Monthly day/night changes and seasonal daily rhythms of sexual steroids in Senegal sole (Solea senegalensis) under natural fluctuating or controlled environmental conditions

In this paper we attempted to investigate the existence of daily fluctuations on plasma sexual steroids (17beta-estradiol, E(2) and testosterone, T) in Senegal sole (Solea senegalensis) females. We described the monthly day/night concentrations and seasonal daily rhythms in animals reared under natural photo- and thermo-period. In addition, the influence of the natural annual fluctuation of the water temperature on the plasma concentration of these steroids was investigated, using one group of Senegal sole under a natural photoperiod, but with an attenuated thermal cycle (around 17-20 degrees C) for one year. Although no significant day/night differences were detected in monthly samplings, the existence of an annual rhythm of E(2) and T (p<0.01) with an acrophase in February was revealed by COSINOR analysis. Maximum values were reached in March for both steroids (6.1+/-1.7 ng mL(-1) at mid-dark, MD and 4.0+/-0.6 ng mL(-1) at mid-light, ML for E2 and 1.4+/-0.4 ng mL(-1) at MD and 0.8+/-0.1 ng mL(-1) at ML for T) in anticipation of the spawning season (May-June). As regards seasonal daily rhythms, the presence of daily oscillations was revealed. At the spring solstice (21st March) a daily rhythm was observed for both steroids (COSINOR, p<0.01), with an acrophase at 20:00 h (E(2)) and at 21:08 h (T). In summer, autumn and winter no daily rhythms were observed due to the low steroid levels at those seasons. When Senegal sole females were submitted to an attenuated annual thermal cycle, the steroid rhythm disappeared (there was no surge in spring, as in the control group) and these fish did not spawn, despite being subjected to natural photoperiod conditions. This result underlined the importance of the natural annual fluctuation of water temperature and photoperiod on the synchronization of the spawning season and on the onset of steroidogenesis.     

Changes in cultured endothelial cell glycosaminoglycans under hyperglycemic conditions and the effect of insulin and heparin

Background

Diabetic cardiomyopathy (DCM) contributes to cardiac failure in diabetic patients. It is characterized by excessive lipids accumulation, with increased triacylglycerol (TAG) stores, and fibrosis in left ventricle (LV). The mechanisms responsible are incompletely known and no specific treatment is presently defined. We evaluated the possible usefulness of two molecules promoting lipid oxidation, fenofibrate and metformin, in an experimental model of DCM, the Zucker diabetic rat (ZDF).

Methods

ZDF and controls (C) rats were studied at 7, 14 and 21 weeks. After an initial study at 7 weeks, ZDF rats received no treatment, metformin or fenofibrate until final studies (at 14 or 21 weeks). C rats received no treatment. Each study comprised measurements of metabolic parameters (plasma glucose, TAG, insulin levels) and sampling of heart for histology and measurements of TAG content and relevant mRNA concentration.

Results

ZDF rats were insulin-resistant at 7 weeks, type 2 diabetic at 14 weeks and diabetic with insulin deficiency at 21 weeks. Their plasma TAG levels were increased. ZDF rats had at 7 weeks an increased LV TAG content with some fibrosis. LV TAG content increased in untreated ZDF rats at 14 and 21 weeks and was always higher than in C. Fibrosis increased also moderately in untreated ZDF rats. Metformin and fenofibrate decreased plasma TAG concentrations. LV TAG content was decreased by metformin (14 and 21 weeks) and by fenofibrate (14 weeks). Fibrosis was reduced by fenofibrate only and was increased by metformin. Among the mRNA measured, fenofibrate increased Acyl-CoA Oxidase mRNA level, metformin decreased Acyl-CoA Synthase and increased AdipoR1 and pro-inflammatory mRNA levels.

Conclusion

Fenofibrate had favourable actions on DCM. Metformin had beneficial effect on TAG content but not on fibrosis. PPARα agonists could be useful for the prevention and treatment of DCM.     

Quantitative proteomic analysis by iTRAQ<Superscript>®</Superscript> for the identification of candidate biomarkers in ovarian cancer serum

Background

Ovarian cancer is the most lethal gynecologic malignancy, with the majority of cases diagnosed at an advanced stage when treatments are less successful. Novel serum protein markers are needed to detect ovarian cancer in its earliest stage; when detected early, survival rates are over 90%. The identification of new serum biomarkers is hindered by the presence of a small number of highly abundant proteins that comprise approximately 95% of serum total protein. In this study, we used pooled serum depleted of the most highly abundant proteins to reduce the dynamic range of proteins, and thereby enhance the identification of serum biomarkers using the quantitative proteomic method iTRAQ^®.

Results

Medium and low abundance proteins from 6 serum pools of 10 patients each from women with serous ovarian carcinoma, and 6 non-cancer control pools were labeled with isobaric tags using iTRAQ^® to determine the relative abundance of serum proteins identified by MS. A total of 220 unique proteins were identified and fourteen proteins were elevated in ovarian cancer compared to control serum pools, including several novel candidate ovarian cancer biomarkers: extracellular matrix protein-1, leucine-rich alpha-2 glycoprotein-1, lipopolysaccharide binding protein-1, and proteoglycan-4. Western immunoblotting validated the relative increases in serum protein levels for several of the proteins identified.

Conclusions

This study provides the first analysis of immunodepleted serum in combination with iTRAQ^® to measure relative protein expression in ovarian cancer patients for the pursuit of serum biomarkers. Several candidate biomarkers were identified which warrant further development.

     

Electron microscopic analysis of glucose-induced endothelial damage in primary culture: possible mechanism and prevention

We previously reported that high glucose treated cultured endothelial cells (ECs) showed intercellular gaps by transmission electron microscopy (TEM). These gaps were abrogated with insulin and/or heparin treatment. Our aims were to assess the severity of injury in ECs treated with high glucose for variable duration, and to further study the protective effects of insulin and/or heparin. Cells were also treated with L-buthionine sulfoximine (BSO), a glutathione inhibitor, to help understand the mechanism of high glucose injury. Primary porcine ECs were treated with high glucose (30 mM) for 2, 6 or 10 days; and glucose plus insulin (1 U/ml), glucose plus heparin (5 microg/ml), glucose plus insulin plus heparin for 6 days. ECs were treated with BSO (0.001-0.05 mM) for 2 days. Pellets from trypsinized cells were processed for TEM. High glucose treatment revealed apoptosis or necrosis showing variable cell size, abnormal nuclei, condensation of nuclear chromatin, few mitochondria, cell membrane disruption and needle-shaped structures. Changes increased with duration of exposure. In high glucose plus heparin or insulin treated cultures at least one-half of the cells appeared normal. Most ECs were intact when treated with high glucose plus insulin plus heparin. BSO treatment showed dose-dependent changes with low doses showing apoptosis whereas higher doses revealed necrosis similar to high glucose treatment for 6 or 10 days. High glucose-induced EC injury increased with duration of exposure. These data demonstrate that high glucose injury resembles that of BSO treatment, suggesting that glutathione depletion may be involved in EC injury. Insulin and/or heparin protect against high glucose-induced injury.     

Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation

Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation.Key words: MS-HRM, pyrosequencing, digital PCR, heterogeneous DNA methylation, DAPK1, chronic lymphocytic leukemia     

Gut bacteria profiles of Mus musculus at the phylum and family levels are influenced by saturation of dietary fatty acids

BackgroundMammalian gut microbiota have been implicated in a variety of functions including the breakdown of ingested nutrients, the regulation of energy intake and storage, the control of immune system development and activity, and the synthesis of novel chemicals. Previous studies have shown that feeding mammalian hosts a high-fat diet shifts gut bacteria at the phylum level to reduce the ratio of Bacteroidetes-to-Firmicutes, while feeding hosts a fat-restricted diet increases this ratio. However, few studies have investigated the differential effects of fatty acid type on gut bacterial profile.MethodsOver a 14-week period, Mus musculus were fed a diet rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs), omega-6 polyunsaturated fatty acids (n-6 PUFAs), or saturated fatty acids (SFAs). Fecal pellets were collected before and after the treatment period from 12 randomly selected mice (4 per treatment group). Bacterial DNA was extracted from the pellets and characterized by analysis of the hypervariable V3 region of the 16S rRNA. Nominal logistic regression models were used to assess shifts in microbial profile at the phylum and family levels in response to diet.ResultsA significant decrease in the proportion of phylum Bacteroidetes species was observed for mice fed any of the three diets over time. However, the SFA-rich diet group showed a significantly greater decrease in Bacteroidetes proportion (?28%) than did either the n-3 PUFA group (?10%) or the n-6 PUFA group (?12%). At the family level, a significant decrease in proportion of Porphyromonadaceae was observed for mice fed the n-6 PUFA-rich diet, and a significant decrease in proportion of Lachnospiraceae was observed for mice fed the SFA-rich diet. There was no significant effect of diet type on body mass change.ConclusionOur results indicate that SFAs have stronger effects than PUFAs in shifting gut microbiota profiles toward those typical of obese individuals, and that dietary fatty acid saturation influences shifts in gut microbiota independently of changes in body mass.     

Cell surface expression and orientation in membranes of the 44-amino-acid SH protein of simian virus 5.

Antiserum was raised against a synthetic peptide containing the N-terminal hydrophilic domain of the small hydrophobic protein (SH) of simian virus 5 (SV5) and used to characterize properties of the SH protein. SH demonstrated properties of an integral membrane protein. Indirect immunofluorescence experiments showed that the protein is involved in the exocytotic pathway, and isolation of plasma membranes from SV5-infected cells showed an enrichment of SH, indicating that SH is transported to the infected-cell surface. Biochemical analysis of the orientation of SH in membranes by proteolysis of intact SV5-infected cell surfaces and intracellular microsomal vesicles indicated that SH is oriented in membranes with its N-terminal hydrophilic domain exposed on the cytoplasmic face of the plasma membrane and the C terminus of approximately five amino acid residues exposed at the cell surface. These data are discussed with respect to positive-acting signals being necessary in the ectodomain of SH for cell surface expression.     

A pilot study of rivastigmine in the treatment of delirium after stroke: A safe alternative

Background

Delirium is a common disorder in the early phase of stroke. Given the presumed cholinergic deficiency in delirium, we tested treatment with the acetylcholinesterase inhibitor rivastigmine.

Methods

This pilot study was performed within an epidemiological study. In 527 consecutive stroke patients presence of delirium was assessed during the first week with the confusion assessment method. Severity was scored with the delirium rating scale (DRS). Sixty-two patients developed a delirium in the acute phase of stroke. Only patients with a severe and persistent delirium (defined as a DRS of 12 or more for more than 24 hours) were enrolled in the present study. In total 26 fulfilled these criteria of whom 17 were treated with orally administered rivastigmine with a total dose between 3 and 12 mg a day. Eight patients could not be treated because of dysphagia and one because of early discharge.

Results

No major side effects were recorded. In 16 patients there was a considerable decrease in severity of delirium. The mean DRS declined from 14.8 on day one to 8.5 after therapy and 5.6 after tapering. The mean duration of delirium was 6.7 days (range; 2–17).

Conclusion

Rivastigmine is safe in stroke patients with delirium even after rapid titration. In the majority of patients the delirium improved after treatment. A randomized controlled trial is needed to establish the usefulness of rivastigmine in delirium after stroke.

Trial registration

Nederlands Trial Register NTR1395