Associations between 24-h movement behaviors and self-rated health: a representative sample of school-aged children and adolescents in Okinawa, Japan.

OBJECTIVES: This study aimed to determine the associations between adherence to 24-h movement behavior guidelines and self-rated health (SRH) among Japanese adolescents according to their age group. STUDY DESIGN: This was a cross-sectional study. METHODS: Probability proportional sampling data, which were collected from six regions of Okinawa Prefecture, Japan, considering the number of schools, included 2408 fifth-grade students (aged 10-11 years) in 31 elementary schools and 4360 eighth-grade students (aged 13-14 years) in 30 junior high schools. SRH, moderate-to-vigorous physical activity (MVPA), screen time (ST), sleep duration, and confounding factors (sex, weight status, family affluence, parental support, school satisfaction, and school demands) were self-reported. RESULTS: The logistic regression models showed that adherence to ST and sleep recommendations in elementary school students was associated with a high prevalence of good health only, whereas adherence to only MVPA, only sleep, ST and sleep, MVPA and sleep, and all three recommendations were associated with a high prevalence of good health among junior high school students. All combinations that included achievement of the recommended sleep duration were associated with SRH. CONCLUSIONS: Achieving 24-h movement behavior guidelines, particularly sleep recommendations, is associated with better perceived health in school-aged children, especially in adolescents.

Nerve growth factor regulation and production by macrophages in osteoarthritic synovium.

Nerve growth factor (NGF) functions to modulate osteoarthritis (OA)-associated pain. Although recent studies suggest that tumour necrosis factor (TNF)-α and interleukin (IL)-1ß mediate NGF activity in human synovial fibroblasts, the regulation of NGF expression in human synovial macrophages remains unclear. Here, we examined the role of macrophages in the production and regulation of synovial (SYN) NGF in osteoarthritic knee joints by examining the mRNA expression of TNF-α and IL-1ß in freshly isolated CD14-positive (macrophage-rich fraction) and CD14-negative cells (fibroblast-rich fraction) in synovial tissue from OA patients by quantitative polymerase chain reaction. We also examined the effects of IL-1ß and TNF-α on NGF mRNA expression in cultured CD14-positive (macrophage-rich fraction) and CD14-negative cells (fibroblast-rich fraction). In addition, to examine the contribution of macrophages to NGF, TNF-α and IL-1ß expression, we injected clodronate liposomes systemically into STR/Ort mice, an osteoarthritis animal model, to deplete macrophages. TNF-α and IL-1ß mRNA levels in CD14-positive cells from the SYN of OA patients was significantly higher than that in CD14-negative cells, while NGF expression did not differ markedly between the two cell fractions. In addition, treatment of human cultured CD14-positive and -negative cells with IL-1ß and TNF-α enhanced NGF mRNA and protein levels. Expression of NGF, IL-1ß and TNF-α was also reduced significantly in STR/Ort mice upon macrophage depletion. These findings suggest that IL-1ß and TNF-α regulate NGF expression and production in synovial macrophages and fibroblasts in osteoarthritic joints.

Site-Specific Quantification of Lysine Acetylation Using Isotopic Labeling.

Acetylation of ɛ-amino group of lysine is one of the most common protein posttranslational modifications. The modification is reversible and catalyzed by lysine acetyltransferases in one direction and lysine deacetylases in the other direction. Although numerous lysine acetylation sites have been identified in many proteins involved in a diverse range of cellular processes, little has been revealed about the roles of this modification at the level of individual sites. To understand better the site-specific roles of this modification, it is important to investigate what fraction of each modified site is actually acetylated (stoichiometry of acetylation) in vivo in different physiological conditions. Here we describe a method that allows us to determine the site-specific stoichiometry of lysine acetylation. The method chemically acetylates all of the lysine residues in proteins that are not endogenously acetylated with an isotopically labeled acetyl (13C2-acetyl) group. The chemical treatment enables to determine the stoichiometry of acetylation at individual sites by measuring the abundance of the endogenously acetylated group (carrying a natulally abundant 12C2-acetyl group) and the chemically introduced acetyl group (carrying an isotopically labeled 13C2-acetyl group) in the subsequent mass spectrometry analysis. The method is most suitable to apply to pure proteins or relatively simple protein mixtures.

Monitoring Protein Synthesis in Caenorhabditis elegans Using SILAC.

The static levels of proteins are the net results of their production and clearance regulated by the activities of proteins involved in their synthesis, degradation, and transportation. Therefore, the information on the rates of protein synthesis and clearance is needed to understand the underlying dynamic nature of a proteome. In this chapter, the experimental technique, we use in our laboratory for monitoring the synthesis of individual proteins in Caenorhabditis elegans (C. elegans) is described. The technique utilizes a preisotopically labeled amino acid (13C6-Lys) as a precursor for protein synthesis and monitors the kinetics of the precursor incorporation into the newly synthesized proteins. C. elegans is a powerful animal model in various fields of biomedical science such as aging, developmental biology, and neurobiology. The experimental technique would, therefore, be useful for research laboratories using C. elegans as an animal model.

Synovial macrophage-derived IL-1ß regulates the calcitonin receptor in osteoarthritic mice.

Recent studies have reported that calcitonin gene-related peptide (CGRP) contributes to joint pain. However, regulation of the CGRP/CGRP receptor signalling in osteoarthritis (OA) is not fully understood. To investigate the regulation of CGRP/CGRP receptor signalling by macrophages in the synovial tissue (ST) of OA joints, we characterized the gene expression profiles of CGRP and CGRP receptors in the ST of OA mice (STR/Ort). In addition, we examined whether macrophage depletion by the systemic injection of clodronate-laden liposomes affected the expression of CGRP and CGRP receptors in ST. CD11c(+) macrophages in the ST of STR/Ort and C57BL/6J mice were analysed by flow cytometry. Real-time polymerase chain reaction (PCR) was used to evaluate the expression of interleukin (IL)-1ß, CGRP, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in F4/80(+) and F4/80(-) cells. The effects of IL-1ß on the expression of CGRP and CLR by cultured synovial cells were also examined. The percentage of CD11c(+) macrophages in the ST of STR/Ort was higher than that in C57/BL6J mice. Notably, the F4/80(+) cell fraction expressed IL-1ß highly, whereas the F4/80(-) cell fraction expressed CGRP, CLR, and RAMP1 highly. In addition, expression of the IL-1ß and CLR genes was increased in ST, but was decreased upon macrophage depletion, and the IL-1ß treatment of cultured synovial cells up-regulated CLR. Taken together, the present findings suggest that synovial macrophages are the major producers of IL-1ß and regulators of CLR in OA mice. Therefore, macrophages and IL-1ß may be suitable therapeutic targets for treating OA pain.

CD11c(+) macrophages and levels of TNF-α and MMP-3 are increased in synovial and adipose tissues of osteoarthritic mice with hyperlipidaemia.

To understand more clearly the link between osteoarthritis and hyperlipidaemia, we investigated the inflammatory macrophage subsets and macrophage-regulated matrix metalloprotease-3 (MMP-3) and A disintegrin and metalloprotease with thrombospondin motifs-4 (ADAMTS4) in synovial (ST) and adipose tissues (AT) of osteoarthritic mice with hyperlipidaemia (STR/Ort). CD11c(+) F4/80(+) CD11b(+) macrophage populations in the ST and AT of 9-month-old STR/Ort and C57BL/6J mice were characterized and compared by flow cytometry and real-time polymerase chain reaction (PCR) analyses. Expression of tumour necrosis factor (TNF)-α, MMP-3 and ADAMTS4, and the response of these factors to anionic liposomal clodronate induced-macrophage depletion were also evaluated by real-time PCR. Expression of TNF-α in CD11c(+) cells, which were isolated by magnetic beads, was compared to CD11c(-) cells. In addition, the effect of TNF-α on cultured synovial fibroblasts and adipocytes was investigated. CD11c(+) F4/80(+) CD11b(+) macrophages were increased in ST and AT of STR/Ort mice. The CD11c(+) cell fraction highly expressed TNF-α. Expression of TNF-α and MMP3 was increased in ST and AT, and was decreased upon macrophage depletion. TNF-α treatment of cultured synovial fibroblasts and adipocytes markedly up-regulated MMP-3. CD11c(+) F4/80(+) CD11b(+) macrophages were identified as a common inflammatory subset in the AT and ST of STR/Ort mice with hyperlipidaemia. The induction of inflammation in AT and ST may be part of a common mechanism that regulates MMP3 expression through TNF-α. Our findings suggest that increased numbers of CD11c(+) macrophages and elevated levels of TNF-α and MMP-3 in AT and ST may explain the relationship between hyperlipidaemia and OA.

[Decrease in allogenic transfusions due to the spread of use of postoperative retransfusion systems in knee replacement surgery]. / Disminución de las transfusiones alogénicas por la generalización del uso de recuperadores de sangre postoperatorios en cirugía de prótesis de rodilla.

OBJECTIVES: Surgical teams have several tools in order to reduce the need for postoperative allogenic transfusion. Postoperative autotransfusion of unwashed shed blood has become common practice for total knee replacement surgery since 2006 in our hospital. This study was designed to evaluate if this practice has reduced allogenic blood transfusions. MATERIAL AND METHODS: A retrospective study comparing two cohorts, group 2004 with patients operated on for total knee replacement during the year 2004, before the use of the retransfusion system, and group 2008, patients operated on in the year 2008, with regular use of the retransfusion system. Gender, preoperative and postoperative haemoglobin levels, total amount of calculated erythrocytes lost, reinfusion of shed blood and allogenic blood transfusion during hospital stay were recorded. RESULTS: Both groups were similar as regards gender, preoperative and postoperative hemoglobin levels, and total amount of erythrocytes lost. The proportion of transfused patients was significantly lower in group 2008 versus group 2004 (20.18% versus 42.19%), with a relative risk of being transfused of 0.47 and a NNT of 4.54. P=.0017. CONCLUSIONS: In our hospital the use of postoperative retransfusion systems has reduced the proportion of transfused patients during hospitalization for total knee replacement surgery, although this result cannot be generalized due to the lack of a fixed transfusion trigger.

BMP2, BMP4, noggin, BMPRIA, BMPRIB, and BMPRII are differentially expressed in the adult rat spinal cord.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor ß (TGF-ß) superfamily. BMPs, such as BMP2 and BMP4, exert its biological functions by interacting with membrane bound receptors belonging to the serine/threonine kinase family including bone morphogenetic protein receptor I (BMPRIA, BMPRIB) and type II (BMPRII). Functions of BMPs are also regulated in the extracellular space by secreted antagonistic regulators such as noggin. Although BMP2, BMP4, noggin, BMPRIA, BMPRIB, and BMPRII expressions have been well described in the central nervous system, little information is available for their expressions in the spinal cord. We, thus, investigated these protein expressions in the adult rat spinal cord using immunohistochemistry. Here, we show that BMP2, BMP4, noggin, BMPRIA, BMPRIB, and BMPRII are widely and differentially expressed in the spinal cord. Besides abundant BMP2, BMP4, noggin, BMPRIA, BMPRIB, and BMPRII protein expressions in neurons, we detected them also in astrocytes, oligodendrocytes, and ependymal cells. In addition, we found BMPRIA, BMPRIB, and BMPRII protein expressions in microglia. Interestingly, we also observed that these proteins are strongly expressed in many kinds of axons in both ascending and descending tracts. These data indicate that BMP2, BMP4, noggin, BMPRIA, BMPRIB, and BMPRII proteins are more widely expressed in the adult spinal cord than previously reported, and their continued abundant expressions in the adult spinal cord strongly support the idea that BMP signaling plays pivotal roles in the adult spinal cord.

Quantitative evaluation and visualization of lumbar foraminal nerve root entrapment by using diffusion tensor imaging: preliminary results.

BACKGROUND AND PURPOSE: DTI can provide valuable structural information that may become an innovative tool in evaluating lumbar foraminal nerve root entrapment. The purpose of this study was to visualize the lumbar nerve roots and to measure their FA in healthy volunteers and patients with lumbar foraminal stenosis by using DTI and tractography with 3T MR imaging. MATERIALS AND METHODS: Eight patients with lumbar foraminal stenosis and 8 healthy volunteers underwent 3T MR imaging. In all subjects, DTI was performed with echo-planar imaging at a b-value of 800 s/mm(2) and the lumbar nerve roots were visualized with tractography. Mean FA values in the lumbar nerve roots were quantified on DTI images. RESULTS: In all subjects, the lumbar nerve roots were clearly visualized with tractography. In all patients, tractography also showed abnormalities such as tract disruption, nerve narrowing, and indentation in their course through the foramen. Mean FA values were significantly lower in entrapped roots than in intact roots. CONCLUSIONS: We demonstrated that DTI and tractography of human lumbar nerves can visualize and quantitatively evaluate lumbar nerve entrapment with foraminal stenosis. We believe that DWI is a potential tool for the diagnosis of lumbar nerve entrapment.

Ototoxicity of Povidone-Iodine applied to the middle ear cavity of guinea pigs.

OBJECTIVE: Povidone-Iodine preparation is used as a disinfectant in otological surgeries. The ototoxicity of Povidone-Iodine preparation was evaluated using infant, young and adult guinea pigs. The effects of different concentrations and of different exposure durations on compound action potentials were also studied. MATERIALS & METHODS: Povidone-Iodine was used to fill one middle ear cavity of the guinea pig, and the compound action potential (CAP) was measured from the round window membrane at 24h, 7 days, and 28 days. The contralateral side was filled with saline as control. Test sounds used were clicks and tone bursts of 2, 4, and 8 kHz. RESULTS: At 24h, Povidone-Iodine solution showed a significant toxic effect in the infant group. In the young animal group, no toxic effect was seen. In the adult group, a mild degree of deafness for 2 kHz was found. At 7 days, the young group showed significant hearing loss for all frequencies, but the adult group did not show any hearing loss. With a half strength solution, both young and adult group did not show hearing loss. At 28 days, with a full strength solution, hearing loss became prominent for all sound stimulation. With 1/8th dilution, the young group showed a moderate hearing loss, but the adult group did not. CONCLUSION: The thicker round window membrane in human is expected to provide more protection to the human cochlea than in the guinea pig model that we have studied. Mild hearing loss at 24h and 7 days using 10% solution, but no hearing loss with 5% solution at 7 days may indicate that rinsing of the middle ear cavity with saline during surgery should minimize the ototoxic effect of this product. The age of the animals does influence the outcome of the ototoxicity experiment. From this experiment, Povidone-Iodine preparations in the infant should be used with caution. Povidone scrub should not be used for otologic surgery.

Bone morphogenetic protein receptor expressions in the adult rat brain.

Bone morphogenetic proteins (BMP) are members of the transforming growth factor ß (TGF-ß) superfamily. BMPs exert its biological functions by interacting with membrane bound receptors belonging to the serine/threonine kinase family including bone morphogenetic protein receptor I (BMPRIA, BMPRIB) and type II (BMPRII). Although BMPR expressions have been well described in the early development of the CNS, little information is available for their expressions in the adult CNS. We, thus, investigated BMPR expressions in the adult rat CNS using immunohistochemistry. Here, we show that BMPRIA, IB and II proteins are widely expressed throughout the adult CNS. Interestingly, we observed that BMPRIA, IB and II proteins are abundantly expressed in many kinds of axons. In addition, we found that BAMRIB-IR was preferentially expressed in dendrites of many neurons throughout the CNS, while BMPRIA was mainly expressed in cell bodies, showing that BMPRIA and BMPRIB are differentially targeted in a single neuron. In addition, besides abundant BMPR expressions in neurons, we exhibited BMPR expressions in astrocytes and ependymal cells. These data indicate that BMPRs are more widely expressed throughout the adult CNS than previously reported, and their continued abundant expressions in the adult brain strongly support the idea that BMPRs play pivotal roles also in the adult brain.

Safety of ofloxacin (OFLX) and fosfomycin sodium (FOM) ear drops.

OBJECTIVE: The objective of this study is to evaluate the safety of two ear drops, Ofloxacin (OFLX: Taribid Otic Solution, Daiichi Seiyaku) and Fosfomycin sodium (FOM: Fosmicin S, Meiji Seiyaku). METHODS: Albino guinea pigs were used as experimental animals, and the ototoxicity was evaluated by means of threshold changes in the compound action potentials (CAP), when topically applied to the middle ear cavity of the guinea pig. The sound stimuli applied were; click sound, with tone bursts of 8 kHz, 4 kHz, and 2 kHz. In one group of animals, after one application of the ear drops in the right middle ear cavity, the change in CAP was compared with a contralateral saline control at 24h, one week, and four weeks. In other group of animals, the ear drops were applied into the middle ear cavity for seven consecutive days and the CAP was measured at 24h. RESULTS: At 24h the CAP threshold for click, 8 and 4 kHz elevated significantly for both the saline and ear drop treatment, but the threshold returned to normal when measured at 7 days and 28 days. Seven consecutive days of ear drops administration resulted in no reduction in the CAP for either ear drops. CONCLUSIONS: Based on the lack of changes in the CAP, these two ear drops studied did not show any significant ototoxicities.

Use of hollow-core fibers to deliver nanosecond Nd:YAG laser pulses to form sparks in gases.

We report what is to our knowledge the first delivery of nanosecond laser pulses through flexible fibers to produce optical sparks in atmospheric-pressure gases. Our work employs a Nd:YAG laser beam (1.064 microm) delivered through a cyclic olefin polymer-coated silver hollow fiber. We studied the beam properties at the fiber exit as a function of the fiber launch geometry. We found that for a low-angle launch (approximately 0.01 rad half-angle), the exit beam has relatively high optical intensity (approximately 2 GW/cm2) and low light divergence (approximately 0.01 rad half-angle) and allows downstream spark formation. The effect of fiber bending on the exit beam and on the ability to make sparks is also investigated.

Reversibility from delayed hyperacute rejection in ABO-incompatible renal transplantation: histopathological findings of renal allograft biopsy.

ABO-incompatible renal transplantation (ABOIRTx) tend to lead to blood type antibody-mediated rejection, the so-called delayed hyperacute rejection (DHAR), which results in short-term graft loss. To clarify the accurate incidence and prognostic value of DHAR among ABOIRTx, we reviewed biopsy specimens obtained from ABOKTx allografts with abrupt dysfunction during the early period after transplantation. Among 74 ABOIRTx patients, 34 patients displayed allograft dysfunction within 14 days following transplantation. The biopsy specimens were classified based on the Banff schema. The pathological diagnosis of ABO blood type antibody-mediated humoral rejection (ABO-AMHR) was made by the following 3 findings: Specimens with all of above-mentioned findings were categorized as severe ABO-AMHR; those with at least one findings, were categorized as mild ABO-AMHR. All patients were treated with steroid pulse therapy and/or modification of other immunosuppressants. Group 1 consisted of severe ABO-AMHR (n = 6); group 2 consisted of mild ABO-AMHR (n = 5); group 3 consisted of acute cellular rejection (n = 3); group 4 consisted of recovery phase of ATN (n = 11); group 5 consisted of calcineurin inhibitor toxicity (n = 2); and group 6 consisted of normal histology (n = 5). One of 6 patients (16%) in group 1 lost the graft because of DHAR irreversible by antirejection and anticoagulation therapy. However, there has been no clear definition of histpathological criteria for DHAR after ABO-incompatible kidney transplantation. The definition must prognosticate whether the rejection process is reversible.

Baseline glomerular sclerosis influences morphological changes, but not level of serum creatinine.

The aim of this study was to investigate whether glomerular sclerosis (GS) at the time of engraftment affects subsequent morphology and clinical course of renal allografts. Eighty-one renal transplant recipients were recruited for this study. Protocol biopsies of the renal allografts were performed at engraftment, as well as at 1, 3, 5, and 7 years after transplantation. All cases were divided into 2 groups based on the presence of GS at engraftment, namely, non-GS and GS groups. Morphological changes in the renal allografts were graded from 0 to 3+ based on the severity of chronic allograft nephropathy (CAN) of the Banff classification based on 5 factors: percentage of GS, extent of interstitial fibrosis, tubular atrophy, arterial intimal thickening, and arteriolar hyalinosis. Furthermore, the level of serum creatinine (s-Cr) at each year was examined by recipient age and gender, donor age and gender, type of donor (living/cadaver), delayed graft function, acute rejection within 1 year after transplantation, mean blood pressure, and use of calcineurin inhibitors as well as the presence of GS at engraftment. The extent of GS at engraftment significantly correlated with donor age (P = .0038) but with a weak correlation coefficient. Although the severity of CAN developed gradually in both non-GS and GS groups, differences in morphological changes at engraftment between the 2 groups persisted throughout 7 years. Donor age and recipient gender influenced s-Cr significantly. In conclusion, the presence of GS at engraftment aggravates subsequent morphological changes and affects short-term but not long-term allograft prognosis.

Construction of bacterial artificial chromosome libraries and their application in developing PCR-based markers closely linked to a major locus conditioning bruchid resistance in mungbean (Vigna radiata L. Wilczek).

Bacterial artificial chromosome (BAC) libraries have been widely used in different aspects of genome research. In this paper we report the construction of the first mungbean (Vigna radiata L. Wilczek) BAC libraries. These BAC clones were obtained from two ligations and represent an estimated 3.5 genome equivalents. This correlated well with the screening of nine random single-copy restriction fragment length polymorphism probes, which detected on average three BACs each. These mungbean clones were successfully used in the development of two PCR-based markers linked closely with a major locus conditioning bruchid (Callosobruchus chinesis) resistance. These markers will be invaluable in facilitating the introgression of bruchid resistance into breeding programmes as well as the further characterisation of the resistance locus.

Proteomic method identifies proteins nitrated in vivo during inflammatory challenge.

Inflammation in asthma, sepsis, transplant rejection, and many neurodegenerative diseases associates an up-regulation of NO synthesis with increased protein nitration at tyrosine. Nitration can cause protein dysfunction and is implicated in pathogenesis, but few proteins that appear nitrated in vivo have been identified. To understand how this modification impacts physiology and disease, we used a proteomic approach toward targets of protein nitration in both in vivo and cell culture inflammatory disease models. This approach identified more than 40 nitrotyrosine-immunopositive proteins, including 30 not previously identified, that became modified as a consequence of the inflammatory response. These targets include proteins involved in oxidative stress, apoptosis, ATP production, and other metabolic functions. Our approach provides a means toward obtaining a comprehensive view of the nitroproteome and promises to broaden understanding of how NO regulates cellular processes.

Evaluation of the epidermal growth factor receptor (EGFR) and c-erbB-2 in superspreading-type and penetrating-type gastric carcinoma.

The expression of epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), transforming growth factor alpha (TGF alpha), and of c-erbB-2 was immunohistochemically investigated in resected gastric carcinoma (in 39 cases of superspreading type and in 11 cases of penetrating type), to understand the differential biological features of these two types of gastric carcinoma. EGF, EGFR and c-erbB-2 positive cases were preferentially found in penetrating type rather than in superspreading type (p < 0.05, p < 0.01, and p < 0.05, respectively). The positive rates of EGFR and c-erbB-2 were significantly higher in submucosal gastric carcinoma than in intramucosal gastric carcinoma (p < 0.01, p < 0.05, respectively). These results suggested that the autocrine mechanism of the growth factors and the expression of c-erbB-2 were correlated to the degree of gastric wall invasion.

Angiotensin-converting enzyme (ACE) I/D genotype and renal ACE gene expression.

BACKGROUND: The angiotensin-converting enzyme (ACE) I/D genotype affects serum ACE levels and the onset and progression of renal disease, but little is known about the mechanism. We investigated a possible association between the ACE I/D genotype and renal ACE mRNA levels in healthy subjects. METHODS: Renal biopsy samples were obtained from 50 healthy kidney donors. The ACE I/D genotype was determined by polymerase chain reaction (PCR). Renal ACE mRNA quantification was performed by competitive RNA-PCR. In situ hybridization (ISH) for ACE mRNA on renal biopsy specimens was also performed. RESULTS: The number of ACE transcripts in 100 ng of total RNA was significantly (P < 0.01) lower in subjects with II genotype (5.6 +/- 5.3 x 10(5), N = 20) compared with those with the ID (17.9 +/- 13.6 x 10(5), N = 23) or the DD genotype (36.9 +/- 14.6 x 10(5), N = 7) in healthy donors. The ISH studies showed that both tubular and glomerular ACE mRNA expressions were weak in subjects with the II genotype, intermediate in subjects with ID genotype, and strong in subjects with DD genotype. CONCLUSIONS: It is suggested that renal ACE gene expression is associated with the ACE I/D genotype in healthy Japanese subjects.