Chronic OVA allergen challenged Siglec-F deficient mice have increased mucus,remodeling, and epithelial Siglec-F ligands which are up-regulated by IL-4 and IL-13

Background

In this study we examined the role of Siglec-F, a receptor highly expressed on eosinophils, in contributing to mucus expression, airway remodeling, and Siglec-F ligand expression utilizing Siglec-F deficient mice exposed to chronic allergen challenge.

Methods

Wild type (WT) and Siglec-F deficient mice were sensitized and challenged chronically with OVA for one month. Levels of airway inflammation (eosinophils), Siglec-F ligand expresion and remodeling (mucus, fibrosis, smooth muscle thickness, extracellular matrix protein deposition) were assessed in lung sections by image analysis and immunohistology. Airway hyperreactivity to methacholine was assessed in intubated and ventilated mice.

Results

Siglec-F deficient mice challenged with OVA for one month had significantly increased numbers of BAL and peribronchial eosinophils compared to WT mice which was associated with a significant increase in mucus expression as assessed by the number of periodic acid Schiff positive airway epithelial cells. In addition, OVA challenged Siglec-F deficient mice had significantly increased levels of peribronchial fibrosis (total lung collagen, area of peribronchial trichrome staining), as well as increased numbers of peribronchial TGF-β1+ cells, and increased levels of expression of the extracellular matrix protein fibronectin compared to OVA challenged WT mice. Lung sections immunostained with a Siglec-Fc to detect Siglec-F ligand expression demonstrated higher levels of expression of the Siglec-F ligand in the peribronchial region in OVA challenged Siglec-F deficient mice compared to WT mice. WT and Siglec-F deficient mice challenged intranasally with IL-4 or IL-13 had significantly increased levels of airway epithelial Siglec-F ligand expression, whereas this was not observed in WT or Siglec-F deficient mice challenged with TNF-α. There was a significant increase in the thickness of the peribronchial smooth muscle layer in OVA challenged Siglec-F deficient mice, but this was not associated with significant increased airway hyperreactivity compared to WT mice.

Conclusions

Overall, this study demonstrates an important role for Siglec-F in modulating levels of chronic eosinophilic airway inflammation, peribronchial fibrosis, thickness of the smooth muscle layer, mucus expression, fibronectin, and levels of peribronchial Siglec-F ligands suggesting that Siglec-F may normally function to limit levels of chronic eosinophilic inflammation and remodeling. In addition, IL-4 and IL-13 are important regulators of Siglec-F ligand expression by airway epithelium.     

Inhibition of dynamin‐2 confers endothelial barrier dysfunctions by attenuating nitric oxide production

Hypoxia induces barrier dysfunctions in endothelial cells. Nitric oxide is an autacoid signalling molecule that confers protection against hypoxia‐mediated barrier dysfunctions. Dyn‐2 (dynamin‐2), a large GTPase and a positive modulator of eNOS (endothelial nitric oxide synthase), plays an important role in maintaining vascular homeostasis. The present study aims to elucidate the role of dyn‐2 in hypoxia‐mediated leakiness of the endothelial monolayer in relation to redox milieu. Inhibition of dyn‐2 by transfecting the cells with K44A, a dominant negative construct of dyn‐2, elevated leakiness of the endothelial monolayer under hypoxia. Sodium nitroprusside (nitric oxide donor) and uric acid (peroxynitrite quencher) were used to evaluate the role of nitric oxide and peroxynitrite in maintaining endothelial barrier functions under hypoxia. Administration of nitric oxide and uric acid recovered hypoxia‐mediated leakiness of K44A‐overexpressed endothelial monolayer. Our study confirms that inhibition of dyn‐2 induces leakiness in the endothelial monolayer by increasing the load of peroxynitrite under hypoxia.     

Bio-transformation of artemisinin using soil microbe: Direct C-acetoxylation of artemisinin at C-9 by Penicillium simplissimum

Potent antimalarial compound artemisinin, 1 was bio-transformed to C-9 acetoxy artemisinin, 2 using soil microbe Penicillium simplissimum along with C-9 hydroxy derivative 3. The products were characterized using high field NMR and MS–MS data. The absolute stereochemistry of the newly generated chiral centers has been ascertained by COSY and 1D NOESY experiments. This is the first Letter of direct C-acetoxylation of artemisinin using microbial strains.     

Substrate Based Ablation of Ventricular Tachycardia Through An Epicardial Approach

Ventricular tachycardia (VT) occurring late after myocardial infarction is often due to reentry circuit in the peri-infarct zone. The circuit is usually located in the sub-endocardium, though subepicardial substrates are known. Activation mapping during VT to identify target regions for ablation can be difficult if VT is non inducible or poorly tolerated. In the latter, a substrate based approach of mapping during sinus rhythm in conjunction with pace mapping helps to define the reentry circuit and select target sites for ablation in majority of patients with hemodynamically unstable VT. Percutaneous epicardial catheter ablation has been attempted as an approach where ablation by a conventional endocardial access has been unsuccessful. We report a case of post myocardial infarction scar VT which could be successfully ablated with a substrate based approach from the epicardial aspect.     

Condensin Regulates the Stiffness of Vertebrate Centromeres

When chromosomes are aligned and bioriented at metaphase, the elastic stretch of centromeric chromatin opposes pulling forces exerted on sister kinetochores by the mitotic spindle. Here we show that condensin ATPase activity is an important regulator of centromere stiffness and function. Condensin depletion decreases the stiffness of centromeric chromatin by 50% when pulling forces are applied to kinetochores. However, condensin is dispensable for the normal level of compaction (rest length) of centromeres, which probably depends on other factors that control higher-order chromatin folding. Kinetochores also do not require condensin for their structure or motility. Loss of stiffness caused by condensin-depletion produces abnormal uncoordinated sister kinetochore movements, leads to an increase in Mad2(+) kinetochores near the metaphase plate and delays anaphase onset.     

Anti-hyperlipidemic and insulin sensitizing activities of fenofibrate reduces aortic lipid deposition in hyperlipidemic Golden Syrian hamster

Cholesterol ester transfer protein (CETP) and apolipoprotein (apo) E are important in peroxisome proliferation activated receptor-α (PPAR-α)-mediated regulation of lipoprotein metabolism. Therefore, popularly used apolipoprotein E knockout mice are not suitable to evaluate PPAR-α agonists. In this study, we aimed to: a) evaluate hamster as a model for insulin resistance, hyperlipidemia and atherosclerosis; and b) investigate the effect of a PPAR-α activator, fenofibrate, in this model. A high fat high cholesterol (HFHC) diet increased serum cholesterol and triglycerides, but inclusion of fenofibrate in the diet decreased cholesterol and proatherogenic lipoproteins, VLDL and LDL, in a time-dependent manner. Concomitantly, serum levels of triglycerides also decreased. These reductions were attributed, in part, to the down-regulation of lipogenic genes and upregulation of lipoprotein lipase. The HFHC diet caused body weight gain and mild insulin resistance, both of which were prevented following the treatments with fenofibrate. Insulin resistance was further investigated in high fructose-fed hamsters. Fenofibrate prevented both hyperinsulinemia and hypertriglyceridemia. The insulin sensitizing activity of fenofibrate appeared to occur via reductions in protein tyrosine phophatase-1B. To determine whether lowering of lipids by fenofibrate treatment contributed to the reduced risks of developing atherosclerosis in hyperlipidemic hamsters, we measured lipid deposition in the aorta. Our results showed that fenofibrate treatment reduced aortic lipid deposition by 70%. These findings suggest that hamster may be an adequate animal model to evaluate the efficacy of lipid lowering, insulin sensitizing and antiatherosclerotic agents. We also show that fenofibrate is an effective antiatherosclerotic agent in hyperlipidemic hamster model.     

Human embryonic stem cells express an immunogenic nonhuman sialic acid

Human embryonic stem cells (HESC) can potentially generate every body cell type, making them excellent candidates for cell- and tissue-replacement therapies. HESC are typically cultured with animal-derived 'serum replacements' on mouse feeder layers. Both of these are sources of the nonhuman sialic acid Neu5Gc, against which many humans have circulating antibodies. Both HESC and derived embryoid bodies metabolically incorporate substantial amounts of Neu5Gc under standard conditions. Exposure to human sera with antibodies specific for Neu5Gc resulted in binding of immunoglobulin and deposition of complement, which would lead to cell killing in vivo. Levels of Neu5Gc on HESC and embryoid bodies dropped after culture in heat-inactivated anti-Neu5Gc antibody-negative human serum, reducing binding of antibodies and complement from high-titer sera, while allowing maintenance of the undifferentiated state. Complete elimination of Neu5Gc would be likely to require using human serum with human feeder layers, ideally starting with fresh HESC that have never been exposed to animal products.     

Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution

Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4″-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4″-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.