Big flies, small repeats: the "Thr-Gly" region of the period gene in Diptera

The region of the clock gene period (per) that encodes a repetitive tract of threonine-glycine (Thr-Gly) pairs has been compared between Dipteran species both within and outside the Drosophilidae. All the non- Drosophilidae sequences in this region are short and present a remarkably stable picture compared to the Drosophilidae, in which the region is much larger and extremely variable, both in size and composition. The accelerated evolution in the repetitive region of the Drosophilidae appears to be mainly due to an expansion of two ancestral repeats, one encoding a Thr-Gly dipeptide and the other a pentapeptide rich in serine, glycine, and asparagine or threonine. In some drosophilids the expansion involves a duplication of the pentapeptide sequence, but in Drosophila pseudoobscura both the dipeptide and the pentapeptide repeats are present in larger numbers. In the nondrosophilids, however, the pentapeptide sequence is represented by one copy and the dipeptide by two copies. These observations fulfill some of the predictions of recent theoretical models that have simulated the evolution of repetitive sequences.      

In vivo consequences of cholesterol-24S-hydroxylase (CYP46A1) inhibition by voriconazole on cholesterol homeostasis and function in the rat retina

Cholesterol 24S-hydroxylase (CYP46A1) converts cholesterol into 24S-hydroxycholesterol in neurons and participates in cholesterol homeostasis in the central nervous system, including the retina. We aimed to evaluate the consequences of CYP46A1 inhibition by voriconazole on cholesterol homeostasis and function in the retina. Rats received daily intraperitoneal injections of voriconazole (60 mg/kg), minocycline (22 mg/kg), voriconazole plus minocycline, or vehicle during five consecutive days. The rats were submitted to electroretinography to monitor retinal functionality. Cholesterol and 24S-hydroxycholesterol were measured in plasma, brain and retina by gas chromatography-mass spectrometry. The expression of CYP46A1, and GFAP as a marker for glial activation was analyzed in the retina and brain. Cytokines and chemokines were measured in plasma, vitreous, retina and brain. Voriconazole significantly impaired the functioning of the retina as exemplified by the reduced amplitude and increased latency of the b-wave of the electroretinogram, and altered oscillary potentials. Voriconazole decreased 24S-hydroxycholesterol levels in the retina. Unexpectedly, CYP46A1 and GFAP expression was increased in the retina of voriconazole-treated rats. ICAM-1 and MCP-1 showed significant increases in the retina and vitreous body. Minocycline did not reverse the effects of voriconazole. Our data highlighted the cross talk between retinal ganglion cells and glial cells in the retina, suggesting that reduced 24S-hydroxycholesterol concentration in the retina may be detected by glial cells, which were consequently activated.     

Dissection of a metastatic gene expression signature into distinct components

Background

Metastasis, the process whereby cancer cells spread, is in part caused by an incompletely understood interplay between cancer cells and the surrounding stroma. Gene expression studies typically analyze samples containing tumor cells and stroma. Samples with less than 50% tumor cells are generally excluded, thereby reducing the number of patients that can benefit from clinically relevant signatures.

Results

For a head-neck squamous cell carcinoma (HNSCC) primary tumor expression signature that predicts the presence of lymph node metastasis, we first show that reduced proportions of tumor cells results in decreased predictive accuracy. To determine the influence of stroma on the predictive signature and to investigate the interaction between tumor cells and the surrounding microenvironment, we used laser capture microdissection to divide the metastatic signature into six distinct components based on tumor versus stroma expression and on association with the metastatic phenotype. A strikingly skewed distribution of metastasis associated genes is revealed.

Conclusion

Dissection of predictive signatures into different components has implications for design of expression signatures and for our understanding of the metastatic process. Compared to primary tumors that have not formed metastases, primary HNSCC tumors that have metastasized are characterized by predominant down-regulation of tumor cell specific genes and exclusive up-regulation of stromal cell specific genes. The skewed distribution agrees with poor signature performance on samples that contain less than 50% tumor cells. Methods for reducing tumor composition bias that lead to greater predictive accuracy and an increase in the types of samples that can be included are presented.     

24S-hydroxycholesterol and cholesterol-24S-hydroxylase (CYP46A1) in the retina: from cholesterol homeostasis to pathophysiology of glaucoma

Free cholesterol is the predominant form of cholesterol in the neural retina. The vertebrate neural retina exhibits its own capacity to synthesize cholesterol and meets its demand also by taking it from the circulation. Defects in cholesterol synthesis and trafficking in the neural retina has detrimental consequences on its structure and function, highlighting the crucial importance of maintaining cholesterol homeostasis in the retina. Our purpose was to give a review on the functioning of the retina, the role of cholesterol and cholesterol metabolism therein, with special emphasis on cholesterol-24S-hydroxylase (CYP46A1). Similar to the brain, the retina expresses cholesterol-24S-hydroxylase (CYP46A1) and is enriched in its metabolic product, 24S-hydroxycholesterol. We recently published that one single nucleotide polymorphism in CYP46A1 gene, designated as rs754203, was a risk factor for glaucoma. Glaucoma is the second leading cause of blindness worldwide, affecting more than 60 million people. Glaucoma is characterized by the loss of retinal ganglion cells, which show high CYP46A1 expression. These data suggest the potential involvement of CYP46A1 and 24S-hydroxycholesterol in the pathophysiology of glaucoma.     

Erythrocyte Phospholipid and Polyunsaturated Fatty Acid Composition in Diabetic Retinopathy

Background

Long chain polyunsaturated fatty acids (LCPUFAs) including docosahexaenoic acid and arachidonic acid are suspected to play a key role in the pathogenesis of diabetes. LCPUFAs are known to be preferentially concentrated in specific phospholipids termed as plasmalogens. This study was aimed to highlight potential changes in the metabolism of phospholipids, and particularly plasmalogens, and LCPUFAs at various stages of diabetic retinopathy in humans.

Methodology and Principal Findings

We performed lipidomic analyses on red blood cell membranes from controls and mainly type 2 diabetes mellitus patients with or without retinopathy. The fatty acid composition of erythrocytes was determined by gas chromatography and the phospholipid structure was determined by liquid chromatography equipped with an electrospray ionisation source and coupled with a tandem mass spectrometer (LC-ESI-MS/MS). A significant decrease in levels of docosahexaenoic acid and arachidonic acid in erythrocytes of diabetic patients with or without retinopathy was observed. The origin of this decrease was a loss of phosphatidyl-ethanolamine phospholipids esterified with these LCPUFAs. In diabetic patients without retinopathy, this change was balanced by an increase in the levels of several phosphatidyl-choline species. No influence of diabetes nor of diabetic retinopathy was observed on the concentrations of plasmalogen-type phospholipids.

Conclusions and Significance

Diabetes and diabetic retinopathy were associated with a reduction of erythrocyte LCPUFAs in phosphatidyl-ethanolamines. The increase of the amounts of phosphatidyl-choline species in erythrocytes of diabetic patients without diabetic retinopathy might be a compensatory mechanism for the loss of LC-PUFA-rich phosphatidyl-ethanolamines.