In Situ Routes to Catalytically Active Ru(0) Species by Reduction of Readily Available, Air-Stable Precursors

Cross-dimerization of a conjugated diene with a substituted alkene catalyzed by in situ reduction of an air-stable Ru­(II) catalyst precursor has been achieved. Reaction of 2,3-dimethylbutadiene with styrene is catalyzed by [Ru­(acac)₂(η⁴-1,5-COD)] (<b>2a</b>) (5 mol %) with BuLi (10 mol %) at 50 °C for 6 h in hexane, giving the cross-dimers in 99% yield ((<i>E</i>)-4,5-dimethyl-1-phenylhexa-1,4-diene (<b>3a</b>)/(<i>E</i>)-4,5-dimethyl-1-phenylhexa-2,4-diene (<b>3b</b>)/isomers = 84/9/7). Because neither <b>2a</b> nor BuLi separately catalyzes the cross-dimerization and reduction of <b>2a</b> with BuLi in the presence of naphthalene produces [Ru­(η⁶-naphthalene)­(η⁴-1,5-COD)] (<b>1a</b>), the active species in this catalysis is considered to be a Ru(0) compound. Interestingly, this in situ reduction method of Ru­(II) using BuLi can be applied to the cross-dimerization using an ester such as methyl acrylate. Alternatively, an air-stable Ru­(II) complex having a labile arene ligand such as [RuCl₂(η⁶-anisole)]₂ (<b>5c</b>) (5 mol %) with Na₂CO₃ (40 mol %) in the presence of 1,5-COD (20 mol %) at 100 °C for 6 h in 2-butanol also catalyzes the same cross-dimerization in 62% yield. These protocols provide facile methods for production of unsaturated linear compounds by the cross-dimerization using air-stable Ru­(II) catalyst precursors

Investigation of Encephalopathy Caused by Shiga Toxin 2c-Producing <em>Escherichia coli</em> Infection in Mice

<div><p>A large outbreak of Shiga toxin (Stx)-producing enteroaggregative <i>Escherichia coli</i> (EAEC) O104:H4 occurred in northern Germany. From this outbreak, at least 900 patients developed hemolytic uremic syndrome (HUS), resulting in more than 50 deaths. Thirty percent of the HUS patients showed encephalopathy. We previously established a mouse model with encephalopathy associated with blood brain barrier (BBB) damage after oral infection with the Shiga toxin (Stx) 2c-producing <i>Escherichia coli</i> O157: H- strain E32511 (E32511). In this model, we detected high expression of the Stx receptor synthase enzyme, glycosphingolipid globotriaosylceramide (Gb3) synthase, in endothelial cells (ECs) and neurons in the reticular formation of the medulla oblongata by <i>in situ</i> hybridization. Caspase-3 was activated in neurons in the reticular formation of the medulla oblongata and the anterior horn of the spinal cord. Astrocytes (ASTs) were activated in the medulla oblongata and spinal cord, and a decrease in aquaporin 4 around the ECs suggested that BBB integrity was compromised directly by Stx2c or through the activation of ASTs. We also report the effectiveness of azithromycin (AZM) in our model. Moreover, AZM strongly inhibited the release of Stx2c from E32511 <i>in vitro</i>.</p> </div

Microglial activation in i.p. LPS-injected and E32511 model mice.

<p>(A) Microglia in the medulla oblongata and the spinal cord were not activated in the control (uninfected) and E32511-infected mice, but were activated in i.p. LPS-injected mice. Scale bars: 2 µm. (B) The number of activated microglia significantly increased in the LPS-injected mice but not in the control and E32511-infected mice (LPS vs. control, <i>p</i><0.0001; LPS vs. E32511, <i>p</i><0.0001 and control vs. E32511, <i>p</i> = 1). Microglia were observed and counted manually from 5 non-overlapping fields. Statistical difference was measured by student's t test. **<i>p</i><0.001.</p

ISH of Gb3 synthase in brains of E32511 model and i.p.-injected LPS mice.

<p>(A) The brain of E32511-infected mice after hybridization of Gb3 synthase using Gb3syt anti-sense and sense probes. Scale bars: 1.5 mm. (A.1) Expression of Gb3 synthase was observed with anti-sense, but not sense probes in E32511-infected mice in the reticular formation of the medulla oblongata. Scale bars: 200 µm. (A.2) Magnification and expression of Gb3 synthase in endothelial cells (scale bars: 50 µm) and neurons (scale bars: 20 µm) of E32511-infected mouse brain, detected with anti-sense, but not sense probes. (B) The brain of i.p. LPS-injected mice after hybridization of Gb3 synthase using probe Gb3syt anti-sense and sense probes. Scale bars: 1.5 mm. (C) Expression of Gb3 synthase was observed in i.p. LPS-injected mice with the anti-sense, but not the sense probe in the spinal cord. Scale bars: 100 µm. (C.1) Magnification and expression of Gb3 synthase in endothelial cells (scale bars: 30 µm) and neurons (scale bars: 20 µm) of i.p. LPS-injected mice, detected with the anti-sense, but not the sense probe.</p

Bacterial count at sub-minimum inhibitory concentration (MIC) of AZM and other antimicrobial agents against E32511. AZM, azithromycin; FOM, fosfomycin; NFLX, norfloxacin; KM, kanamycin; OFLX, orfloxacin; CPFX, ciprofloxacin.

<p>AZM, azithromycin; FOM, fosfomycin; NFLX, norfloxacin; KM, kanamycin; OFLX, orfloxacin; CPFX, ciprofloxacin.</p

Effectiveness of a single dose of AZM in the E32511 model.

<p>(A) The administration of a single dose of AZM 2 h after infection significantly increased the BW of E32511-infected mice. (B) Mouse BW for AZM 2 h vs. AZM 6 h, <i>p</i> = 0.015 at day 14 after inoculation. Student's t test, *<i>p</i><0.05; **<i>p</i><0.001). (C) The administration of a single dose of AZM was significantly effective in the survival mice when given 2 h after infection, compared with 6 and 24 h after infection, (for AZM 2 h after infection vs. untreated, <i>p</i> = 0.012; AZM 2 h after infection vs. 24 h after infection, <i>p</i> = 0.013 and AZM 6 h and 24 h after infection vs. untreated, <i>p</i>>0.05. Log rank and χ² test, (*<i>p</i><0.05) (D) The administration of a single dose of 200–1.6 µg/g AZM 2 h after infection significantly increased the BW of E32511-infected mice. (E) The correlation between the dose of AZM and the increase in BW of E32511-infected mice was statistically significant, as shown by the Spearman's ρ (rho) = 0.887 and the <i>p</i> value, <i>p</i><0.0001. Black-filled circle = AZM 100 µg/g; grey-filled circle = AZM 25 µg/g; empty circle = AZM 6.25 µg/g. (F) 1.6 µg/g AZM 2 h after infection with E32511 (10¹¹ CFU/ mouse) was still effective in treating the E32511-infected mice, with a significant effect on the survival curve (200–6.25 µg/g AZM vs. untreated, <i>p</i> = 0.002; 3.1 µg/g AZM vs. untreated, <i>p</i> = 0.008 and 1.6 µg/g AZM vs. untreated, <i>p</i> = 0.016. Log rank and χ² test, **<i>p</i><0.01, *<i>p</i><0.05.</p

Caspase-3 activation in E32511 model mouse brain.

<p>Lesions were not detected in the brains of control (uninfected), whereas lesions were detected in the brains of E32511-infected. Scale bars: 1.5 mm. (A) Caspase-3 activation was detected in the neurons of the mouse spinal cord. Scale bars: 750 µm and 200 µm (inset). (B) Caspase-3 activation was also detected in neurons of the mouse reticular formation, scale bars: 300 µm and 200 µm (inset) and endothelial cells of the midbrain. Scale bars: 25 µm. (C) Four week-old female ICR outbred mice after inoculation. The representative clinical features of flaccid paralysis of the extremities were observed in ICR mice 3 to 5 days after orogastric challenge with E32511 10¹¹ CFU/0.5 mL per mouse (green arrow). (D) Four week-old female ICR mice after inoculation. The representative clinical features of spinal deformity were observed in ICR mice 3 to 5 days after orogastric challenge with E32511/HSC 10¹¹ CFU/0.5 mL per mouse (green arrow).</p

AZM compared with other antimicrobial agents in the E32511 model.

<p>(A) The concentration of Stx2c in NFLX, KM, OFLX, and CPFX treatment was significantly increased compared with control (no antibiotic) (NFLX vs. control, <i>p</i> = 0.023; KM vs. control, <i>p</i> = 0.02; OFLX vs. control, <i>p</i> = 0.023 and CPPFX vs. control, <i>p</i> = 0.017), while this was not the case for FOM. On the other hand, AZM statistically decreased Stx2c concentration compared with control (AZM vs. control, <i>p</i> = 0.007). Data were collected from the supernatants of cultures with 2-fold serial dilutions of each antimicrobial agent and are shown as mean and standard deviations of two independent experiments. Statistical difference was measured by Student's t test, *<i>p</i><0.05. (B) AZM had a statistically significant effect on the mice survival, with 100% survival; NFLX and KM had a statistically significant effect on the mice survival, with 80% survival (AZM vs. untreated, <i>p</i> = 0.002; AZM vs. FOM, <i>p</i> = 0.003; AZM vs. CPFX, <i>p</i> = 0.014; AZM vs. NFLX and KM, <i>p</i>>0.05). Log Rank and χ² test, *<i>p</i><0.05.</p

AST activation and AQP4 expression in E32511 model and i.p. LPS-injected mice.

<p>(A) Severe diffuse astrogliosis was observed in spinal cord of E32511-infected but not in control and LPS-injected mice at low (400×) and high (1000×) magnifications. The black arrow shows a degenerating neuron adjacent to an activated astrocyte. (B) More intense staining of AQP4 was observed surrounding the ECs which comprised the BBB in the spinal cord in control and LPS-injected mice, but not in E32511-infected mice at low (400×) and high (1000×) magnifications. (C) Severe diffuse astrogliosis was observed in the medulla oblongata of E32511-infected but not in control and LPS-injected mice at low (400×) and high (1000×) magnification. The black arrow shows a degenerating neuron adjacent to an activated astrocyte. (D) More intense staining of AQP4 was observed surrounding the blood vessels that comprise the BBB in the medulla oblongata in control and LPS-injected mice, but not in E32511-infected mice at low (400×) and high (1000×) magnifications. Scale bars (400×): 2 µm and (1000×): 1 µm.</p