Quantitative Proteomics in Yeast : From bSLIM and Proteome Discoverer Outputs to Graphical Assessment of the Significance of Protein Quantification Scores.

Simple light isotope metabolic labeling (bSLIM) is an innovative method to accurately quantify differences in protein abundance at the proteome level in standard bottom-up experiments. The quantification process requires computation of the ratio of intensity of several isotopologs in the isotopic cluster of every identified peptide. Thus, appropriate bioinformatic workflows are required to extract the signals from the instrument files and calculate the required ratio to infer peptide/protein abundance. In a previous study (Sénécaut et al., J Proteome Res 20:1476-1487, 2021), we developed original open-source workflows based on OpenMS nodes implemented in a KNIME working environment. Here, we extend the use of the bSLIM labeling strategy in quantitative proteomics by presenting an alternative procedure to extract isotopolog intensities and process them by taking advantage of new functionalities integrated into the Minora node of Proteome Discoverer 2.4 software. We also present a graphical strategy to evaluate the statistical robustness of protein quantification scores and calculate the associated false discovery rates (FDR). We validated these approaches in a case study in which we compared the differences between the proteomes of two closely related yeast strains.

Factors influencing acceptability of voluntary HIV testing among pregnant women in Gamboma, Republic of Congo.

BACKGROUND: This study was carried out to identify factors affecting the acceptability of voluntary HIV testing among pregnant women in a semi-rural city, Gamboma, Republic of Congo. METHODS: A cross-sectional study was conducted between January and September 2012. Pregnant women attending antenatal heath care at an integrated health center were enrolled after informed consent and followed through voluntary HIV testing. RESULTS: Among 136 participants, 98 women (72 %) accepted voluntary HIV testing after pre-test counseling. Women with basic education, those who cited blood transfusion as a mode of transmission and prevention of mother-to-child transmission (MTCT) were more likely to accept testing as well those informed about free HIV testing. Interestingly, pregnant women who had heard about HIV/AIDS from hospital setting were less likely to accept testing. CONCLUSIONS: Our data indicate that increasing general education on HIV transmission/prevention modes is crucial for increasing acceptability of screening. Furthermore, HIV/AIDS knowledge disseminated to patients in hospital settings should be carefully monitored. Lastly, scaling-up MTCT services along with a better and larger community information, may address accessibility barriers observed in the present study.

Prostaglandin E2 release from astrocytes triggers gonadotropin-releasing hormone (GnRH) neuron firing via EP2 receptor activation.

Astrocytes in the hypothalamus release prostaglandin E(2) (PGE(2)) in response to cell-cell signaling initiated by neurons and glial cells. Upon release, PGE(2) stimulates the secretion of gonadotropin-releasing hormone (GnRH), the neuropeptide that controls reproduction, from hypothalamic neuroendocrine neurons. Whether this effect on GnRH secretion is accompanied by changes in the firing behavior of these neurons is unknown. Using patch-clamp recording we demonstrate that PGE(2) exerts a dose-dependent postsynaptic excitatory effect on GnRH neurons. These effects are mimicked by an EP2 receptor agonist and attenuated by protein kinase A (PKA) inhibitors. The acute blockade of prostaglandin synthesis by indomethacin (INDO) or the selective inhibition of astrocyte metabolism by fluoroacetate (FA) suppresses the spontaneous firing activity of GnRH neurons in brain slices. Similarly, GnRH neuronal activity is reduced in mice with impaired astrocytic PGE(2) release due to defective erbB signaling in astrocytes. These results indicate that astrocyte-to-neuron communication in the hypothalamus is essential for the activity of GnRH neurons and suggest that PGE(2) acts as a gliotransmitter within the GnRH neurosecretory system.

Phenotypic and molecular characterization of proliferating and differentiated GnRH-expressing GnV-3 cells.

GnRH neurons provide the primary driving force upon the neuroendocrine reproductive axis. Here we used GnV-3 cells, a model of conditionally immortalized GnRH-expressing neurons, to perform an analysis of cell cycle and compare the gene expression profile of proliferating cells with differentiated cells. In the proliferation medium, 45 ± 1.5% of GnV-3 cells are in S-phase by FACS analysis. In the differentiation medium, only 9 ± 0.9% of them are in S-phase, and they acquire the characteristic bipolar shape displayed by preoptic GnRH neurons in vivo. In addition, GnV-3 cells in the differentiated state exhibit electrophysiological properties characteristic of neurons. Transcriptomic analysis identified up-regulation of 1931 genes and down-regulation of 1270 genes in cells grown in the differentiation medium compared to cells in the proliferation medium. Subsequent gene ontology study indicated that genes over-expressed in proliferating GnV-3 cells were mainly involved in cell cycle regulations, whereas genes over-expressed in differentiated cells were mainly involved in processes of differentiation, neurogenesis and neuronal morphogenesis. Taken together, these data demonstrate the occurrence of morphological and physiological changes in GnV-3 cells between the proliferating and the differentiated state. Moreover, the genes differentially regulated between these two different states are providing novel pathways potentially important for a better understanding of the physiology of mature GnRH neurons.

A new method for class prediction based on signed-rank algorithms applied to Affymetrix microarray experiments.

BACKGROUND: The huge amount of data generated by DNA chips is a powerful basis to classify various pathologies. However, constant evolution of microarray technology makes it difficult to mix data from different chip types for class prediction of limited sample populations. Affymetrix(R) technology provides both a quantitative fluorescence signal and a decision (detection call: absent or present) based on signed-rank algorithms applied to several hybridization repeats of each gene, with a per-chip normalization. We developed a new prediction method for class belonging based on the detection call only from recent Affymetrix chip type. Biological data were obtained by hybridization on U133A, U133B and U133Plus 2.0 microarrays of purified normal B cells and cells from three independent groups of multiple myeloma (MM) patients. RESULTS: After a call-based data reduction step to filter out non class-discriminative probe sets, the gene list obtained was reduced to a predictor with correction for multiple testing by iterative deletion of probe sets that sequentially improve inter-class comparisons and their significance. The error rate of the method was determined using leave-one-out and 5-fold cross-validation. It was successfully applied to (i) determine a sex predictor with the normal donor group classifying gender with no error in all patient groups except for male MM samples with a Y chromosome deletion, (ii) predict the immunoglobulin light and heavy chains expressed by the malignant myeloma clones of the validation group and (iii) predict sex, light and heavy chain nature for every new patient. Finally, this method was shown powerful when compared to the popular classification method Prediction Analysis of Microarray (PAM). CONCLUSION: This normalization-free method is routinely used for quality control and correction of collection errors in patient reports to clinicians. It can be easily extended to multiple class prediction suitable with clinical groups, and looks particularly promising through international cooperative projects like the "Microarray Quality Control project of US FDA" MAQC as a predictive classifier for diagnostic, prognostic and response to treatment. Finally, it can be used as a powerful tool to mine published data generated on Affymetrix systems and more generally classify samples with binary feature values.

Activation of neuronal nitric oxide release inhibits spontaneous firing in adult gonadotropin-releasing hormone neurons: a possible local synchronizing signal.

The activation of nitric oxide (NO) signaling pathways in hypothalamic neurons plays a key role in the control of GnRH secretion that is central to reproductive function. It is unknown whether NO directly modulates the firing behavior of GnRH neurons in the preoptic region of the mature brain. Using patch-clamp recordings from GnRH neurons expressing green fluorescent protein in adult mice brain slices, we demonstrate that the NO precursor, L-arginine (Arg), or the NO donor, diethylamine/NO, induced a robust and reversible reduction in the spontaneous firing activity of GnRH neurons, including bursting activity. The effects of L-Arg were prevented by the NO synthase inhibitor N omega-nitro-L-Arg methyl ester hydrochloride. Histochemical studies revealing a close anatomical relationship between neurons producing NO and GnRH perikarya, together with the loss of the L-Arg-mediated inhibition of GnRH neuronal activity via the selective blockade of neuronal NO synthase, suggested that the primary source of local NO production in the mouse preoptic region was neuronal. Synaptic transmission uncoupling did not alter the effect of NO, suggesting that NO affects the firing pattern of GnRH neurons by acting at a postsynaptic site. We also show that the NO-mediated changes in membrane properties in the GnRH neurons require soluble guanylyl cyclase activity and may involve potassium conductance. By revealing that NO is a direct modulator of GnRH neuronal activity, our results introduce the intriguing possibility that this gaseous neurotransmitter may be used by the sexual brain to modulate burst firing patterns. It may set into phase the bursting activity of GnRH neurons at key stages of reproductive physiology.

Motor cortical control of cardiovascular bulbar neurones projecting to spinal autonomic areas.

There is evidence that the motor cortex is involved in cardiovascular adjustments associated with somatic motor activity, as it has functional connections with the ventrolateral medulla, a brainstem region critically involved in the control of blood pressure and the regulation of plasma catecholamine levels. The ventrolateral medulla sends projections to the spinal intermediolateral nucleus, where preganglionic neurones controlling heart and blood vessels (T2 segment) and adrenal medulla (T8 segment) are found. The aim of the present study was to determine whether electrical stimulation of the rat motor cortex induces cardiovascular responses and Fos expression in ventrolateral medulla neurones projecting to the T2 and T8 segments. After a set of experiments designed to record cardiovascular parameters (blood pressure and plasma catecholamine levels), injections of retrograde tracer (Fluorogold) were performed in the intermediolateral nucleus of two groups of rats, at the T2 or at the T8 segmental levels. Five days later, the motor cortex was stimulated in order to induce Fos expression in the ventrolateral medulla. Stimulation of the motor cortex induced: (1). hypotension and a significant decrease in plasma noradrenaline levels, and (2). a significant increase in the number of the double-labelled neurones in the rostral ventrolateral medulla projecting to T2. These data demonstrate that cardiovascular adjustments, preparatory to, or concomitant with, motor activity may be initiated in the motor cortex and transmitted to cardiac and vasomotor spinal preganglionic neurones, via the ventrolateral medulla.

A confocal microscopic study of gonadotropin-releasing hormone (GnRH) neuron inputs to dopaminergic neurons containing estrogen receptor alpha in the arcuate nucleus of GnRH-green fluorescent protein transgenic mice.

The purpose of the present study was to determine whether the septo-preoptico-tuberoinfundibular gonadotropin-releasing hormone (GnRH) pathway comes in close juxtaposition with tyrosine hydroxylase immunoreactive (TH-IR) neurons in the arcuate nucleus of female mice. Immunohistochemical staining with a TH monoclonal antibody coupled with confocal microscopy was employed on vibratome-cut brain sections of female GnRH-green fluorescent protein (GFP) transgenic mice to evaluate possible appositions between GnRH and tuberoinfundibular dopaminergic (TIDA) neurons. TH-IR neurons of the arcuate nucleus received GnRH neuronal appositions in adult female mice at proestrus and estrus stages. In contrast, no GnRH appositions were observed in adult females at diestrus. Subsequently, double immunohistochemical staining for TH and estrogen receptor-alpha (ERalpha) was performed to examine the role of estradiol on this relationship. We found that most TH-IR neurons contacted by GnRH fibers were immunoreactive for ERalpha. Our observations suggest that GnRH neurons communicate directly with TIDA neurons in the adult female. Furthermore, ERalpha activation in TIDA neurons may be involved in the formation of connections between GnRH neurons and TIDA neurons.