TET enzymes and 5hmC epigenetic mark: new key players in carcinogenesis and progression in gynecological cancers.

DNA methylation is an epigenetic mechanism involving the transfer of a methyl group onto the C5 position of the cytosine to form 5-methylcytosine (5mC). In general, DNA methylation in cancer is associated with the repression of the expression of tumor suppressor genes (TSG) and the demethylation with the overexpression of oncogenes. DNA methylation was considered a stable modification for a long time, but in 2009, it was reported that DNA methylation is a dynamic modification. The Ten-Eleven-Translocations (TET) enzymes include TET1, TET2, and TET3 and participate in DNA demethylation through the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC). The 5hmC oxidates to 5-formylcytosine (5fC) and 5-carboxylcitosine (5caC), which are replaced by unmodified cytosines via Thymine-DNA Glycosylase (TDG). Several studies have shown that the expression of TET proteins and 5hmC levels are deregulated in gynecological cancers, such as cervical (CC), endometrial (EC), and ovarian (OC) cancers. In addition, the molecular mechanisms involved in this deregulation have been reported, as well as their potential role as biomarkers in these types of cancers. This review shows the state-of-art TET enzymes and the 5hmC epigenetic mark in CC, EC, and OC.

Microbiome-induced Increases and Decreases in Bone Tissue Strength can be Initiated After Skeletal Maturity.

Recent studies in mice have indicated that the gut microbiome can regulate bone tissue strength. However, prior work involved modifications to the gut microbiome in growing animals and it is unclear if the same changes in the microbiome, applied later in life, would change matrix strength. Here we changed the composition of the gut microbiome before and/or after skeletal maturity (16 weeks of age) using oral antibiotics (ampicillin + neomycin). Male and female mice (n=143 total, n=12-17/group/sex) were allocated into five study groups:1) Unaltered, 2) Continuous (dosing 4-24 weeks of age), 3) Delayed (dosing only 16-24 weeks of age), 4) Initial (dosing 4-16 weeks of age, suspended at 16 weeks), and 5) Reconstituted (dosing from 4-16 weeks following by fecal microbiota transplant from Unaltered donors). Animals were euthanized at 24 weeks of age. In males, bone matrix strength in the femur was 25-35% less than expected from geometry in mice from the Continuous (p= 0.001), Delayed (p= 0.005), and Initial (p=0.040) groups as compared to Unaltered. Reconstitution of the gut microbiota, however, led to a bone matrix strength similar to Unaltered animals (p=0.929). In females, microbiome-induced changes in bone matrix strength followed the same trend as males but were not significantly different, demonstrating sex-related differences in the response of bone matrix to the gut microbiota. Minor differences in chemical composition of bone matrix were observed (Raman spectroscopy). Our findings indicate that microbiome-induced impairment of bone matrix in males can be initiated and/or reversed after skeletal maturity. The portion of the femoral cortical bone formed after skeletal maturity (16 weeks) is small; however, this suggests that microbiome-induced changes in bone matrix occur without osteoblast/osteoclast turnover using an, as of yet unidentified mechanism. These findings add to evidence that the mechanical properties of bone matrix can be altered in the adult skeleton.

Gut microbial-derived short-chain fatty acids and bone: a potential role in fracture healing.

Bone healing complications such as delayed healing or non-union affect 5-10 % of patients with a long-bone fracture and lead to reduced quality of life and increased health-care costs. The gut microbiota and the metabolites they produce, mainly short-chain fatty acids (SCFAs), have been shown to impact nearly all organs of the human body including bone. SCFAs show broad activity in positively influencing bone healing outcomes either by acting directly on cell types involved in fracture healing, such as osteoblasts, osteoclasts, chondrocytes and fibroblasts, or indirectly, by shaping an appropriate anti-inflammatory and immune regulatory response. Due to the ability of SCFAs to influence osteoblast and osteoclast differentiation, SCFAs may also affect the integration of orthopaedic implants in bone. In addition, SCFA-derivatives have already been used in a variety of tissue engineering constructs to reduce inflammation and induce bone tissue production. The present review summarises the current knowledge on the role of the gut microbiota, in particular through the action of SCFAs, in the individual stages of bone healing and provides insights into how SCFAs may be utilised in a manner beneficial for fracture healing and surgical reconstruction.

From Beyond the Pale to the Pale Riders: The Emerging Association of Bacteria with Oral Cancer.

Oral cancer, predominantly oral squamous cell carcinoma (OSCC), is the eighth-most common cancer worldwide, with a 5-y survival rate <50%. There are numerous risk factors for oral cancer, among which periodontal disease is gaining increasing recognition. The creation of a sustained dysbiotic proinflammatory environment by periodontal bacteria may serve to functionally link periodontal disease and oral cancer. Moreover, traditional periodontal pathogens, such as Porphyromonas gingivalis, Fusobacterium nucleatum, and Treponema denticola, are among the species most frequently identified as being enriched in OSCC, and they possess a number of oncogenic properties. These organisms share the ability to attach and invade oral epithelial cells, and from there each undergoes its own unique molecular dialogue with the host epithelium, which ultimately converges on acquired phenotypes associated with cancer, including inhibition of apoptosis, increased proliferation, and activation of epithelial-to-mesenchymal transition leading to increased migration of epithelial cells. Additionally, emerging properties of structured bacterial communities may increase oncogenic potential, and consortia of P. gingivalis and F. nucleatum are synergistically pathogenic within in vivo oral cancer models. Interestingly, however, some species of oral streptococci can antagonize the phenotypes induced by P. gingivalis, indicating functionally specialized roles for bacteria in oncogenic communities. Transcriptomic data support the concept that functional, rather than compositional, properties of oral bacterial communities have more relevance to cancer development. Collectively, the evidence is consistent with a modified polymicrobial synergy and dysbiosis model for bacterial involvement in OSCC, with driver mutations generating a conducive microenvironment on the epithelial boundary, which becomes further dysbiotic by the synergistic action of bacterial communities.

PINK1 Silencing Modifies Dendritic Spine Dynamics of Mouse Hippocampal Neurons.

PTEN-induced kinase 1 (PINK1) mutations can cause early-onset Parkinson's disease and patients are likely to develop cognitive decline, depression, and dementia. Several neurophysiological studies have demonstrated PINK1 deficiency impairs striatal and hippocampal presynaptic plasticity. Dendritic spine postsynaptic abnormalities are common in neurological diseases; however, whether PINK1 silencing modifies dendritic spine dynamics of hippocampal neurons is unclear. To address this question, confocal images of mouse cultured hippocampal neurons transfected with plasmids to silence PINK1 were analyzed. These studies revealed that PINK1 silencing increased density of thin spines and reduced head size of stubby spines. Immunoblotting analysis uncovered that PINK1 silencing decreased expression of postsynaptic density proteins (PSD95 and Shank) and glutamate receptors (NR2B and mGluR5). We also found PINK1 silencing regulated dendritic spine morphology by actin regulatory proteins (RhoGAP29 and ROCK2) and regulated neuronal survival by decreased Akt activation. These results suggest PINK1 may regulate postsynaptic plasticity in hippocampal neurons generating presymptomatic alterations in dendritic spines that eventually could lead to the neurodegeneration and cognitive decline often seen in Parkinson's disease.

PRL -1149T allele (rs1341239) is associated with decreased risk of rheumatoid arthritis in population from southern Mexico: analysis of mRNA expression and PRL serum levels.

INTRODUCTION: Prolactin (PRL) is a sex hormone with immunomodulatory properties, and it is associated with the clinical activity of rheumatoid arthritis (RA). The -1149G>T polymorphism at the prolactin (PRL) gene has been associated with autoimmune diseases, but its functional effect is unclear. OBJECTIVE: To analyze the association of the PRL -1149G>T polymorphism with disease susceptibility, mRNA, and protein expression of PRL in RA patients from Southern Mexico. METHODS: We included 300 RA patients and 300 control subjects (CS). Genotypes were identified by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, the PRL mRNA expression was determined by real-time PCR, and PRL serum levels were measured by enzyme-linked immunosorbent assay. RESULTS: Applying genetic models of inheritance (dominant, recessive, and additive), we found an association between the T allele and decreased RA susceptibility (OR = 0.55, 95% CI 0.35-0.87, p = 0.009; OR = 0.09, 95% CI 0.012-0.76, p = 0.011; OR = 0.49, 95% CI 0.32-0.76, p = 0.001, respectively). RA patients had higher mRNA expression and soluble levels of PRL than CS (p < 0.05). The PRL serum levels were similar in RA and CS according to genotypes. However, in CS, carriers of GT and TT genotypes showed lower PRL mRNA expression than GG genotype carriers (7.1-fold and 20-fold respectively, p = 0.006). CONCLUSIONS: This study demonstrated that the PRL -1149T allele is a genetic marker of decrease risk to RA in population from Southern Mexico, and it is associated with low PRL mRNA. KEY POINTS: • PRL -1149T allele is a marker of decreased RA susceptibility in population from southern Mexico. • PRL -1149TT genotype is associated with low PRL mRNA expression. • RA patients have higher mRNA expression and soluble levels of PRL than healthy subjects. • PRL serum levels are higher in those RA patients with < 2 years of disease evolution.

Can quantitative computed tomography detect bone morphological changes associated with catastrophic proximal sesamoid bone fracture in Thoroughbred racehorses?

BACKGROUND: Fracture of the proximal sesamoid bones continues to be the most common fatal musculoskeletal injury in US racehorses. Identifying factors that influence fracture risk could lead to screening techniques to reduce catastrophic injury rates and improve animal welfare. OBJECTIVES: To identify morphological differences between proximal sesamoid bones of the contralateral limb of fracture and control horses and assess the feasibility of computed tomography (CT) to detect traits associated with proximal sesamoid bone fracture. We hypothesised that horses with proximal sesamoid bone fracture would have greater bone density. STUDY DESIGN: Cross-sectional cadaver morphological study. METHODS: Proximal sesamoid bone morphology was measured using high-resolution micro-CT images from 16 Thoroughbred racehorses (eight fracture, eight control) euthanised on New York racetracks. Nominal logistic regression models and receiver operating characteristic curves were created to assess the ability of CT-derived morphological traits to accurately classify fracture horses vs. controls. RESULTS: Bone volume fraction was greater in the fracture group (90.39 ± 1.76%) as compared to controls (87.20 ± 2.79%, P<0.0001). Bone volume fraction, bone width, trabecular thickness and degree of anisotropy were significantly different between fracture and control horses. Receiver operating characteristic curves showed that a combined model that incorporates bone volume fraction and width can identify fracture from control horses with an area under the curve of 0.938, indicating high accuracy at classifying fracture horses from controls. MAIN LIMITATIONS: The number of horses per group is small, although the total number of sesamoids imaged is reasonable (n = 62). In vivo CT at the resolution performed in this study is currently unattainable; however, density and width could be measured with quantitative CT. CONCLUSIONS: Differences in proximal sesamoid bone morphology were identified between fracture and control horses. As improved technology becomes accessible, quantitative CT could potentially be used as a clinical imaging technique to estimate proximal sesamoid bone fracture risk in Thoroughbred racehorses.

The effects of metabolic syndrome, obesity, and the gut microbiome on load-induced osteoarthritis.

OBJECTIVE: Metabolic syndrome is characterized by obesity, hyperglycemia, hypertension, insulin resistance, and dyslipidemia. Metabolic syndrome is associated with osteoarthritis (OA), but it is unclear if the association is attributable to increased mechanical loading on joints caused by obesity or other aspects of metabolic syndrome. Here we examined the effects of altered metabolism, obesity, and the gut microbiome on load-induced OA. DESIGN: Cartilage damage was induced through cyclic compressive loading in four groups of adult male mice: Toll-like receptor-5 deficient (TLR5KO) mice that develop metabolic syndrome due to alterations in the gut microbiome, TLR5KO mice submitted to chronic antibiotics to prevent metabolic syndrome (TLR5KOΔMicrobiota), C57BL/6J mice fed a high fat diet to cause obesity (HFD), and untreated C57BL/6J mice (WT). Loading was applied for 2 weeks (n = 10-11/group) or 6 weeks (n = 10-11/group). RESULTS: After 2 weeks of loading, cartilage damage (OARSI score) was not different among groups. After 6 weeks of loading, HFD mice had increased load-induced cartilage damage, while TLR5KO mice had cartilage damage comparable to WT mice. TLR5KOΔMicrobiota mice had less cartilage damage than other groups. HFD mice had elevated serum inflammatory markers. Each group had a distinct gut microbiome composition. CONCLUSIONS: Severe obesity increased load-induced cartilage damage, while milder changes in adiposity/metabolic syndrome seen in TLR5KO mice did not. Furthermore, the effects of systemic inflammation/obesity on cartilage damage depend on the duration of mechanical loading. Lastly, reduced cartilage damage in the TLR5KOΔMicrobiota mice suggests that the gut microbiome may influence cartilage pathology.

Expression of MIF and TNFA in psoriatic arthritis: relationship with Th1/Th2/Th17 cytokine profiles and clinical variables.

Psoriatic arthritis (PsA) is an autoimmune inflammatory disease associated with psoriasis. The cause of this pathology is still unknown, but research suggests the diseases are caused by a deregulated cytokine production. MIF is a cytokine associated with immunomodulation of Th1, Th2, and Th17 cytokine profiles in inflammatory diseases. Based on this knowledge, the aim of this study was to determine the association of MIF and TNFA expression with Th1, Th2, and Th17 cytokine profiles in serum levels of PsA patients. A cross-sectional study was performed in 50 PsA patients and 30 control subjects (CS). The cytokine profiles were quantified by BioPlex MagPix system and the mRNA expression levels by real-time PCR. TNFA mRNA expression was 138.81-folds higher in PsA patients than CS (p < 0.001). Regarding MIF mRNA expression, no significant differences were observed; however, a positive correlation was identified between MIF mRNA expression and PsA time of evolution (r = - 0.53, p = 0.009). An increase of Th1 (IFNγ: PsA = 37.1 pg/mL vs. CS = 17 pg/mL, p < 0.05; TNFα: PsA = 24.6 pg/mL vs. CS = 9.8 pg/mL, p < 0.0001) and Th17 cytokine profiles (IL-17: PsA = 6.4 pg/mL vs. CS = 2.7 pg/mL, p < 0.05; IL-22: PsA = 8.4 pg/mL vs. CS = 1.8 pg/mL, p < 0.001), were found in PsA patients. Th2 cytokines were not significantly different in both groups. In conclusion, a high expression of TNFA mRNA, as well as an increase of Th1 and Th17 cytokine profiles evaluated by IFNγ, TNFα, IL-17, and IL-22 cytokines, was observed in PsA patients.

Bone Mechanical Function and the Gut Microbiota.

The primary function of bone in the body is to resist mechanical forces. Impairment of the mechanical performance of bone is therefore the primary clinical challenge presented by bone disease. Failure to resist forces associated with activities of daily living leads to fragility fracture. In this chapter we review the characteristics of bone that influence mechanical performance and fracture risk, how bone remodeling and modeling alter mechanically relevant characteristics of bone, and the potential for the gut microbiome to alter bone mechanical performance and risk of fragility fracture. The ability of bone to resist fragility fracture is determined by characteristics of bone tissue at scales ranging from nanometers to centimeters. Bone remodeling and modeling can influence whole bone shape and density, but the effects of bone remodeling on bone tissue mechanical properties are not as well understood. The gut microbiome may influence bone by altering nutrient absorption, stimulating the immune system or through translocation of microbes and microbial products across the gut endothelium. Although there is evidence that the microbiome can alter bone density and bone remodeling, the ability of the microbiome to cause changes in tissue material properties, whole bone strength, and fragility fracture remains to be determined.

Spatial relationships between bone formation and mechanical stress within cancellous bone.

Bone adapts to mechanical stimuli. While in vivo mechanical loading has been shown to increase the density of cancellous bone, theory suggests that the relationship between tissue stress/strain and subsequent bone formation occurs at the scale of individual trabeculae. Here we examine bone formation one week following mechanical stimulus. Three bouts of cyclic loading (300 cycles/day on 3 consecutive days) were applied to caudal vertebrae of female rats (n=7). Bone formation was determined using three-dimensional images of fluorescent markers of bone formation (0.7×0.7×5.0µm(3)) and local tissue stress/strain was determined using high-resolution finite element models. Three days of mechanical stimuli resulted in an increase in mineralizing surface (loaded: 17.68±2.17%; control: 9.05±3.20%; mean±SD) and an increase in the volume of bone formed (loaded: 7.09±1.97%; control: 1.44±0.50%). The number of bone formation sites was greater in loaded animals (650.71±118.54) than pinned not loaded controls (310.71±91.55), a difference that was explained by the number of formation sites at regions with large local tissue strain energy density (SED). In addition, the probability of observing bone formation was greater at locations of the microstructure experiencing greater SED, but did not exceed 32%, consistent with prior work. Our findings demonstrate that bone formation in the week following a short term mechanical stimulus occurs near regions of bone tissue experiencing high tissue SED, although the ability of finite element models to predict the locations of bone formation remains modest and further improvements may require accounting for additional factors such as osteocyte distribution or fluid flow.

Finite element models predict the location of microdamage in cancellous bone following uniaxial loading.

High-resolution finite element models derived from micro-computed tomography images are often used to study the effects of trabecular microarchitecture and loading mode on tissue stress, but the degree to which existing finite element methods correctly predict the location of tissue failure is not well characterized. In the current study, we determined the relationship between the location of highly strained tissue, as determined from high-resolution finite element models, and the location of tissue microdamage, as determined from three-dimensional fluoroscopy imaging, which was performed after the microdamage was generated in-vitro by mechanical testing. Fourteen specimens of human vertebral cancellous bone were assessed (8 male donors, 2 female donors, 47-78 years of age). Regions of stained microdamage, were 50-75% more likely to form in highly strained tissue (principal strains exceeding 0.4%) than elsewhere, and generally the locations of the regions of microdamage were significantly correlated (p<0.05) with the locations of highly strained tissue. This spatial correlation was stronger for the largest regions of microdamage (≥1,000,000µm(3) in volume); 87% of large regions of microdamage were located near highly strained tissue. Together, these findings demonstrate that there is a strong correlation between regions of microdamage and regions of high strain in human cancellous bone, particularly for the biomechanically more important large instances of microdamage.

Fatigue-induced microdamage in cancellous bone occurs distant from resorption cavities and trabecular surfaces.

Impaired bone toughness is increasingly recognized as a contributor to fragility fractures. At the tissue level, toughness is related to the ability of bone tissue to resist the development of microscopic cracks or other tissue damage. While most of our understanding of microdamage is derived from studies of cortical bone, the majority of fragility fractures occur in regions of the skeleton dominated by cancellous bone. The development of tissue microdamage in cancellous bone may differ from that in cortical bone due to differences in microstructure and tissue ultrastructure. To gain insight into how microdamage accumulates in cancellous bone we determined the changes in number, size and location of microdamage sites following different amounts of cyclic compressive loading. Human vertebral cancellous bone specimens (n=32, 10 male donors, 6 female donors, age 76 ± 8.8, mean ± SD) were subjected to sub-failure cyclic compressive loading and microdamage was evaluated in three-dimensions. Only a few large microdamage sites (the largest 10%) accounted for 70% of all microdamage caused by cyclic loading. The number of large microdamage sites was a better predictor of reductions in Young's modulus caused by cyclic loading than overall damage volume fraction (DV/BV). The majority of microdamage volume (69.12 ± 7.04%) was located more than 30 µm (the average erosion depth) from trabecular surfaces, suggesting that microdamage occurs primarily within interstitial regions of cancellous bone. Additionally, microdamage was less likely to be near resorption cavities than other bone surfaces (p<0.05), challenging the idea that stress risers caused by resorption cavities influence fatigue failure of cancellous bone. Together, these findings suggest that reductions in apparent level mechanical performance during fatigue loading are the result of only a few large microdamage sites and that microdamage accumulation in fatigue is likely dominated by heterogeneity in tissue material properties rather than stress concentrations caused by micro-scale geometry.

The effects of misalignment during in vivo loading of bone: techniques to detect the proximity of objects in three-dimensional models.

Theories of mechanical adaptation of bone suggest that mechanical loading causes bone formation at discrete locations within bone microstructure experiencing the greatest mechanical stress/strain. Experimental testing of such theories requires in vivo loading experiments and high-resolution finite element models to determine the distribution of mechanical stresses. Finite element models of in vivo loading experiments typically assume idealized boundary conditions with applied load perfectly oriented on the bone, however small misalignments in load orientation during an in vivo experiment are unavoidable, and potentially confound the ability of finite element models to predict locations of bone formation at the scale of micrometers. Here we demonstrate two different three-dimensional spatial correlation methods to determine the effects of misalignment in load orientation on the locations of high mechanical stress/strain in the rodent tail loading model. We find that, in cancellous bone, the locations of tissue with high stress are maintained under reasonable misalignments in load orientation (p<0.01). In cortical bone, however, angular misalignments in the dorsal direction can alter the locations of high mechanical stress, but the locations of tissue with high stress are maintained under other misalignments (p<0.01). We conclude that, when using finite element models of the rodent tail loading model, small misalignments in loading orientation do not affect the predicted locations of high mechanical stress within cancellous bone.

Quantitative relationships between microdamage and cancellous bone strength and stiffness.

Microscopic tissue damage (microdamage) is an aspect of bone quality associated with impaired bone mechanical performance. While it is clear that bone tissue submitted to more severe loading has greater amounts of microdamage (as measured through staining), how microdamage influences future mechanical performance of the bone has not been well studied, yet is necessary for understanding the mechanical consequences of the presence of microdamage. Here we determine how stained microdamage generated by a single compressive overload affects subsequent biomechanical performance of cancellous bone. Human vertebral cancellous bone specimens (n=47) from 23 donors (14 males, 9 females, 64-92years of age) were submitted to a compressive overload, stained for microdamage, then reloaded in compression to determine the relationship between the amount of microdamage caused by the initial load and reductions in mechanical performance during the reload. Damage volume fraction (DV/BV) caused by the initial overload was related to reductions in Young's modulus, yield strength, ultimate strength, and yield strain upon reloading (p<0.05, R(2)=0.18-0.34). The regression models suggest that, on average, relatively small amounts of microdamage are associated with large reductions in reload mechanical properties: a 1.50% DV/BV caused by a compressive overload was associated with an average reduction in Young's modulus of 41.0±3.2% (mean±SE), an average reduction in yield strength of 63.1±4.5% and an average reduction in ultimate strength of 52.7±4.0%. Specimens loaded beyond 1.2% (1.2-4.0% apparent strain) demonstrated a single relationship between reload mechanical properties (Young's modulus, yield strength, and ultimate strength) and bone volume fraction despite a large range in amounts of microdamage. Hence, estimates of future mechanical performance of cancellous bone can be achieved using the bone volume fraction and whether or not a specimen was previously loaded beyond ultimate strain. The empirical relationships provided in this study make it possible to estimate the degree of impaired mechanical performance resulting from an observed amount of stained microdamage.

Theoretical consideration of the effect of drug holidays on BMD and tissue age.

UNLABELLED: It has been suggested that some patients undergoing prolonged treatment for osteoporosis with anti-resorptive agents may benefit from discontinuing treatment. Here we use a computer simulation of bone cell activity to estimate changes in bone mineral density (BMD) and tissue age when treatment is discontinued. INTRODUCTION: Although anti-resorptive agents are effective at reducing fracture risk, questions remain regarding how long patients should continue treatment and how long treatment should be discontinued. Suspending treatment as part of a drug holiday may reduce the risk of adverse effects, but may also lead to reduced BMD. METHODS: We use a computer simulation of the bone remodeling process to estimate how BMD and mean tissue age are changed after treatment is suspended. Mean tissue age is studied because increased tissue age has been associated with impaired bone quality and has been linked to the risk of adverse effects. RESULTS: Our simulations suggest that BMD gains from anti-resorptive therapy can be lost over time, especially with anti-resorptive agents that have little residual effects. With regard to mean tissue age, the simulations suggest that increases in tissue age from anti-resorptive treatment are long lasting; increases in mean tissue age caused by treatment may remain for as long as 15 years after treatment is suspended. After stopping treatment, reductions in BMD are expected to occur long before mean tissue age returns to normal. CONCLUSIONS: Our simulations suggest that, when using a long-lasting anti-resorptive agent, 1- to 5-year drug holidays may have little effect on BMD in most patients but that drug holiday intervals that maintain BMD are unlikely to reverse alterations in tissue age caused by treatment. Our analysis echoes recent reviews suggesting patient selection and monitoring when anti-resorptive treatment is discontinued.

Anti-resorptive agents reduce the size of resorption cavities: a three-dimensional dynamic bone histomorphometry study.

Alterations in resorption cavities and bone remodeling events during anti-resorptive treatment are believed to contribute to reductions in fracture risk. Here, we examine changes in the size of individual remodeling events associated with treatment with a selective estrogen receptor modulator (raloxifene) or a bisphosphonate (risedronate). Adult female rats (6months of age) were submitted to ovariectomy (n=17) or sham surgery (SHAM, n=5). One month after surgery, the ovariectomized animals were separated into three groups: untreated (OVX, n=5), raloxifene treated (OVX+Ral, n=6) and risedronate treated (OVX+Ris, n=6). At 10months of age, the lumbar vertebrae were submitted to three-dimensional dynamic bone histomorphometry to examine the size (depth, breadth and volume) of individual resorption cavities and formation events. Maximum resorption cavity depth did not differ between the SHAM (23.66±1.87µm, mean±SD) and OVX (22.88±3.69µm) groups but was smaller in the OVX+Ral (14.96±2.30µm) and OVX+Ris (14.94±2.70µm) groups (p<0.01). Anti-resorptive treatment was associated with reductions in the surface area of resorption cavities and the volume occupied by each resorption cavity (p<0.01 each). The surface area and volume of individual formation events (double-labeled events) in the OVX+Ris group were reduced as compared to other groups (p<0.02). Raloxifene treated animals showed similar amounts of bone remodeling (ES/BS and dLS/BS) compared to sham-operated controls but smaller cavity size (depth, breadth and volume). The current study shows that anti-resorptive agents influence the size of resorption cavities and individual remodeling events and that the effect of anti-resorptives on individual remodeling events may not always be directly related to the degree of suppression of bone remodeling.

Sodium tungstate modulates ATM function upon DNA damage.

Both radiotherapy and most effective chemotherapeutic agents induce different types of DNA damage. Here we show that tungstate modulates cell response to DNA damaging agents. Cells treated with tungstate were more sensitive to etoposide, phleomycin and ionizing radiation (IR), all of which induce DNA double-strand breaks (DSBs). Tungstate also modulated the activation of the central DSB signalling kinase, ATM, in response to these agents. These effects required the functionality of the Mre11-Nbs1-Rad50 (MRN) complex and were mimicked by the inhibition of PP2A phosphatase. Therefore, tungstate may have adjuvant activity when combined with DNA-damaging agents in the treatment of several malignancies.

Three-dimensional characterization of resorption cavity size and location in human vertebral trabecular bone.

The number and size of resorption cavities in cancellous bone are believed to influence rates of bone loss, local tissue stress and strain and potentially whole bone strength. Traditional two-dimensional approaches to measuring resorption cavities in cancellous bone report the percent of the bone surface covered by cavities or osteoclasts, but cannot measure cavity number or size. Here we use three-dimensional imaging (voxel size 0.7×0.7×5.0 µm) to characterize resorption cavity location, number and size in human vertebral cancellous bone from nine elderly donors (7 male, 2 female, ages 47-80 years). Cavities were 30.10 ± 8.56 µm in maximum depth, 80.60 ± 22.23∗10(3) µm(2) in surface area and 614.16 ± 311.93∗10(3) µm(3) in volume (mean ± SD). The average number of cavities per unit tissue volume (N.Cv/TV) was 1.25 ± 0.77 mm(-3). The ratio of maximum cavity depth to local trabecular thickness was 30.46 ± 7.03% and maximum cavity depth was greater on thicker trabeculae (p<0.05, r(2)=0.14). Half of the resorption cavities were located entirely on nodes (the intersection of two or more trabeculae) within the trabecular structure. Cavities that were not entirely on nodes were predominately on plate-like trabeculae oriented in the cranial-caudal (longitudinal) direction. Cavities on plate-like trabeculae were larger in maximum cavity depth, cavity surface area and cavity volume than cavities on rod-like trabeculae (p<0.05). We conclude from these findings that cavity size and location are related to local trabecular microarchitecture.