The mechanical stimulation of cells in 3D culture within a self-assembling peptide hydrogel

The aim of this present study was to provide a scaffold as a tool for the investigation of the effect of mechanical stimulation on three-dimensionally cultured cells. For this purpose, we developed an artificial self-assembling peptide (SPG-178) hydrogel scaffold. The structural properties of the SPG-178 peptide were confirmed by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and transmission electron microscopy (TEM). The mechanical properties of the SPG-178 hydrogel were studied using rheology measurements. The SPG-178 peptide was able to form a stable, transparent hydrogel in a neutral pH environment In the SPG-178 hydrogel, mouse skeletal muscle cells proliferated successfully (increased by 12.4 +/- 1.5 times during 8 days of incubation; mean +/- SEM). When the scaffold was statically stretched, a rapid phosphorylation of ERK was observed (increased by 2.8 +/- 0.2 times; mean +/- SEM). These results demonstrated that the developed self-assembling peptide gel is non-cytotoxic and is a suitable tool for the investigation of the effect of mechanical stimulation on three-dimensional cell culture

Marine integrons containing novel integrase genes, attachment sites, attI, and associated gene cassettes in polluted sediments from Suez and Tokyo Bays

In order to understand the structure and biological significance of integrons and associated gene cassettes in marine polluted sediments, metagenomic DNAs were extracted from sites at Suez and Tokyo Bays. PCR amplicons containing new integrase genes, intI, linked with novel gene cassettes, were recovered and had sizes from 1.8 to 2.5 kb. This approach uncovered, for the first time, the structure and diversity of both marine integron attachment site, attI, and the first gene cassette, the most efficiently expressed integron-associated gene cassette. The recovered 13 and 20 intI phylotypes, from Suez and Tokyo Bay samples, respectively, showed a highly divergence, suggesting a difference in integron composition between the sampling sites. Some intI phylotypes showed similarity with that from Geobacter metallireducens, belonging to Deltaproteobacteria, the dominant class in both sampling sites, as determined by 16S rRNA gene analysis. Thirty distinct families of putative attI site, as determined by the presence of an attI-like simple site, were recovered. A total of 146 and 68 gene cassettes represented Suez and Tokyo Bay unsaturated cassette pools, respectively. Gene cassettes, including a first cassette, from both sampling sites encoded two novel families of glyoxalase/bleomycin antibiotic-resistance protein. Gene cassettes from Suez Bay encoded proteins similar to haloacid dehalogenases, protein disulfide isomerases and death-on-curing and plasmid maintenance system killer proteins. First gene cassettes from Tokyo Bay encoded a xenobiotic-degrading protein, cardiolipin synthetase, esterase and WD40-like β propeller protein. Many of the first gene cassettes encoded proteins with no ascribable function but some of them were duplicated and possessed signal functional sites, suggesting efficient adaptive functions to their bacterial sources. Thus, each sampling site had a specific profile of integrons and cassette types consistent with the hypothesis that the environment shapes the genome

コウレイシャ テンカン ノ モンシンヒョウ ニヨル ソウキ ハッケン : ニンチショウ トノ カンレン ニオイテ

本研究の目的は高齢者てんかんを問診票の活用により、物忘れ外来を含む高齢者複合施設における高齢者てんかんの実態を明らかにすることにある。物忘れ外来を含む高齢者複合施設を調査期間中に利用した417名を対象に、高齢者てんかん問診票を用い、てんかん有病率を算出した。また、新規に高齢者てんかんと判定された者の特徴を記述した。その結果、すでにてんかんの診断ありが7名（1.7%）、問診票の回答内容からてんかんの疑いありは33名（7.9%）、その中から医師の診断により新規に高齢者てんかんが判定された者は14名（3.0%）であった。施設別有病率では、物忘れ外来7名（5.0%）、ショートステイ利用者、グループホーム入所者は2割前後であった。新規に高齢者てんかんと判定された者の問診項目では、意識減損がもっとも多かった。さらに、新規にてんかんが判明したすべての者が何らかの認知症を有していた。本研究により物忘れ外来を含む高齢者複合施設において、高齢者てんかんが潜在していることが明らかとなり、これらの早期発見のため高齢者てんかんの問診票の必要性が示された

Rab39a Interacts with Phosphatidylinositol 3-Kinase and Negatively Regulates Autophagy Induced by Lipopolysaccharide Stimulation in Macrophages

<div><p>Rab39a has pleiotropic functions in phagosome maturation, inflammatory activation and neuritogenesis. Here, we characterized Rab39a function in membrane trafficking of phagocytosis and autophagy induction in macrophages. Rab39a localized to the periphery of LAMP2-positive vesicles and showed the similar kinetics on the phagosome to that of LAMP1. The depletion of Rab39a did not influence the localization of LAMP2 to the phagosome, but it augments the autophagosome formation and LC3 processing by lipopolysaccharide (LPS) stimulation. The augmentation of autophagosome formation in Rab39a-knockdown macrophages was suppressed by Atg5 depletion or an inhibitor for phosphatidylinostol 3-kinase (PI3K). Immunoprecipitation analysis revealed that Rab39a interacts with PI3K and that the amino acid residues from 34^th to 41^st in Rab39a were indispensable for this interaction. These results suggest that Rab39a negatively regulates the LPS-induced autophagy in macrophages.</p> </div

Prediction of 24-h and 6-h Periods before Calving Using a Multimodal Tail-Attached Device Equipped with a Thermistor and 3-Axis Accelerometer through Supervised Machine Learning

In this study, we developed calving prediction models for 24-h and 6-h periods before calving using data on physiological (tail skin temperature) and behavioral (activity intensity, lying time, posture change, and tail raising) parameters obtained using a multimodal tail-attached device (tail sensor). The efficiencies of the models were validated under tethering (tie-stall) and untethering (free-stall and individual pen) conditions. Data were collected from 33 and 30 pregnant cattle under tethering and untethering conditions, respectively, from approximately 15 days before the expected calving date. Based on pre-calving changes, 40 features (8 physiological and 32 behavioral) were extracted from the sensor data, and one non-sensor-based feature (days to the expected calving date) was added to develop models using a support vector machine. Cross-validation showed that calving within the next 24 h under tethering and untethering conditions was predicted with a sensitivity of 97% and 93% and precision of 80% and 76%, respectively, while calving within the next 6 h was predicted with a sensitivity of 91% and 90% and precision of 88% and 90%, respectively. Calving prediction models based on the tail sensor data with supervised machine learning have the potential to achieve effective calving prediction, irrespective of the cattle housing conditions

Aerobic exercise training-induced irisin secretion is associated with the reduction of arterial stiffness via nitric oxide production in adults with obesity

This study aimed to clarify whether muscle-derived irisin secretion induced by aerobic exercise training is involved in reduction of arterial stiffness via arterial nitric oxide (NO) productivity in obesity. In animal study, 16 Otsuka Long-Evans Tokushima Fatty (OLETF) rats with obesity were randomly divided into 2 groups: sedentary control (OLETF-CON) and 8-week aerobic treadmill training (OLETF-EX) groups. In human study, 15 subjects with obesity completed 8-week aerobic exercise training for 45 min at 60%–70% peak oxygen uptake intensity for 3 days/week. As a result of animal study, carotid-femoral pulse wave velocity (cfPWV) was decreased, and arterial phosphorylation levels of AMP-activated protein kinase (AMPK), protein kinase B (Akt), and endothelial NO synthase (eNOS), circulating levels of nitrite/nitrate (NOx) and irisin, and muscle messenger RNA expression of fibronectin type III domain containing 5 (Fndc5) were increased in the OLETF-EX group compared with OLETF-CON group. In a human study, regular aerobic exercise reduced cfPWV and elevated circulating levels of NOx and irisin. Furthermore, change in circulating irisin levels by regular exercise was positively correlated with circulating NOx levels and was negatively correlated with cfPWV. Thus, aerobic exercise training-induced increase in irisin secretion may be related to reduction of arterial stiffness achieved by NO production via activated arterial AMPK–Akt–eNOS signaling pathway in obesity. Novelty Aerobic exercise training promoted irisin secretion with upregulation of muscle Fndc5 gene expression in rats with obesity. Irisin affected the activation of arterial AMPK–Akt–eNOS signaling by aerobic exercise training. Increased serum irisin level by aerobic exercise training was associated with reduction of arterial stiffness in obese adults.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Augmentation of autophagy induced by LPS in Rab39a-KD macrophages.

<p>(A) Immunostaining analysis of LPS-induced autophagy in Rab39a-KD macrophages. Raw264.7 macrophages transfected with control or Rab39a siRNA were treated with LPS for 24 h and immunostained with anti-LC3 antibody. An arrowhead indicates an LC3-dot fluorescence. (B) Proportion of Raw264.7 macrophages with LC3-dots induced by LPS stimulation for 24 h. Data represent the mean and SD of three or four independent experiments. *<i>p</i> < 0.05 (unpaired Student’s <i>t</i>-test). (C) Immunoblot analysis of LC3 processing in macrophages treated with LPS. Raw264.7 macrophages transfected with control or Rab39a siRNA were treated with LPS for 24 h and subjected to immunoblot analysis using indicated antibodies. (D) Autophagic flux in Rab39a-KD macrophages treated with LPS. Raw264.7 macrophages treated with control or Rab39a siRNA were treated with LPS in the absence or presence of protease inhibitors, E64d and pepstatin A, for 24 h. Cell lysates were subjected to immunoblot analysis using indicated antibodies.</p

Autophagy induced by TLR2 or TLR7/8 ligand in Rab39a-KD macrophages.

<p>(A) Immunostaining analysis of Rab39a-KD macrophages stimulated by TLR2 or TLR7/8 ligand. Raw264.7 macrophages transfected with control or Rab39a siRNA were treated with TLR2 ligand Pam3CSK4 at 10 ng/ml or TLR7 ligand R848 at 1 μg/ml for 24 h and immunostained with anti-LC3 antibody. (B) Proportion of Raw264.7 macrophages with LC3-dots induced by Pam3CSK4 or R848 for 24 h. (C) Immunoblot analysis of LC3 processing in macrophages treated with Pam3CSK4 or R848. Raw264.7 macrophages transfected with control or Rab39a siRNA were treated with Pam3CSK4 or R848 for 24 h and subjected to immunoblot analysis using indicated antibodies. Data represent the mean and SD of three or four independent experiments. *<i>p</i> < 0.05 (unpaired Student’s <i>t</i>-test).</p

Interaction of Rab39a with Beclin1.

<p>(A) HEK293T cells were transfected with plasmids for Myc-Beclin1, Myc-Vps34, Myc-UVRAG or Myc-Atg14L and EGFP-Rab39a or EGFP. Whole cell lysates (WCL) were used for immunoprecipitation (IP) with anti-GFP antibody, followed by immunoblot analysis (IB) with anti-Myc antibody. For detection of input, aliquots of 15 μg of WCL were used. (B) ClustalW alignment of the amino acid sequences of Rab39a, Rab39a_M1 and Rab39b is shown. (C) HEK293T cells were transfected with plasmids for Myc-Beclin1 and EGFP-Rab39a, EGFP-Rab39a_M1, EGFP-Rab39b or EGFP. WCL were used for IP with anti-GFP antibody followed by IB with anti-Myc antibody. For detection of input, aliquots of 15 μg of WCL were used.</p