A novel underuse model shows that inactivity but not ovariectomy determines the deteriorated material properties and geometry of cortical bone in the tibia of adult rats

Our goal in this study was to determine to what extent the physiologic consequences of ovariectomy (OVX) in bones are exacerbated by a lack of daily activity such as walking. We forced 14-week-old female rats to be inactive for 15 weeks with a unique experimental system that prevents standing and walking while allowing other movements. Tibiae, femora, and 4th lumbar vertebrae were analyzed by peripheral quantitative computed tomography (pQCT), microfocused X-ray computed tomography (micro-CT), histology, histomorphometry, Raman spectroscopy, and the three-point bending test. Contrary to our expectation, the exacerbation was very much limited to the cancellous bone parameters. Parameters of femur and tibia cortical bone were affected by the forced inactivity but not by OVX: (1) cross-sectional moment of inertia was significantly smaller in Sham-Inactive rat bones than that of their walking counterparts; (2) the number of sclerostin-positive osteocytes per unit cross-sectional area was larger in Sham-Inactive rat bones than in Sham-Walking rat bones; and (3) material properties such as ultimate stress of inactive rat tibia was lower than that of their walking counterparts. Of note, the additive effect of inactivity and OVX was seen only in a few parameters, such as the cancellous bone mineral density of the lumbar vertebrae and the structural parameters of cancellous bone in the lumbar vertebrae/tibiae. It is concluded that the lack of daily activity is detrimental to the strength and quality of cortical bone in the femur and tibia of rats, while lack of estrogen is not. Our inactive rat model, with the older rats, will aid the study of postmenopausal osteoporosis, the etiology of which may be both hormonal and mechanical

Standalone gamma knife radiosurgery for brain metastasis of malignant peripheral nerve sheath tumor

Background: Brain metastasis (BM) of malignant peripheral nerve sheath tumor (MPNST) is extremely rare. The importance of aggressive surgical resection with or without radiation therapy has been emphasized, while the significance of other local therapies has been rarely reported. Three reports documented patients treated with stereotactic radiosurgery, while no previous study reported a BM case of MPNST treated with standalone Gamma Knife (Elekta AB, Stockholm, Sweden) radiosurgery (GKRS). The aim of this report is to describe the detailed clinical course of a patient with BM of MPNST and its radiographic response to standalone GKRS. Case Description: The patient was a 29-year-old male with a history of MPNST. Seven years prior to the current presentation, he experienced respiratory symptoms, and a chest CT showed multiple pulmonary nodules. A bronchoscopic biopsy confirmed the diagnosis of metastatic MPNST. His disease was refractory to chemotherapy, and 1 month prior to this presentation, the chest and abdominal CT scans showed systemic progression. He developed worsening headache and left-side weakness. A brain MRI revealed a large right parietal (10.1 cm3) enhancing mass. Considering his poor general condition and the large size of the lesion, the tumor was treated with standalone GKRS. The 2-month follow-up MRI revealed the lesion was smaller and associated with surrounding ill-defined heterogeneous enhancements, compatible with asymptomatic radiation necrosis. This lesion showed a low cerebral blood volume, consistent with non-viable tumor. Unfortunately, the patient died from his primary disease before his second follow-up. Conclusions: This is the first case report describing the details of the post-treatment course and radiographic responses following standalone GKRS in a patient with BMs from MPNST

Regulation of FOXOs and p53 by SIRT1 modulators under oxidative stress.

Excessive reactive oxygen species (ROS) induce apoptosis and are associated with various diseases and with aging. SIRT1 (sirtuin-1), an NAD+-dependent protein deacetylase, decreases ROS levels and participates in cell survival under oxidative stress conditions. SIRT1 modulates the transcription factors p53, a tumor suppressor and inducer of apoptosis, and the forkhead O (FOXO) family, both of which play roles for cell survival and cell death. In this study, we aimed to know which is working greatly among p53 and FOXOs transcription factors in SIRT1's cell protective functions under oxidative stress conditions. The antimycin A-induced increase in ROS levels and apoptosis was enhanced by SIRT1 inhibitors nicotinamide and splitomicin, whereas it was suppressed by a SIRT1 activator, resveratrol, and a SIRT1 cofactor, NAD+. SIRT1-siRNA abolished the effects of splitomicin and resveratrol. p53-knockdown experiment in C2C12 cells and experiment using p53-deficient HCT116 cells showed that splitomicin and resveratrol modulated apoptosis by p53-dependent and p53-independent pathways. In p53-independent cell protective pathway, we found that FOXO1, FOXO3a, and FOXO4 were involved in SOD2's upregulation by resveratrol. The knockdown of these three FOXOs by siRNAs completely abolished the SOD2 induction, ROS reduction, and anti-apoptotic function of resveratrol. Our results indicate that FOXO1, FOXO3a and FOXO4, are indispensable for SIRT1-dependent cell survival against oxidative stress, although deacetylation of p53 has also some role for cell protective function of SIRT1

A Novel Association between the 27-bp Deletion and 538G\u3eA Mutation in the ABCC11 Gene

A single nucleotide polymorphism in the ABCC11 gene, 538G\u3eA (rs17822931), is known to determine human ear wax type. The G/G and G/A genotypes correspond to the wet type, while the A/A genotype corresponds to the dry type. Another earwax determinant, a 27-bp deletion (Δ27) downstream from the rs17822931 site, is a rare variant that leads to the dry phenotype. In a previous report, we found an individual with the G allele who unexpectedly showed the dry type of earwax, leading to the identification of Δ27. We also demonstrated that the Δ27 allele was present in individuals of Japanese, Thai, native North American, Andean, and Bolivian ancestry but absent in those of European and African ancestry. Here, we assessed the Δ27 allele frequency among Japanese and Ukrainian individuals and identified a novel association between the Δ27 and 538G\u3eA mutations. The Δ27 allele frequency was 0.002 (3/1,520; one individual is heterozygous, and another is homozygous) among Japanese individuals and 0 (0/794) among Ukrainians. We also found a previously unreported homozygous genotype for both the Δ27 and A alleles. Our findings suggest that the Δ27 deletion may have occurred in an ABCC11 gene with the 538G\u3eA mutation

The effect of resveratrol on AMPK activity in C2C12 cells.

<p>(A) Representative immunoblots of phospho-AMPK (Thr172), total AMPK, phospho-acetyl-CoA carboxylase (ACC) (Ser79), total ACC, and GAPDH in C2C12 cells treated with vehicle or 60 µM antimycin A (AA) with or without 30 µM resveratrol (RSV). (B) Quantification of phospho-AMPK (Thr172) level normalized to GAPDH (N = 5). (C) Quantification of phospho-ACC (Ser79) level normalized to GAPDH (N = 5). n.s. = not significant.</p

FOXOs mediate the anti-apoptotic effect of resveratrol.

<p>(A) (Left) Representative images of nuclear staining with Hoechst33342 in C2C12 cells. Cells transfected with control-siRNA (Ctrl-siRNA) or a mixture of siRNAs against all three FOXOs (FOXO1, FOXO3a, FOXO4) (FOXOs-siRNA) were pretreated with vehicle or resveratrol (RSV, 30 µM) for 3 hours and then incubated with antimycin A (AA, 50 µM) for 24 hours under serum-free conditions. Scale bar: 50 µm. (Right) Quantification of apoptotic cells defined by nuclear condensation. Data are from three independent experiments. (B) (Left) Representative images of immunostaining for BAX (green) in C2C12 cells treated as in A. Scale bar: 10 µm. (Right) Quantification of cells with BAX accumulation. Data are from three independent experiments. (C) Representative immunofluorescence images for acetylated p53 (red) in C2C12 cells treated as in A. The acetylation of p53 was detected by immunostaining using an antibody against acetyl-p53. Data are from three independent experiments. Scale bar: 20 µm. ***p<0.001, **p<0.01, n.s. = not significant.</p

Effects of p53 knockdown on the functions of SIRT1 modulators.

<p>(A) RT-PCR analysis for p53 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in C2C12 cells transfected with siRNA against negative control (Ctrl) or p53. (B) C2C12 cells transfected with control- (Ctrl-si) or p53-siRNA (p53-si) were treated with vehicle or AA (50 µM) for 24 hours. Apoptotic cells with condensed nuclei were quantified. Data are from three independent experiments. (C and D) The percentage of nuclear-condensed C2C12 cells was analyzed. C2C12 cells transfected with control- (Ctrl-si) or p53-siRNA (p53-si) were pretreated with vehicle or either 60 µM splitomicin (SP in C) or 10 µM RSV in D for 3 hours followed by incubation with 50 µM antimycin A (AA) for 24 hours. Data are from three independent experiments. (E) RT-PCR analysis for p53 mRNA in wild type (p53+/+) or p53-deficient (p53−/−) HCT116 cells. (F) Quantification of apoptotic cells with nuclear condensation in wild-type (p53+/+) or p53-null (p53−/−) HCT116 cells treated with vehicle or AA for 24 hours. Data are from three independent experiments. (G and H) Analysis of apoptosis in HCT116 cells treated with SIRT1 modulators. Wild-type (p53+/+) or p53-deficient (p53−/−) HCT116 cells were pretreated with 60 µM SP (G) or 10 µM RSV (H) for 3 hours and then incubated with 50 µM AA for 24 hours. Data are from three independent experiments. ***p<0.001, **p<0.01, *p<0.05. (I) Representative immunoblots of acetyl-p53 (K379) and p53 in C2C12 cells treated with vehicle or antimycin A (AA, 50 µM) with or without pretreatment with resveratrol (RSV) or Ex527. All cells were treated in the presence of 50 nM of trichostatin A. (J) Representative images of immunostaining for acetyl-p53 (Lys379) in C2C12 cells treated as in A. Scale bar: 20 µm.</p

Effects of SIRT1 knockdown on functions of SIRT1 modulators.

<p>(A) Immunoblot analysis for SIRT1 in C2C12 cells transfected with control (Ctrl)- or SIRT1-siRNA. (B) Representative images of CM-H₂DCFDA fluorescence. C2C12 cells transfected with control- (Ctrl-) or SIRT1-siRNA were treated with antimycin A (AA) (100 µM) for 3 hours after pretreatment with vehicle or resveratrol (RSV, 10 µM) for 6 hours. Scale bar: 10 µm. (C) Quantification of CM-H₂DCFDA (DCF) fluorescence. (D and E) Quantification of cells with condensed nuclei. C2C12 cells transfected with control- (Ctrl-) or SIRT1-siRNA were treated with AA (200 µM) for 24 hours after pretreatment with vehicle (Ctrl), 10 µM RSV, or 60 µM splitomicin (SP) for 3 hours. Data are from three independent experiments. **p<0.01, *p<0.05. a.u. = arbitrary unit. n.s. = not significant.</p

Effects of SIRT1 modulators on H₂O₂-induced ROS generation and apoptosis.

<p>C2C12 cells were treated with 50 µM H₂O₂ for 24 hours after their pretreatment with vehicle (Ctrl), 5 mM nicotinamide (NA), 60 µM splitomicin (SP), 10 µM resveratrol (RSV), or 1 mM NAD⁺ for 3 hours. (A) Intracellular levels of reactive oxygen species (ROS) were detected by CM-H₂DCFDA (DCF). Data are from three independent experiments. (B and C) Cell death was analyzed by nuclear condensation (B), or by immunostaining for cleaved (active) caspase-3 (C). Data in each panel are from three independent experiments. **p<0.01, *p<0.05. a.u. = arbitrary unit.</p