Isolation and whole-genome sequencing of a novel aviadenovirus from owls in Japan.

Adenoviruses have been reported to infect a variety of birds. Here, we isolated a novel adenovirus from the liver of a dead owl chick (Bengal eagle owl; Bubo bengalensis) at a raptor-breeding facility in Japan and determined the complete genome sequence of the virus. We performed necropsies on the dead owl chicks and found that they had enlarged livers, pericardial edema, and focal necrosis of the liver tissue. Transmission electron microscopy of the liver tissue revealed a virus-like structure, appearing as paracrystalline arrays in the nucleus, and immunohistochemical staining with anti-adenovirus antibodies showed positive reactions in hepatocytes and other cells. Attempts to isolate the virus from homogenized liver tissue of a dead owl chick showed a cytopathic effect on chicken-derived cultured cells after multiple blind passages. Further, we determined the complete genome sequence of this virus and performed phylogenetic analysis, revealing that this adenovirus belongs to the genus Aviadenovirus, forming a cluster with fowl and turkey aviadenoviruses. The amino acid sequence divergence between the DNA polymerase of this virus and its closest known adenovirus relative is approximately 29%, implying that this virus can be assigned to a new species in the genus Aviadenovirus. Based on our data, this novel owl adenovirus is a likely cause of fatal infections in owls, which may threaten wild and captive owl populations. Further, this virus is unique among raptor adenoviruses in that it infects chicken-derived cultured cells, raising the importance of further investigations to evaluate interspecies transmission of this virus.

Novel small molecule SIRT2 inhibitors induce cell death in leukemic cell lines.

BACKGROUND: Sirtuin 2 (SIRT2) is a member of the sirtuin family, nicotinamide adenine dinucleotide+-dependent deacylases, which participates in modulation of cell cycle control, neurodegeneration, and tumorigenesis. SIRT2 expression increases in acute myeloid leukemia blasts. Downregulation of SIRT2 using siRNA causes apoptosis of HeLa cells. Therefore, selective inhibitors of SIRT2 are candidate therapeutic agents for cancer. Adult T-cell leukemia/lymphoma (ATL) is a T-cell malignancy that has a poor prognosis and develops after long-term infection with human T-cell leukemia virus (HTLV)-1. Sirtuin 1 inhibition has been shown to induce apoptosis and autophagy in HTLV-1-infected cell lines, whereas the effects of SIRT2 inhibition alone have not been elucidated. METHODS: We assessed the efficacy of our small molecule selective SIRT2 inhibitors NCO-90/141 to induce leukemic cell death. Cell viability was examined using the cell proliferation reagent Cell Count Reagent SF. Apoptotic cells were detected by annexin V-FITC and terminal deoxynucleotidyl transferase dUTP nick end labeling assays by flow cytometry. Caspase activity was detected using an APOPCYTO Intracellular Caspase Activity Detection Kit. The presence of autophagic vacuoles was assessed using a Cyto-ID Autophagy Detection Kit. RESULTS: Our novel small molecule SIRT2-specific inhibitors NCO-90/141 inhibited cell growth of leukemic cell lines including HTLV-1-transformed T-cells. NCO-90/141 induced apoptosis via caspase activation and mitochondrial superoxide generation in leukemic cell lines. However, a caspase inhibitor did not prevent this caspase-associated cell death. Interestingly, NCO-90/141 increased the LC3-II level together with autophagosome accumulation, indicating autophagic cell death. Thus, NCO-90/141 simultaneously caused apoptosis and autophagy. CONCLUSIONS: These results suggest that NCO-90/141 are highly effective against leukemic cells in caspase-dependent or -independent manners via autophagy, and they may have a novel therapeutic potential for treatment of leukemias including ATL.

Differences in leg muscle activity and body sway between elderly adults able and unable to maintain one-leg stance for 1 min: the effect of hand support.

BACKGROUND: One-leg stance (OLS) training is often used to prevent falls in the elderly. The burden imposed on the supporting lower limb during OLS may differ depending on whether hand support is used, particularly in patients with decreased lower-limb strength. AIMS: Here we examined the effect of hand support on leg muscle activity and body sway during OLS in elderly subjects able to maintain OLS for 1 min unaided [able group (AG), n = 13] and those who were unable to do so [unable group (UG), n = 11]. METHODS: All subjects performed OLS unaided and OLS with front support (OLS-FS) using one hand for 1 min each. We estimated leg muscle activity [mean and maximum % root mean square (%RMS)] and body sway (total, X-axis, and Y-axis path lengths) for both tests. %RMS was calculated according to the results of the maximum voluntary isometric contraction test. RESULT: The overall average mean and maximum %RMS for the tibialis anterior muscle was larger in UG than in AG. In AG, tibialis anterior muscle mean and maximum %RMS and body sway was larger during OLS than during OLS-FS. Total and X-axis path lengths were larger during the first 20 s OLS phase in AG and the first 20 s OLS-FS phase in UG. CONCLUSION: These results highlight the need to differentiate between patients able and unable to perform OLS unaided for training because of differences in leg muscle activity.

Stimulus tempos and the reliability of the successive choice reaction test.

In ball game sports, players demand agility, which is a quick and adequate reaction to various changing stimuli. We developed a "successive choice reaction test" that evaluates such agility. This test requires subjects to be exposed to successive stimuli. Hence, it is very important to select appropriate stimulus tempos. In addition, it is necessary to examine the reliability of a new test. This study examined adequate stimulus tempos and test reliability. Fifteen healthy university students participated in this study. All the subjects conducted a successive choice reaction test with 3 kinds of tempos (1.3, 1.5, and 2.0 seconds). To examine its reliability, the test was conducted again on another day. It was found that any tempo has a high reliability, that their relationships are close, and insignificant time differences exist among the tempos. In conclusion, the successive choice reaction test was judged to be useful with any of the above tempos.

SRT1720 induces SIRT1-independent cell death in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) develops after a long period of human T-cell leukemia virus (HTLV)-1 infection and is associated with host aging in addition to genetic abnormalities in HTLV-1 infected cells. SIRT1 is a histone deacetylase involved in cell cycle and apoptosis. We previously showed the high expression of SIRT1 protein in peripheral blood mononuclear cells from patients with ATL. There have been many reports that SIRT1 inhibitors show tumor-suppressive effects. On the other hand, SIRT1 activator SRT1720 induces the cell death of multiple myeloma and breast cancer cells. However, the effect of SRT1720 on ATL is unknown. This study aimed to evaluate the effect of SRT1720 on cell death in leukemic cell lines in vitro and freshly isolated ATL cells ex vivo and in an ATL in vivo mouse model. SRT1720 reduced cell viability in vitro and ex vivo. Additionally, SRT1720 increased the number of apoptotic cells, as shown by annexin V positive cells, cleaved poly (ADP-ribose) polymerase 1, cleaved caspase-3, and fragmented DNA. SRT1720 also induced mitochondrial outer membrane permeabilization with the generation of mitochondrial reactive oxygen species and autophagy. However, SIRT1 knockdown did not attenuate SRT1720-induced cell death in leukemic cell lines. Finally, SRT1720 treatment decreased the growth of human ATL tumor xenografts in immunodeficient mice. Our study shows that while SRT1720 does not target SIRT1, it induces cell death in ATL cells via apoptosis and autophagy and has antitumor activity.

Effectiveness of the 1RM estimation method based on isometric squat using a back-dynamometer.

This study aimed to clarify the relationships between isometric squat (IS) using a back dynamometer and 1 repetition maximum (1RM) squat for maximum force and muscle activities and to examine the effectiveness of a 1RM estimation method based on IS. The subjects were 15 young men with weight training experience (mean age 20.7 ± 0.8 years, mean height 171.3 ± 4.4 cm, mean weight 64.4 ± 8.4 kg). They performed the IS with various stance widths and squat depths. The measured data of exerted maximum force and the action potential of the agonist muscles were compared with the 1RM squat data. The exerted maximum force during IS was significantly larger in wide stance (140% shoulder width) than in narrow stance (5-cm width). The maximum force was significantly larger with decreased knee flexion. As for muscle activity, the % root mean square value of muscle electric potential of the rectus femoris and the vastus lateralis tended to be higher in wide stance. As for exerted maximum force, wide stance and parallel depth in IS showed a significant and high correlation (r = 0.73) with 1RM squat. Simple linear regression analysis revealed a significant estimated regression equation [Y = 0.992X + 30.3 (Y:1RM, X:IS)]. However, the standard error of an estimate value obtained by the regression equation was very large (11.19 kg). In conclusion, IS with wide stance and parallel depth may be useful for the estimation of 1RM squat. However, estimating a 1RM by IS using a back dynamometer may be difficult.

Amyloid-specific extraction using organic solvents.

Typing of amyloidosis by mass spectrometry (MS) based proteomic analysis contribute to the diagnosis of amyloidosis. For MS analysis, laser microdissection (LMD) is used for amyloid specific sampling. This study aimed to establish a method for selectively extracting amyloids from formalin-fixed, paraffin-embedded (FFPE) specimens by organic solvent instead of LMD. The extracts using dimethyl sulfoxide (DMSO), dimethylformamide (DMF), methanol, trifluoroethanol (TFE) or hexafluoro-2-propanol from FFPE brain of alzheimer's disease mouse model generated protein bands on SDS-PAGE, and Aß was identified in the extract of DMF using mass spectrometry. The extract using DMSO from the kidney of a AA amyloidosis patient produced a protein band in SDS-PAGE. This protein band was identified to be serum amyloid A (SAA) by Western blotting and mass spectrometry. Circular dichroism spectrometry revealed that the secondary structures of Aß and transthyretin were converted to α-helices from ß-sheets in TFE. Our results suggest that organic solvents can extract amyloids from FFPE specimens by converting their secondary structure. This method could eliminate the LMD step and simplified amyloid typing by MS analysis. •DMSO, DMF, methanol, TFE and HFIP can extract Aß specifically from the FFPE brain of a Alzheimer' disease mouse model.•DMSO can extract SAA specifically from a FFPE section of AA amyloidosis.•Secondary structures of Aß and transthyretin converted from ß-sheet to α-helix in TFE.

High expression of NAMPT in adult T-cell leukemia/lymphoma and anti-tumor activity of a NAMPT inhibitor.

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature T lymphocytes induced by human T-cell leukemia virus-1 and has a poor outcome. New molecular targets for the prevention and treatment of ATL are needed urgently. We previously reported high expression of Sirtuin 1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone/protein deacetylase, in primary acute-type ATL cells. NAD+ biosynthesis via nicotinamide phosphoribosyltransferase (NAMPT) modulates Sirtuin 1 activity. Here, we examined the expression and effects of inhibiting NAMPT, a rate-limiting enzyme in NAD+ biosynthesis, in ATL cells. We found that peripheral blood mononuclear cells from patients with acute-type ATL expressed significantly higher levels of NAMPT protein than cells from healthy subjects. FK866, a NAMPT inhibitor, induced apoptosis of freshly isolated ATL cells ex vivo and HTLV-1-infected T-cell lines in vitro, which was accompanied by activation of caspases, DNA fragmentation, and disruption of mitochondrial transmembrane potential. However, a pan-caspase inhibitor failed to prevent this FK866-induced cell death, while FK866 increased the caspase-independent cell death mediator endonuclease G. Intriguingly, FK866 also activated autophagy, as demonstrated by increases in protein levels of autophagosome marker LC3-II. Thus, FK866 simultaneously activated apoptosis and autophagy. Finally, FK866 treatment markedly decreased the growth of human ATL tumor xenografts in immunodeficient mice. We showed that NAMPT is highly expressed in primary ATL cells ex vivo, and that FK866 induces autophagy and caspase-dependent and -independent cell death pathways in vitro and has an anti-tumor activity in vivo. These results suggest a novel therapeutic strategy for patients with this fatal disease.

Body sway and muscle activity during one-leg stance with help using a hand.

The authors examined the effects of the direction (front or lateral) and method (light touch [LT] or support [LS]) of hand help on body sway and muscle activity during 1-leg stance (OLS). Fifteen subjects performed OLS with hand help under 4 conditions (front LT, front LS, lateral LT, lateral LS) after conducting normal OLS. Center of pressure (COP) path length and electromyography (EMG) were recorded. When using the lateral help, the COP and EMG were significantly smaller with LS than with LT. When using the front help, a difference in help methods did not affect the COP, but the EMG of gastrocnemius was significantly smaller with LS than with LT. In conclusion, body sway and muscle activity during OLS markedly decrease with lateral LS.

Sex and age-level differences of walking time in preschool children on an obstacle frame.

BACKGROUND: Stepping over an obstacle is a kind of compound movement that makes walking more difficult, especially for preschool children. This study examines sex and age-level differences in walking time in preschool children on an obstacle frame. METHODS: The participants included 324 healthy preschool children: four-year-old boys (51) and girls (51), five-year-old boys (50) and girls (60), and six-year-old boys (62) and girls (50). A 5 cm- or 10 cm-high obstacle (depth 11.5 cm, width 23.5 cm) was set at the halfway point of a 200 cm × 10 cm walking course. RESULTS: The participants walked to the end of the course and back as fast as possible under three conditions: no obstacle, low obstacle and high obstacle. Walking time showed age-level differences in all conditions, but there were no differences in sex. Age levels were divided into two groups, with one group within the first six months of their birthday, and the second group within the last six months of that year. Walking time for children in the first half of their fourth year was longer than that of the five- and six-year-old children. In addition, for children in the last half of their fourth year, walking time was longer than both sexes in the last half of their fifth and sixth years. The children in the latter half of their fifth year had a longer walking time in the high obstacle condition than those in the last half of their sixth year. In the four-year-old participants, walking time was shorter with no obstacles than with a high obstacle frame. CONCLUSIONS: In the above data, obstacle course walking time does not show a gender difference, except that the four-year-old participants needed longer than the five- and six-year-old children. Setting the obstacle 10 cm high also produced a different walking time in the five- and six-year-old participants. The high obstacle step test (10 cm) best evaluated the dynamic balance of preschool children.