Distribution and Diversity of Archaeal and Bacterial Ammonia Oxidizers in Salt Marsh Sediments

Diversity and abundance of ammonia-oxidizing Betaproteobacteria (β-AOB) and archaea (AOA) were investigated in a New England salt marsh at sites dominated by short or tall Spartina alterniflora (SAS and SAT sites, respectively) or Spartina patens (SP site). AOA amoA gene richness was higher than β-AOB amoA richness at SAT and SP, but AOA and β-AOB richness were similar at SAS. β-AOB amoA clone libraries were composed exclusively of Nitrosospira-like amoA genes. AOA amoA genes at SAT and SP were equally distributed between the water column/sediment and soil/sediment clades, while AOA amoA sequences at SAS were primarily affiliated with the water column/sediment clade. At all three site types, AOA were always more abundant than β-AOB based on quantitative PCR of amoA genes. At some sites, we detected 109 AOA amoA gene copies g of sediment−1. Ratios of AOA to β-AOB varied over 2 orders of magnitude among sites and sampling dates. Nevertheless, abundances of AOA and β-AOB amoA genes were highly correlated. Abundance of 16S rRNA genes affiliated with Nitrosopumilus maritimus, Crenarchaeota group I.1b, and pSL12 were positively correlated with AOA amoA abundance, but ratios of amoA to 16S rRNA genes varied among sites. We also observed a significant effect of pH on AOA abundance and a significant salinity effect on both AOA and β-ΑΟΒ abundance. Our results expand the distribution of AOA to salt marshes, and the high numbers of AOA at some sites suggest that salt marsh sediments serve as an important habitat for AOA

Total Linking Numbers of Torus Links and Klein Links

We investigate characteristics of two classes of links in knot theory: torus links and Klein links. Formulas are developed and confirmed to determine the total linking numbers of links in these classes. We find these relations by examining the general braid representations of torus links and Klein links

Adenosine 2A receptor antagonist prevented and reversed liver fibrosis in a mouse model of ethanol-exacerbated liver fibrosis.

The effect of moderate alcohol consumption on liver fibrosis is not well understood, but evidence suggests that adenosine may play a role in mediating the effects of moderate ethanol on tissue injury. Ethanol increases the concentration of adenosine in the liver. Adenosine 2A receptor (A2AR) activation is known to enhance hepatic stellate cell (HSC) activation and A2AR deficient mice are protected from fibrosis in mice. Making use of a novel mouse model of moderate ethanol consumption in which female C57BL/6J mice were allowed continued access to 2% (vol/vol) ethanol (11% calories) or pair-fed control diets for 2 days, 2 weeks or 5 weeks and superimposed with exposure to CCl4, we tested the hypothesis that moderate ethanol consumption increases fibrosis in response to carbon tetrachloride (CCl4) and that treatment of mice with an A2AR antagonist prevents and/or reverses this ethanol-induced increase in liver fibrosis. Neither the expression or activity of CYP2E1, required for bio-activation of CCl4, nor AST and ALT activity in the plasma were affected by ethanol, indicating that moderate ethanol did not increase the direct hepatotoxicity of CCl4. However, ethanol feeding enhanced HSC activation and exacerbated liver fibrosis upon exposure to CCl4. This was associated with an increased sinusoidal angiogenic response in the liver. Treatment with A2AR antagonist both prevented and reversed the ability of ethanol to exacerbate liver fibrosis.Moderate ethanol consumption exacerbates hepatic fibrosis upon exposure to CCl4. A2AR antagonism may be a potential pharmaceutical intervention to decrease hepatic fibrosis in response to ethanol

Effect of A2AR antagonist in prevention and treatment of CCl₄-induced HSC activation and liver fibrosis.

<p>C57BL/6J mice were allowed free access to 2% (vol/vol) ethanol diet or pair-fed control diet and exposed to intraperitoneal CCl₄ injections with KW-6002 for 2 weeks in the prevention model or with KW-6002 in the last week of the 5 weeks treatment model. (<b>A</b>) α-SMA was used as a marker for activated HSC in the 2 week and 5 week models. Representative images are shown. (<b>B</b>) ECM deposition was measured by Sirius red staining in the 2 week and 5 week models. Representative images are shown. (<b>C, D</b>) Morphometric analysis with Image-Pro-Plus software was used for semi-quantification. Values represent means ± SEM. n = 4 in pair-fed diet, n = 6 in ethanol-fed diet. Values with different alphabetical superscripts were significantly different from each other, p<0.05. (<b>E</b>) Hepatic hydroxyproline concentration was measured using a colorimetric assay in liver acid–hydrolysates in the 5 week model. Values represent means ± SEM. n = 3–8 in each group. Values with different alphabetical superscripts were significantly different from each other, p<0.05.</p

Progression of fibrosis with moderate ethanol intake and superimposed CCl₄ liver injury.

<p>C57BL/6J mice were allowed free access to 2% (vol/vol) ethanol diet or pair-fed control diet and then exposed to intraperitoneal CCl₄ injections for 2 weeks or 5 weeks. (<b>A</b>) Collagen 1 staining was performed on frozen liver sections at 2 weeks and 5 weeks. (<b>B</b>) Paraffin-embedded liver sections were de-paraffinized followed by Sirius red staining to assess ECM deposition at 2 weeks and 5 weeks. Representative images are shown. (Solid arrow: immunostaining pattern consistent with bridging fibrosis. Open arrow: immunostaining pattern less than bridging fibrosis.) Morphometric analysis with Image-Pro-Plus software was used for semi-quantification. Value represents mean ± SEM. n = 4 in pair-fed diet, n = 6 in ethanol-fed diet. Values with different alphabetical superscripts were significantly different from each other, p<0.05.</p

Effect of moderate ethanol intake on CYP2E1 and CCl₄ hepatotoxicity.

<p>C57BL/6J mice were allowed free access to 2% (vol/vol) (11% calories) ethanol diet or pair-fed control diet and then exposed to intraperitoneal CCl₄ injections over 72 hours, 2 weeks or 5 weeks. (<b>A</b>) Plasma ALT was measured in the acute time course to assess hepatic injury. (<b>B</b>) Plasma AST and ALT were measured at different time points. (<b>C</b>) CYP2E1 protein level in liver was measured by Western blot using whole liver extracts. HSC 70 was used as loading control. Insets show representative image of CYP2E1 Western blots. (<b>D</b>) Activity of CYP2E1 in liver was measured by the hydroxylation of <i>p</i>-nitrophenol in whole liver extracts. Values represent means ± SEM. n = 4 in pair-fed diet, n = 6 in ethanol-fed diet. Values with different alphabetical superscripts were significantly different from each other, p<0.05.</p

Effect of A2AR antagonist on hepatic angiogenic response and sinusoidal capillarization.

<p>(<b>A</b>) C57BL/6J wild-type (WT) mice were allowed free access to diets with increasing concentrations of 2% (vol/vol) ethanol or pair-fed controls for 4 days. All animals were injected intraperitoneally with pimonidazole (120 mg/Kg, PMD) 1 hour before euthanasia. Paraffin-embedded liver sections were de-paraffinized and stained for PMD-adducts. Representative images are shown. n = 4 in pair-fed diet, n = 6 in ethanol-fed diet. (Solid black arrow: increased pimonidazole adducts accumulation.) (<b>B</b>) A short time course study using single injection of CCl₄ with pretreatment of KW-6002 or saline in C57BL/6J wild-type (WT) mice. ANGP1, TNF-α and MIP2 mRNA in liver was measured after KW-6002 administration as an intermediate marker of angiogenesis and inflammation. (* indicates statistical significance between KW-6002 and saline control, p<0.05) (<b>C</b>) C57BL/6J mice were allowed free access to 2% (vol/vol) ethanol diet or pair-fed control diet and then exposed to intraperitoneal CCl₄ injections with KW-6002 for 2 weeks in the prevention model or with KW-6002 in the last week of the 5 weeks treatment model. Frozen liver sections were used for CD31 immunofluorescent staining to study sinusoidal capillarization and angiogenic response. (Solid arrow: increased CD31 expression in sinusoidal space.) Representative images are shown. n = 4 in pair-fed diet, n = 6 in ethanol-fed diet.</p

Effect of A2AR antagonist on liver injury.

<p>Plasma AST and ALT levels in pair-fed and ethanol-fed C57BL/6J mice exposed to CCl₄ with KW-6002 or saline control at the 2 week and 5 week time points. Values represent mean ± SEM. n = 4 in pair-fed diet, n = 6 in ethanol-fed diet.</p

Effect of moderate ethanol intake on HSC activation in response to CCl₄.

<p>C57BL/6J mice were allowed free access to 2% (vol/vol) ethanol diet or pair-fed control diet and then exposed to intraperitoneal CCl₄ injections over 72 hours, 2 weeks or 5 weeks. (<b>A</b>) Hepatic accumulation of Col1A1, Col1A2 and α-SMA mRNA was used as intermediate biomarkers of HSC activation and fibrosis at 72 hours. (<b>B, C</b>) Paraffin-embedded liver sections were de-paraffinized followed by α-SMA staining at 2 weeks (<b>B</b>) and 5 weeks (<b>C</b>). Representative images are shown. Morphometric analysis with Image-Pro-Plus software was used for semi-quantification. Values represent means ± SEM. n = 4 in pair-fed diet, n = 6 in ethanol-fed diet. Values with different alphabetical superscripts were significantly different from each other, p<0.05.</p