Prenatal nicotine affects catecholamine gene expression in newborn rat carotid body and petrosal ganglion

Nicotine exposure modifiesthe expression of catecholamine and opioid neurotransmitter systemsinvolved in attenuation of hypoxic chemosensitivity. We used insitu hybridization histochemistry to determine the effect of prenataland early postnatal nicotine exposure on tyrosine hydroxylase (TH),dopamine -hydroxylase (DH), preproenkephalin (PPE), andD₂-dopamine receptor mRNA levels in the rat carotid bodyand petrosal ganglion during postnatal development. In the carotidbody, nicotine increased TH mRNA expression in animals at 0 and 3 postnatal days (both, P < 0.05 vs. control) withoutaffecting TH mRNA levels at 6 and 15 days. At 15 postnatal days, DHmRNA levels were increased in the carotid body of nicotine-exposed animals. Dopamine D₂-receptor mRNA levels in the carotidbody increased with postnatal age but were unaffected by nicotineexposure. PPE was not expressed in the carotid body at any of the agesstudied in control or treated animals. In the petrosal ganglion,nicotine increased the number of ganglion cells expressing TH mRNA inanimals at 3 days (P < 0.01 vs. control). DH mRNAexpression was not induced nor was PPE mRNA expression increased in thepetrosal ganglion in treated animals. Prenatal nicotine exposureupregulates mRNAs involved in the synthesis of two inhibitoryneuromodulators, dopamine and norepinephrine, in peripheral arterialchemoreceptors, which may contribute to abnormalities incardiorespiratory control observed in nicotine exposed animals.

     

Treating Cattle to Protect People? Impact of Footbath Insecticide Treatment on Tsetse Density in Chad

Background

In Chad, several species of tsetse flies (Genus: Glossina ) transmit African animal trypanosomoses (AAT), which represents a major obstacle to cattle rearing, and sleeping sickness, which impacts public health. After the failure of past interventions to eradicate tsetse, the government of Chad is now looking for other approaches that integrate cost-effective intervention techniques, which can be applied by the stake holders to control tsetse-transmitted trypanosomoses in a sustainable manner. The present study thus attempted to assess the efficacy of restricted application of insecticides to cattle leg extremities using footbaths for controlling Glossina m. submorsitans, G . tachinoides and G . f . fuscipes in southern Chad.

Methodology/Principal Findings

Two sites were included, one close to the historical human African trypanosomiasis (HAT) focus of Moundou and the other to the active foci of Bodo and Moissala. At both sites, a treated and an untreated herd were compared. In the treatment sites, cattle were treated on a regular basis using a formulation of deltamethrin 0.005% (67 to 98 cattle were treated in one of the sites and 88 to 102 in the other one). For each herd, tsetse densities were monthly monitored using 7 biconical traps set along the river and beside the cattle pen from February to December 2009. The impact of footbath treatment on tsetse populations was strong (p < 10^-3) with a reduction of 80% in total tsetse catches by the end of the 6-month footbath treatment.

Conclusions/Significance

The impact of footbath treatment as a vector control tool within an integrated strategy to manage AAT and HAT is discussed in the framework of the “One Health” concept. Like other techniques based on the treatment of cattle, this technology should be used under controlled conditions, in order to avoid the development of insecticide and acaricide resistance in tsetse and tick populations, respectively.     

The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.     

Prostasomes from four different species are able to produce extracellular adenosine triphosphate (ATP)

Background

Prostasomes are extracellular vesicles. Intracellularly they are enclosed by another larger vesicle, a so called “storage vesicle” equivalent to a multivesicular body of late endosomal origin. Prostasomes in their extracellular context are thought to play a crucial role in fertilization.

Methods

Prostasomes were purified according to a well worked-out schedule from seminal plasmas obtained from human, canine, equine and bovine species. The various prostasomes were subjected to SDS-PAGE separation and protein banding patterns were compared. To gain knowledge of the prostasomal protein systems pertaining to prostasomes of four different species proteins were analyzed using a proteomic approach. An in vitro assay was employed to demonstrate ATP formation by prostasomes of different species.

Results

The SDS-PAGE banding pattern of prostasomes from the four species revealed a richly faceted picture with most protein bands within the molecular weight range of 10–150 kDa. Some protein bands seemed to be concordant among species although differently expressed and the number of protein bands of dog prostasomes seemed to be distinctly fewer. Special emphasis was put on proteins involved in energy metabolic turnover. Prostasomes from all four species were able to form extracellular adenosine triphosphate (ATP). ATP formation was balanced by ATPase activity linked to the four types of prostasomes.

Conclusion

These potencies of a possession of functional ATP-forming enzymes by different prostasome types should be regarded against the knowledge of ATP having a profound effect on cell responses and now explicitly on the success of the sperm cell to fertilize the ovum.

General significance

This study unravels energy metabolic relationships of prostasomes from four different species.     

Characterization of the peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) fromSilene alba cells

The peptide-N ⁴-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) earlier described [Lhernould S., Karamanos Y., Bourgerie S., Strecker G., Julien R., Morvan H. (1992)Glycoconjugate J 9:191–97] was partially purified from culturedSilene alba cells using affinity chromatography. The enzyme is active between pH 3.0 and 6.5, and is stable in the presence of moderate concentrations of several other protein unfolding chemicals, but is readily inactivated by SDS. Although the enzyme cleaves the carbohydrate from a variety of animal and plant glycopeptides, it does not hydrolyse the carbohydrate from most of the corresponding unfolded glycoproteins in otherwise comparable conditions. The substrate specificity of this plant PNGase supports the hypothesis that this enzyme could be at the origin of the production of unconjugated N-glycans in a suspension medium of culturedSilene alba cells.Abbreviations GlcNAc N-acetylglucosamine - PNGase peptide-N ⁴-(N-acetylglucosaminyl) asparagine amidase - BSA bovine serum albumin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TLC thin layer chromatography - HPAEC-PAD High pH anion exchange chromatography-pulsed amperometric detection     

Silencing of the GDP-D-mannose 3,5-epimerase affects the structure and cross-linking of the pectic polysaccharide rhamnogalacturonan II and plant growth in tomato

L-galactose (L-Gal), a monosaccharide involved in L-ascorbate and rhamnogalacturonan II (RG-II) biosynthesis in plants, is produced in the cytosol by a GDP-D-mannose 3,5-epimerase (GME). It has been recently reported that the partial inactivation of GME induced growth defects affecting both cell division and cell expansion (Gilbert, L., Alhagdow, M., Nunes-Nesi, A., Quemener, B., Guillon, F., Bouchet, B., Faurobert, M., Gouble, B., Page, D., Garcia, V., Petit, J., Stevens, R., Causse, M., Fernie, A. R., Lahaye, M., Rothan, C., and Baldet, P. (2009) Plant J. 60, 499-508). In the present study, we show that the silencing of the two GME genes in tomato leaves resulted in approximately a 60% decrease in terminal L-Gal content in the side chain A of RG-II as well as in a lower capacity of RG-II to perform in muro cross-linking. In addition, we show that unlike supplementation with L-Gal or ascorbate, supplementation of GME-silenced lines with boric acid was able to restore both the wild-type growth phenotype of tomato seedlings and an efficient in muro boron-mediated cross-linking of RG-II. Our findings suggest that developmental phenotypes in GME-deficient lines are due to the structural alteration of RG-II and further underline the crucial role of the cross-linking of RG-II in the formation of the pectic network required for normal plant growth and development.     

Respective Roles of Culturable and Viable-but-Nonculturable Cells in the Heterogeneity of Salmonella enterica Serovar Typhimurium Invasiveness

The existence of Salmonella enterica serovar Typhimurium viable-but-nonculturable (VBNC) cells is a public health concern since they could constitute unrecognized sources of infection if they retain their pathogenicity. To date, many studies have addressed the ability of S. Typhimurium VBNC cells to remain infectious, but their conclusions are conflicting. An assumption could explain these conflicting results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state and that most VBNC cells generated under intense stress could exceed the stage where they are still infectious. Using a Radioselectan density gradient centrifugation technique makes it possible to increase the VBNC-cell/culturable-cell ratio without increasing the exposure to stress and, consequently, to work with a larger proportion of newly VBNC cells. Here, we observed that (i) in the stationary phase, the S. Typhimurium population comprised three distinct subpopulations at 10, 24, or 48 h of culture; (ii) the VBNC cells were detected at 24 and 48 h; (iii) measurement of invasion gene (hilA, invF, and orgA) expression demonstrated that cells are highly heterogeneous within a culturable population; and (iv) invasion assays of HeLa cells showed that culturable cells from the different subpopulations do not display the same invasiveness. The results also suggest that newly formed VBNC cells are either weakly able or not able to successfully initiate epithelial cell invasion. Finally, we propose that at entry into the stationary phase, invasiveness may be one way for populations of S. Typhimurium to escape stochastic alteration leading to cell death.Like several readily culturable pathogenic bacterial species, Salmonella enterica has been shown to enter into a viable-but-nonculturable (VBNC) state in response to environmental stresses (25, 33). In this state, cells display integrity and activities but escape detection by conventional culture-based monitoring (24). The physiological significance of this phenotype is unclear: some authors have proposed that it is part of an adaptive response aimed at long-term survival under adverse conditions (22, 32); others argue that it is a consequence of stochastic cellular deterioration and that VBNC cells are on their way to death (4, 10, 12, 23). In any case, the existence of VBNC pathogens is a public health concern since they may constitute unrecognized sources of infection if they retain their pathogenicity.To date, many studies have addressed the ability of VBNC pathogens to remain infectious, but the conclusions of some investigators are conflicting (15, 36). In vitro experiments have shown that VBNC cells of Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Oranienburg can recover their culturability (13, 27, 30, 31). This phenomenon, called resuscitation, confirms that at least some VBNC cells ultimately remain able to multiply and are therefore potentially infectious. On the other hand, most in vivo studies ruled out the ability of S. Typhimurium VBNC cells to initiate infection in mice and chicken or to resuscitate during their passage in the animal gut (6, 17, 34, 35). However, one study reported evidence of the maintenance of pathogenicity by VBNC cells of S. Oranienburg in a model of morphine-immunosuppressed mice (1). An assumption could explain these apparently opposite results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state (8, 19, 26). Experiments intended for testing the ability of VBNC cells to retain their pathogenicity cannot be fully conclusive if the inocula still contain culturable cells. Therefore, all previously published animal experiments with S. Typhimurium were conducted on populations with VBNC-cell/culturable-cell ratios around 10,000:1. Such populations were obtained after strong exposure to stress, either under intense stressing factors for a short period (e.g., germicidal UV-C for 2 min [6]) or under mild stressing factors for a long period (e.g., starvation for a minimum of 1 week [35]). In such populations, most VBNC cells could exceed the stage where they are still infectious, and the negative outcomes of infection studies could actually reflect their inability to specifically address the fraction of recent VBNC cells.A Radioselectan density gradient centrifugation technique was shown to fractionate stationary-phase populations of Escherichia coli into two subpopulations (10, 12, 18). Interestingly, the VBNC cells formed during a 48-h E. coli culture were specifically recovered in the high-density (HD) subpopulation (12). This technique thus gives the opportunity to increase the VBNC-cell/culturable-cell ratio without increasing exposure to stress and, consequently, to work with a larger proportion of cells having recently entered the VBNC state.Here, this technique was used to discriminate different stationary-phase S. Typhimurium subpopulations. We further investigated the invasiveness of these cell subpopulations by using both gene expression assays of invasion genes and in vitro invasion tests. Thus, the aim of this study was to assess the invasiveness of the cell subpopulations in accordance with their cellular states.     

Structural and dynamic studies of two antigenic loops from haemagglutinin: A relaxation matrix approach

Summary We have investigated the dynamics and structural behaviour of two antigenic peptides using ¹H NMR. The two cyclic peptides mimic the antigenic site A of influenza haemagglutinin protein; they only differ in the way they were cyclized and in the size of their respective linkers. Homonuclear relaxation parameters extracted from a complete NOE matrix were interpreted in terms of local dynamics. A set of distance constraints was deduced from these parameters which allowed 3D models to be constructed using distance geometry. NOE back-calculation was used to check the validity of the final models. Strong variations of internal motion amplitude have been found in both peptides along their backbone. Motions with high amplitudes have been localized in the Gly-Pro-Gly sequence which forms a -turn in both structures.Abbreviations DSS 3-(trimethylsilyl)-1-propanesulfonic acid - D-loop aspartic acid loop - ELISA enzyme-linked immunoabsorbent assay - f.i.d free induction decay - HOHAHA homonuclear Hartmann-Hahn spectroscopy - HPLC high pressure liquid chromatography - K-loop lysine loop - NMR nuclear magnetic resonance - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - r.m.s.d. root-mean-square deviation of atomic positions     

The plant-specific ssDNA binding protein OSB1 is involved in the stoichiometric transmission of mitochondrial DNA in Arabidopsis

Plant mitochondrial genomes exist in a natural state of heteroplasmy, in which substoichiometric levels of alternative mitochondrial DNA (mtDNA) molecules coexist with the main genome. These subgenomes either replicate autonomously or are created by infrequent recombination events. We found that Arabidopsis thaliana OSB1 (for Organellar Single-stranded DNA Binding protein1) is required for correct stoichiometric mtDNA transmission. OSB1 is part of a family of plant-specific DNA binding proteins that are characterized by a novel motif that is required for single-stranded DNA binding. The OSB1 protein is targeted to mitochondria, and promoter-beta-glucuronidase fusion showed that the gene is expressed in budding lateral roots, mature pollen, and the embryo sac of unfertilized ovules. OSB1 T-DNA insertion mutants accumulate mtDNA homologous recombination products and develop phenotypes of leaf variegation and distortion. The mtDNA rearrangements occur in two steps: first, homozygous mutants accumulate subgenomic levels of homologous recombination products; second, in subsequent generations, one of the recombination products becomes predominant. After the second step, the process is no longer reversible by backcrossing. Thus, OSB1 participates in controlling the stoichiometry of alternative mtDNA forms generated by recombination. This regulation could take place in gametophytic tissues to ensure the transmission of a functional mitochondrial genome.     

Candidate Luminal B Breast Cancer Genes Identified by Genome,Gene Expression and DNA Methylation Profiling

Breast cancers (BCs) of the luminal B subtype are estrogen receptor-positive (ER+), highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs), DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15) and UTRN (6q24), were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype.