Fast Structure Determination of Electrode Surfaces for Investigating Electrochemical Dynamics Using Wavelength-Dispersive X‑ray Crystal Truncation Rod Measurements

Determining the atomic structure across electrolyte–electrode interfaces with a sufficient temporal resolution is crucial to understanding how electrochemical reactions proceed. Surface X-ray diffraction is a well-established method for determining interface structures at the atomic scale. However, existing measurement methods are often incapable of quantifying time-dependent structural changes during electrochemical processes because acquiring a diffraction rod profile sufficient for structure determination usually takes a longer time than the rate of the structural changes. This report demonstrates that the wavelength-dispersive method, which can acquire a range of the diffraction rod profile at once, is capable of the time-resolved analysis of electrochemical dynamics on a time scale of seconds and less, using electrochemical reactions on Pt(111) electrode surface as examples. In the case of the electrochemical oxidation of methanol, the quantitative analysis of the transient vertical displacement of the Pt(111) surface atomic layer gives evidence for a structural relaxation of the CO poisoning layer during its oxidative stripping. Present limitations and future prospects of the method are also discussed

Angiotensin II Receptor Blocker Ameliorates Stress-Induced Adipose Tissue Inflammation and Insulin Resistance

<div><p>A strong causal link exists between psychological stress and insulin resistance as well with hypertension. Meanwhile, stress-related responses play critical roles in glucose metabolism in hypertensive patients. As clinical trials suggest that angiotensin-receptor blocker delays the onset of diabetes in hypertensive patients, we investigated the effects of irbesartan on stress-induced adipose tissue inflammation and insulin resistance. C57BL/6J mice were subjected to 2-week intermittent restraint stress and orally treated with vehicle, 3 and 10 mg/kg/day irbesartan. The plasma concentrations of lipid and proinflammatory cytokines [Monocyte Chemoattractant Protein-1 (MCP-1), tumor necrosis factor-α, and interleukin-6] were assessed with enzyme-linked immunosorbent assay. Monocyte/macrophage accumulation in inguinal white adipose tissue (WAT) was observed with CD11b-positive cell counts and mRNA expressions of CD68 and F4/80 using immunohistochemistry and RT-PCR methods respectively. The mRNA levels of angiotensinogen, proinflammatory cytokines shown above, and adiponectin in WAT were also assessed with RT-PCR method. Glucose metabolism was assessed by glucose tolerance tests (GTTs) and insulin tolerance tests, and mRNA expression of insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) in WAT. Restraint stress increased monocyte accumulation, plasma free fatty acids, expression of angiotensinogen and proinflammatory cytokines including MCP-1, and reduced adiponectin. Irbesartan reduced stress-induced monocyte accumulation in WAT in a dose dependent manner. Irbesartan treatment also suppressed induction of adipose angiotensinogen and proinflammatory cytokines in WAT and blood, and reversed changes in adiponectin expression. Notably, irbesartan suppressed stress-induced reduction in adipose tissue weight and free fatty acid release, and improved insulin tolerance with restoration of IRS-1 and GLUT4 mRNA expressions in WAT. The results indicate that irbesartan improves stress-induced adipose tissue inflammation and insulin resistance. Our results suggests that irbesartan treatment exerts additive benefits for glucose metabolism in hypertensive patients with mental stress.</p></div

Establishment of Mouse Model of <i>MYH9</i> Disorders: Heterozygous R702C Mutation Provokes Macrothrombocytopenia with Leukocyte Inclusion Bodies, Renal Glomerulosclerosis and Hearing Disability

<div><p>Nonmuscle myosin heavy chain IIA (NMMHCIIA) encoded by <i>MYH9</i> is associated with autosomal dominantly inherited diseases called <i>MYH9</i> disorders. <i>MYH9</i> disorders are characterized by macrothrombocytopenia and very characteristic inclusion bodies in granulocytes. <i>MYH9</i> disorders frequently cause nephritis, sensorineural hearing disability and cataracts. One of the most common and deleterious mutations causing these disorders is the R702C missense mutation.</p><p>We generated knock-in mice expressing the <i>Myh9</i> R702C mutation. R702C knock-in hetero mice (R702C+/− mice) showed macrothrombocytopenia. We studied megakaryopoiesis of cultured fetal liver cells of R702C+/− mice and found that proplatelet formation was impaired: the number of proplatelet tips was decreased, proplatelet size was increased, and proplatelet shafts were short and enlarged. Although granulocyte inclusion bodies were not visible by May–Grünwald Giemsa staining, immunofluorescence analysis indicated that NMMHCIIA proteins aggregated and accumulated in the granulocyte cytoplasm.</p><p>In other organs, R702C+/− mice displayed albuminuria which increased with age. Renal pathology examination revealed glomerulosclerosis. Sensory hearing loss was indicated by lowered auditory brainstem response.</p><p>These findings indicate that <i>Myh9</i> R702C knock-in mice mirror features of human <i>MYH9</i> disorders arising from the R702C mutation.</p></div

Accumulation of monocytes in inguinal adipose of stressed mice.

<p>Stressed mice were individually subjected to 2 h/day of immobilization stress for two weeks. Animals received oral vehicle, 3, or 10 mg/kg/day of irbesartan during the same period. Inguinal adipose tissues from stressed and control (non-stressed) mice were analyzed by H&E staining (A), CD11b immunostaining (B and C), and quantitative RT-PCR for CD68 and F4/80 (D and E). <b>A:</b> Accumulation of mononuclear cells in inguinal adipose tissues following the 2-week restraint stress. Top panel, ×40 magnification, bar = 250 µm. Inset, ×200 magnification, bar = 50 µm. <b>B:</b> Increased accumulation of CD11b-positive cells (monocytes) in adipose tissue of stressed mice (×200 magnification, bar = 50 µm). <b>C:</b> Quantitative analysis of CD11b-positive cells relative to total nuclear number. Data are mean±SD. n = 10 for all the groups.*<i>P<</i>0.001, compared with the vehicle-treated control mice, **<i>P<</i>0.001, compared with the vehicle-treated and stressed mice. <b>D</b> and <b>E</b>: Quantitative analysis of F4/80 (D) and CD68 (E) expression levels in adipose tissue. Data are mean±SD. n = 10 for all the groups. Values are expressed relative to the vehicle-treated control mice. (<b>D</b>) *<i>P<</i>0.001, compared with the vehicle-treated control mice, **<i>P<</i>0.001, compared with the vehicle-treated and stressed mice, respectively. (<b>E</b>) *<i>P<</i>0.001, compared with the vehicle-treated control mice, ^†<i>P<</i>0.012, compared with vehicle-treated and stressed mice, ^#<i>P<</i>0.02, compared with the stressed mice treated with a lower dose of irbesartan (3 mg/kg/day), respectively.</p

Irbesartan rescued stress-induced decline in insulin sensitivity.

<p><b>A:</b> Glucose tolerance was comparable between the stressed mice treated with vehicle and irbesartan (10 mg/kg/day) after stress. Insulin tolerance showed significant recovery in the irbesartan-treated and stressed mice (lower panel). Data are mean ± SD of 10 mice per group. *<i>P<</i>0.05, and **<i>P<</i>0.02, compared with the vehicle-treated and stressed mice. <b>B:</b> Quantitative analysis of IRS-1 and GLUT4 expression in inguinal adipose tissue and skeletal muscle (adductor muscle) of the stressed mice treated with vehicle or irbesartan (10 mg/kg/day). Data are mean ± SD of 10 mice per group. *<i>P<</i>0.05, compared with the vehicle-treated and stressed mice.</p

Irbesartan restored stress-induced decrease in weight gain and reduced adipose tissue weight.

<p>Body weight and inguinal adipose tissue of the control and stressed mice were weighed before and after the stress period, and the cell size in the collected adipose tissue was estimated under a microscope at×200 magnification using image analysis software. <b>A:</b> Body weight gain in the control mice with or without irbesartan treatment (10 mg/kg/day) and stressed mice with or without irbesartan treatment (3 or 10 mg/kg/day). *<i>P<</i>0.001, compared with the vehicle-treated control mice, **<i>P<</i>0.01, compared with the vehicle-treated and stressed mice, ^†<i>P<</i>0.001, compared with the vehicle-treated and stressed mice. <b>B:</b> Plasma fat and fatty acid composition in the control mice with or without irbesartan treatment (10 mg/kg/day) and stressed mice with or without irbesartan treatment (3 or 10 mg/kg/day). *<i>P<</i>0.01, compared with the vehicle-treated control mice, **<i>P<</i>0.05, compared with the vehicle-treated and stressed mice. <b>C:</b> Inguinal adipose tissue weight in the control mice with or without irbesartan treatment (10 mg/kg/day) and stressed mice with or without irbesartan treatment (3 or 10 mg/kg/day). * <i>P</i><0.03, compared with the vehicle-treated control mice. <b>D:</b> Subcutaneous and inguinal fat pad. Circle dot line: adipose tissue. <b>E:</b> Distribution of adipocyte size in inguinal adipose tissues of stressed mice with or without irbesartan (10 mg/kg/day) treatment. Data are mean ± SD of 10 mice per group.</p

Irbesartan reduced the expression of stress-induced proinflammatory adipokines and restored adiponectin expression in adipose tissue.

<p>Inguinal adipose tissues from control mice treated with vehicle or irbesartan (10 mg/kg/day), and stressed mice treated with vehicle or irbesartan (3 or 10 mg/kg/day) were analyzed by quantitative RT-PCR for angiotensinogen (<b>A</b>), MCP-1 (<b>B</b>), TNF-α (<b>C</b>), IL-6 (<b>D</b>), and adiponectin (<b>E</b>). Values are expressed relative to the vehicle-treated control mice. Plasma levels of MCP-1, TNF-α, and IL-6 from these groups were also measured. Data are mean ± SD of 10 mice for RT-PCR, 6 mice for ELISA per group. (<b>A</b>) *<i>P<</i>0.001, compared with the vehicle-treated control mice, **<i>P<</i>0.046, compared with the vehicle-treated and stressed mice, ^†<i>P<</i>0.042, compared with the stressed mice treated with a lower dose of irbesartan (3 mg/kg/day), respectively. (<b>B</b>) *<i>P<</i>0.001, compared with the vehicle-treated control mice, **<i>P<</i>0.001, compared with the vehicle-treated and stressed mice, ^†<i>P<</i>0.003, compared with the vehicle-treated and stressed mice, respectively. (<b>C</b>) *<i>P<</i>0.001, compared with the vehicle-treated control mice, **<i>P<</i>0.001, compared with the vehicle-treated and stressed mice, ^†<i>P<</i>0.004, compared with the stressed mice treated with a lower dose of irbesartan (3 mg/kg/day), ^††<i>P<</i>0.05, compared with the vehicle-treated and stressed mice, respectively. (<b>D</b>) *<i>P<</i>0.001, compared with the vehicle-treated control mice, **<i>P<</i>0.003, compared with the vehicle-treated and stressed mice, ^†<i>P<</i>0.004, compared with stressed mice treated with a lower dose of irbesartan (3 mg/kg/day), ^††<i>P<</i>0.002, compared with the vehicle-treated control mice, ^#<i>P<</i>0.02, compared with the vehicle-treated and stressed mice, respectively. (<b>E</b>) *<i>P<</i>0.001, compared with the vehicle-treated control mice, **<i>P<</i>0.05, compared with the vehicle-treated and stressed mice, respectively.</p

Auditory Brain-stem Response (ABR).

<p>ABR measurement in R702C+/− mice. Means and standard deviations of ABR thresholds (in dB SPL) in R702C+/− mice and WT mice. ABRs were measured in 10 R702C+/− mice and five WT mice aged approximately 20 weeks, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071187#s2" target="_blank">Materials and Methods</a>.</p

Percentage of glomeruli with sclerosis.

<p>Mean ratio of glomerulus with sclerosis (%): 43.4% (n = 5).</p

Myh9 R702C knock-in strategy.

<p>A) Targeting strategy for R702C knock-in mutation of the murine <i>Myh9</i> gene. The targeting vector p<i>Multi-ND1.0_Myh9^neo</i>, the wild-type <i>Myh9</i> allele and the targeted allele before (<i>Myh9^neo</i>) and after Cre-mediated excitation of the <i>loxP</i> franked <i>neo</i> cassette (<i>Myh9^mt</i>) are schematically represented. The targeting vector also contains the Diphtheria toxin fragment A <i>(DTA)</i> gene outside the flanking homologies. Black boxes in the genomic structures represent exon sequences; the asterisk on exon 16 denotes the R702C mutation; the underlined N (<i>Nde</i>I) denotes the extra diagnostic restriction site created by silent mutation. The expected restriction fragments of the genotyping PCR products are indicated with their relative size accompanied with solid lines. The black box above the wild-type gene represents the 5′ probe used for Southern blot analysis. The open arrowheads under the gene represent the primers used for long-PCR genotyping. (B, C) Confirmation at the DNA level of correct targeting of the <i>Myh9</i> gene. Correct homologous recombination as identified by an additional 4.8-kb band in Southern blot analysis of <i>Hin</i>dIII digested genomic DNA with the 5′ probe, as well as by a 3.8 kb targeted fragment in long-PCR products digested with <i>Nde</i>I (B). Correct Cre-mediated excitation of the <i>loxP</i> franked <i>neo</i> cassette as confirmed by the appearance of a 2.4 kb recombined instead of 3.8 kb targeted fragment in long-PCR products digested with <i>Nde</i>I (C).</p