Distribution of Abeta peptide in whole blood

The measurement of amyloid beta peptides (Abeta) in blood and plasma is expected to be a useful biomarker as potential therapeutics designed to lower Abeta peptide enter clinical trials. Many reports have suggested that Abeta could bind to substances in blood that may influence the recovery of Abeta peptide in plasma, its detection by conventional ELISAs or the actual turnover and half-life of the peptide in blood. In this study we describe a process for analyzing total Abeta in whole blood and plasma using denaturing solid-phase extraction followed by reverse-phase HPLC linked to ELISA. Comparison of total Abeta peptide levels in whole blood and plasma from the same bleed showed that most of the Abeta peptide is captured in the plasma if the samples are first denatured. In contrast, plasma that was assayed without denaturation could show greater than 70% reduction in apparent total Abeta peptide. This suggested that there was a pool of Abeta peptide in non-denatured plasma that is occluded from detection by ELISA, perhaps by binding to plasma proteins.     

Particulate organic carbon in the estuarine turbidity maxima of the Gironde,Loire and Seine estuaries: origin and lability

A study of the particulate organic carbon (POC) in the estuarine turbidity maxima (ETMs) of the three major French macrotidal estuaries shows that the average contents are 1.5, 3.3 and 3.1% (expressed in % of dry suspended sediment) in the Gironde, Loire and Seine Estuaries, respectively. There is no seasonal variation of POC contents in the Gironde Estuary, whereas, they often increase in the Loire and the Seine Estuaries in spring and summer. The lability of the estuarine particulate organic matter was estimated by two analyses: 1/labile organic matter was measured as the organic carbon loss during incubation tests over one month; 2/ the hydrolysable organic fraction was determined after 6N HCl digestion. The organic fractions of the ETMs are mainly refractory. Any increase in the amount of POC as compared to the background levels (cited above) is always correlated to an increase of organic matter lability. The yearly average fluvial contributions by various particulate organic pools (soil and litter organic matter; organic matter of phytoplanktonic and human origin) that enter the three estuaries were quantified. In the Garonne River, soil and litter are the major (90%) POC sources. In the Loire system, due to the eutrophication of the river water, phytoplankton contributes up to 50% of the total POC load. In the Seine river, soil and litter contribute 70% of the total POC input; POC of human origin is also significant (10%), due to the impact of the City of Paris (10 million inhabitants). The lability of the different types of organic matter ranks as follows: phytoplankton ∼litter > human-origin organic matter >> soil. By combining the POC budgets and the lability of each type of organic fraction, it was possible to explain why the POC of the three ETMs is different and characterizes its refractory vs. labile nature.     

High-performance liquid chromatographic determination of glyoxal and methylglyoxal in urine by prederivatization to lumazinic rings using in serial fast scan fluorimetric and diode array detectors

Glyoxal and methylglyoxal are two important markers of oxidative stress and both are involved in the evaluation of several diseases. A new HPLC method for determining glyoxal and methylglyoxal in urine was developed. The method is based on the reaction of alpha-dialdehydes, glyoxal and methylglyoxal, with 5,6-diamino-2,4-hydroxypyrimidine sulfate in basic medium to form highly fluorescent lumazine derivatives. Creatinine was also included in the method even though it does not react with the reagent. The derivatives and creatinine are separated on a C(18) reversed-phase column with a mobile phase consisting of acetonitrile:citrate buffer, pH 6.0 (3:97 v/v). The flow rate was 1.0mLmin(-1) and the effluent was monitored photometrically at 250 nm for determination of creatinine and fluorimetrically at 500 nm (exciting at 330 nm) for determination of glyoxal and methylglyoxal derivatives. Recording time of the separation is less than 10 min. Determination of the analytes is performed in urine after incubation of the sample, with the reagent in alkaline medium, for 30 min at 60 degrees C. Urinary levels of glyoxal and methylglyoxal, expressed as glyoxal/creatinine and methylglyoxal/creatinine ratios, in healthy young women and men were determined. For women, values of 0.80+/-0.37 and 0.60+/-0.22 microg/mg of creatinine were found for glyoxal and methylglyoxal, respectively. For men, values of 0.63+/-0.15 and 0.49+/-0.05 microg/mg of creatinine were found for glyoxal and methylglyoxal, respectively. These results were also related to the body mass index of each individual.     

ENCAPP: elastic-net-based prognosis prediction and biomarker discovery for human cancers

Background

With the explosion of genomic data over the last decade, there has been a tremendous amount of effort to understand the molecular basis of cancer using informatics approaches. However, this has proven to be extremely difficult primarily because of the varied etiology and vast genetic heterogeneity of different cancers and even within the same cancer. One particularly challenging problem is to predict prognostic outcome of the disease for different patients.

Results

Here, we present ENCAPP, an elastic-net-based approach that combines the reference human protein interactome network with gene expression data to accurately predict prognosis for different human cancers. Our method identifies functional modules that are differentially expressed between patients with good and bad prognosis and uses these to fit a regression model that can be used to predict prognosis for breast, colon, rectal, and ovarian cancers. Using this model, ENCAPP can also identify prognostic biomarkers with a high degree of confidence, which can be used to generate downstream mechanistic and therapeutic insights.

Conclusion

ENCAPP is a robust method that can accurately predict prognostic outcome and identify biomarkers for different human cancers.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1465-9) contains supplementary material, which is available to authorized users.     

Plasmacytoid dendritic cells migrate in afferent skin lymph

Conventional dendritic cells enter lymph nodes by migrating from peripheral tissues via the lymphatic route, whereas plasmacytoid dendritic cells (pDC), also called IFN-producing cells (IPC), are described to gain nodes from blood via the high endothelial venules. We demonstrate here that IPC/pDC migrate in the afferent lymph of two large mammals. In sheep, injection of type A CpG oligodinucleotide (ODN) induced lymph cells to produce type I IFN. Furthermore, low-density lymph cells collected at steady state produced type I IFN after stimulation with type A CpG ODN and enveloped viruses. Sheep lymph IPC were found within a minor B(neg)CD11c(neg) subset expressing CD45RB. They presented a plasmacytoid morphology, expressed high levels of TLR-7, TLR-9, and IFN regulatory factor 7 mRNA, induced IFN-gamma production in allogeneic CD4(pos) T cells, and differentiated into dendritic cell-like cells under viral stimulation, thus fulfilling criteria of bona fide pDC. In mini-pig, a CD4(pos)SIRP(pos) subset in afferent lymph cells, corresponding to pDC homologs, produced type I IFN after type A CpG-ODN triggering. Thus, pDC can link innate and acquired immunity by migrating from tissue to draining node via lymph, similarly to conventional dendritic cells.     

RFLP procedure to discriminate between Clausocalanus Giesbrecht, 1888 (Copepoda, Calanoida) species in the Central Cantabrian Sea

Traditionally, zooplankton studies have been carried out on larger species and later developmental stages, at least in the case of copepods. This fact is due to the difficulty of distinguishing between all the morphologically very similar species and genus, especially at immature stages. Nowadays, however molecular techniques allow us to discriminate these species easily. We have developed a RFLP (Restriction Fragment Length Polymorphism) technique to distinguish between the species of the genus Clausocalanus which are present at our study zone, the Central Cantabrian Sea. This genus belongs to the “small” fraction of mesozooplankton (500-200 μm), and it has been scarcely studied even though it is an important component of the mesozooplankton. This technique allows identification of individuals and from small body parts.     

Fe65 interacts with P2X2 subunits at excitatory synapses and modulates receptor function

Ionotropic receptors in the neuronal plasma membrane are organized in macromolecular complexes, which assure their proper localization and regulate signal transduction. P2X receptors, the ionotropic receptors activated by extracellular ATP, have been shown to influence synaptic transmission. Using a yeast two-hybrid approach with the P2X(2) subunit C-terminal domain as bait we isolated the beta-amyloid precursor protein-binding proteins Fe65 and Fe65-like 1 as the first identified proteins interacting with neuronal P2X receptors. We confirmed the direct interaction of Fe65 and the P2X(2) C-terminal domain by glutathione S-transferase pull-down experiments. No interaction was observed between Fe65 and the naturally occurring P2X(2) splice variant P2X(2(b)), indicating that alternative splicing can regulate the receptor complex assembly. We generated two antibodies to Fe65 to determine its subcellular localization using postembedding immunogold labeling electron microscopy. We found labeling for Fe65 at the pre- and postsynaptic specialization of CA1 hippocampal pyramidal cell/Schaffer collateral synapses. By double immunogold labeling, we determined that Fe65 colocalizes with P2X(2) subunits at the postsynaptic specialization of excitatory synapses. Moreover, P2X(2) and Fe65 could be coimmunoprecipitated from brain membrane extracts, demonstrating that the interaction occurs in vivo. The assembly with Fe65 regulates the functional properties of P2X(2) receptors. Thus, the time- and activation-dependent change in ionic selectivity of P2X(2) receptors was inhibited by coexpression of Fe65, suggesting a novel role for Fe65 in regulating P2X receptor function and ATP-mediated synaptic transmission.     

Differential expression of spleen tyrosine kinase Syk isoforms in tissues: effects of the microbial flora

Spleen tyrosine kinase (Syk) is expressed widely in hematopoietic and non-hematopoietic cells. The widespread distribution of Syk and its involvement in host defense and allergic reactions, prompted us analyze the influence of microbial exposure on Syk expression. We compared the distribution of Syk in various tissues of germ-free and conventional mice using immunohistochemistry, Western blot analysis and real time RT-PCR. Total Syk expression was similar between germ-free and conventional mice. Since it has been claimed that Syk isoforms are differentially expressed, we studied the distribution and abundance of Syk (L) and Syk (S) isoforms in tissues from these mice. In contrast to previous reports, we found broad tissue expression of Syk (S). Interestingly, in germ-free mice the amount of Syk (S) but not Syk L protein was selectively increased in lung and spleen. In summary, our study reveals new and broad tissue expression of both Syk isoforms and demonstrates that lack of microbial flora results in selectively increased expression of Syk (S) isoform in lung and spleen.Florentina Duta, Marina Ulanova and Daniel Seidel are joint first authors. Ulrich Steinhoff and A. Dean Befus are joint last authors.M. Ulanova received a postdoctoral fellowship from the Canadian Society of Allergy and Clinical Immunology/Merck Frosst and from the Alberta Heritage Foundation for Medical Research.     

Regulation and function of Cav3.1 T-type calcium channels in IGF-I-stimulated pulmonary artery smooth muscle cells

Arterial smooth muscle cells enter the cell cycle and proliferate in conditions of disease and injury, leading to adverse vessel remodeling. In the pulmonary vasculature, diverse stimuli cause proliferation of pulmonary artery smooth muscle cells (PASMCs), pulmonary artery remodeling, and the clinical condition of pulmonary hypertension associated with significant health consequences. PASMC proliferation requires extracellular Ca(2+) influx that is intimately linked with intracellular Ca(2+) homeostasis. Among the primary sources of Ca(2+) influx in PASMCs is the low-voltage-activated family of T-type Ca(2+) channels; however, up to now, mechanisms for the action of T-type channels in vascular smooth muscle cell proliferation have not been addressed. The Ca(v)3.1 T-type Ca(2+) channel mRNA is upregulated in cultured PASMCs stimulated to proliferate with insulin-like growth factor-I (IGF-I), and this upregulation depends on phosphatidylinositol 3-kinase/Akt signaling. Multiple stimuli that trigger an acute rise in intracellular Ca(2+) in PASMCs, including IGF-I, also require the expression of Ca(v)3.1 Ca(2+) channels for their action. IGF-I also led to cell cycle initiation and proliferation of PASMCs, and, when expression of the Ca(v)3.1 Ca(2+) channel was knocked down by RNA interference, so were the expression and activation of cyclin D, which are necessary steps for cell cycle progression. These results confirm the importance of T-type Ca(2+) channels in proper progression of the cell cycle in PASMCs stimulated to proliferate by IGF-I and suggest that Ca(2+) entry through Ca(v)3.1 T-type channels in particular interacts with Ca(2+)-dependent steps of the mitogenic signaling cascade as a central component of vascular remodeling in disease.     

Ectodermal P2X receptor function plays a pivotal role in craniofacial development of the zebrafish

P2X receptors are non-selective cation channels operated by extracellular ATP. Currently, little is known concerning the functions of these receptors during development. Previous work from our lab has shown that zebrafish have two paralogs of the mammalian P2X3 receptor subunit. One paralog, p2rx3.1, is expressed in subpopulations of neural and ectodermal cells in the embryonic head. To investigate the role of this subunit in early cranial development, we utilized morpholino oligonucleotides to disrupt its translation. Loss of this subunit resulted in craniofacial defects that included malformation of the pharyngeal skeleton. During formation of these structures, there was a marked increase in cell death within the branchial arches. In addition, the epibranchial (facial, glossopharyngeal, and vagal) cranial sensory ganglia and their circuits were perturbed. These data suggest that p2rx3.1 function in ectodermal cells is involved in purinergic signaling essential for proper craniofacial development and sensory circuit formation in the embryonic and larval zebrafish.