant mice without any discernable effects on fetal mice and F1 development. Hence, it is relatively safe for tumor gene therapy.

Exploring the Potential Genetic Heterogeneity in the Incidence of Hoof Disorders in Austrian Fleckvieh and Braunvieh Cattle.

Genetic heterogeneity denotes the situation when different genetic architectures underlying diverse populations result in the same phenotype. In this study, we explore the genetic background underlying differences in the incidence of hoof disorders between Braunvieh and Fleckvieh cattle in the context of genetic heterogeneity between the breeds. Despite potentially higher power of testing due to twice as large sample size, none of the SNPs was significantly associated with the total number of hoof disorders in Fleckvieh, while 15 SNPs were significant in Braunvieh. The most promising candidate genes in Braunvieh were as follows: CBLB on BTA1, which causes arthritis in rats; CAV2 on BTA4, which affects skeletal muscles in mice; PTHLH on BTA5, which causes disease phenotypes related to the skeleton in humans, mice, and zebrafish; and SORCS2 on BTA6, which causes decreased susceptibility to injury in mice. Some of the significant SNPs (BTA1, BTA4, BTA5, BTA13, and BTA16) revealed allelic heterogeneity—i.e., different allele frequencies between Fleckvieh and Braunvieh. Some of the significant regions (BTA1, BTA5, BTA13, and BTA16) correlated to inter-breed differences in linkage disequilibrium (LD) structure and may thus represent false-positive heterogeneity. However, positions on BTA6 (SORCS2), BTA14, and BTA24 mark Braunvieh-specific regions. We hypothesize that the observed genetic heterogeneity of hoof disorders is a by-product of different selection goals defined for the analyzed breeds—toward dairy production in Braunvieh and toward beef production in Fleckvieh. Based on the current dataset, it is not possible to unequivocally confirm or exclude the hypothesis of genetic heterogeneity in the susceptibility to hoof disorders between Fleckvieh and Braunvieh. The main reason for the problem is that the potential heterogeneity was explored through SNP–phenotype associations and not through causal mutations, due to a limited SNP density offered by the SNP-chip. The rationale against genetic heterogeneity comprises a limited power of detection of true associations as well as differences in the length of LD blocks and in linkage phase between breeds. On the other hand, different selection goals defined for the analyzed breeds accompanied by no systematic, genome-wide differences in LD structure between the breeds favor the heterogeneity hypothesis at some smaller genomic regions.

Rapid and longer-term effects of selective breeding for voluntary exercise behavior on skeletal morphology in house mice.

Selection experiments can elucidate the varying course of adaptive changes across generations. We examined the appendicular skeleton of house mice from four replicate High Runner (HR) lines bred for physical activity on wheels and four non-selected Control (C) lines. HR mice reached apparent selection limits between generations 17 and 27, running ~3-fold more than C. Studies at generations 11, 16, and 21 found that HR mice had evolved thicker hindlimb bones, heavier feet, and larger articular surface areas of the knee and hip joint. Based on biomechanical theory, any or all of these evolved differences may be beneficial for endurance running. Here, we studied mice from generation 68, plus a limited sample from generation 58, to test whether the skeleton continued to evolve after selection limits were reached. Contrary to our expectations, we found few differences between HR and C mice for these later generations, and some of the differences in bone dimensions identified in earlier generations were no longer statistically significant. We hypothesize that the loss of apparently coadapted lower-level traits reflects (1) deterioration related to a gradual increase in inbreeding and/or (2) additional adaptive changes that replace the functional benefits of some skeletal changes.