Bacterial Distribution in the Rhizosphere of Wild Barley under Contrasting Microclimates

Background: All plants in nature harbor a diverse community of rhizosphere bacteria which can affect the plant growth. Our samples are isolated from the rhizosphere of wild barley Hordeum spontaneum at the Evolution Canyon (‘EC’), Israel. The bacteria which have been living in close relationship with the plant root under the stressful conditions over millennia are likely to have developed strategies to alleviate plant stress. Methodology/Principal Findings: We studied distribution of culturable bacteria in the rhizosphere of H. spontaneum and characterized the bacterial 1-aminocyclopropane-1-carboxylate deaminase (ACCd) production, biofilm production, phosphorus solubilization and halophilic behavior. We have shown that the H. spontaneum rhizosphere at the stressful South Facing Slope (SFS) harbors significantly higher population of ACCd producing biofilm forming phosphorus solubilizing osmotic stress tolerant bacteria. Conclusions/Significance: The long-lived natural laboratory ‘EC ’ facilitates the generation of theoretical testable and predictable models of biodiversity and genome evolution on the area of plant microbe interactions. It is likely that the bacteria isolated at the stressful SFS offer new opportunities for the biotechnological applications in our agro-ecologica

Porcine circovirus type 2 ORF3 protein induces apoptosis in melanoma cells

Abstract Background The current treatment of malignant melanoma is limited by the lack of effective therapeutic approaches, and alternative treatments are needed. Proliferative diseases such as melanoma and other cancers may be treatable by virally-encoded apoptotic proteins that are targeted to rapidly multiplying cells. Caspase-dependent apoptosis, that is frequently used in chemotherapy, can boost the cell proliferation that caspase-independent cell death does not. Methods In the current study, the porcine circovirus type 2 (PCV2), proapoptotic protein ORF3 was expressed in mouse and human cancer cell lines, and its apoptotic activity was assessed. Results Quantitative assessment of the apoptotic cells by flow cytometry showed that apoptotic cell death was significantly increased in ORF3-expressing malignant cells, compared to ORF3 non-expressing cells. Our data show that PCV2 ORF3 induces apoptosis in a caspase-3 and -8 independent manner. ORF3 expression seems to cause an increase in abnormal mitosis in B16F10 melanoma cells by interacting with centrosomes and thereby disrupting the formation of the mitotic spindle. In addition, we show that ORF3 of PCV2 also exhibits significant anti-tumor effects in vivo. Although the expression of Regulator of G protein Signaling (RGS)-16 by recipient mice inhibited the development of grafted melanoma in vivo, it was not required for the antitumoral activity of ORF3. Conclusion PCV2 ORF3 causes abnormal mitosis in rapidly dividing cells and increases the apoptosis of cancer cells. Apoptin might, therefore, be considered to develop future antitumoral strategies

Endospore forming bacterial distribution on the ‘EC’ SFS and NFS slopes.

1<p>The endospore forming bacterial fraction was isolated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>.</p>2<p>ACC (1-Aminocyclopropane-1-carboxylate) medium was composed of salts for <i>P. polymyxa</i> minimal medium there the nitrogen source is replaced with ACC.</p>3<p>Biofilm formation was estimated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>. The isolates with OD >1 were considered as biofilm formers.</p>4<p>Halophilic growth was determined in the medium containing 2 M NaCl (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>).</p>5<p>P solubilization was estimated using NBRIP medium (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>). The colonies with index >2 were considered as P solubilizers.</p

Cross section of the ‘Evolution Canyon’ indicating the collection sites on South Facing Slope (SFS) 1 and 2 and North Facing Slope (NFS) 5 and 7.

<p>Cross section of the ‘Evolution Canyon’ indicating the collection sites on South Facing Slope (SFS) 1 and 2 and North Facing Slope (NFS) 5 and 7.</p

Partially disordered structure in intravirus coat protein of potyvirus potato virus A.

Potyviruses represent the most biologically successful group of plant viruses, but to our knowledge, this work is the first detailed study of physicochemical characteristics of potyvirus virions. We measured the UV absorption, far and near UV circular dichroism spectra, intrinsic fluorescence spectra, and differential scanning calorimetry (DSC) melting curves of intact particles of a potato virus A (PVA). PVA virions proved to have a peculiar combination of physicochemical properties. The intravirus coat protein (CP) subunits were shown to contain an unusually high fraction of disordered structures, whereas PVA virions had an almost normal thermal stability. Upon heating from 20 °C to 55 °C, the fraction of disordered structures in the intravirus CP further increased, while PVA virions remained intact at up to 55 °C, after which their disruption (and DSC melting) started. We suggest that the structure of PVA virions below 55 °C is stabilized by interactions between the remaining structured segments of intravirus CP. It is not improbable that the biological efficiency of PVA relies on the disordered structure of intravirus CP

Bacterial distribution on the ‘EC’SFS and NFS slopes.

1<p>The data are expressed per gram of fresh weight. Wild barley roots were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>.</p><p>Each data point represents three independent experiments. The culturable aerobic fraction of the total bacterial CFUs was determined on TSA plates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a> s-sun area; sh-shaded area.</p>2<p>Spores were counted using Shaeffer-Fulton staining.</p>3<p>ACC (1-Aminocyclopropane-1-carboxylate) medium was composed of salts for <i>P. polymyxa</i> minimal medium there the nitrogen source is replaced with ACC.</p>4<p>Biofilm formation was estimated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>. The isolates with OD >1 were considered as biofilm formers.</p>5<p>Halophilic growth was determined in the medium containing 2 M NaCl (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>).</p>6<p>P solubilization was estimated using NPRJP medium (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>). The colonies with index >2 were considered as P solubilizers.</p

Endospore forming bacterial phosphorus solubilization.

1<p>Endospore forming bacterial isolates as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#pone-0017968-t002" target="_blank">Table 2</a>. Solubilization index and pH change was studied of each isolate in three independent experiments.</p>2<p>Bacterial phosphorus solubilization index was determined after bacterial growth in NBRIP agar plates for 7 days at 30C (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>).</p>3<p>Change of pH by the bacteria was determined after 7 days of incubation in NBRIP broth (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>).</p><p>*<i>P. polymyxa, B. megaterium, B. cereus</i> and <i>B. pumilus</i> SFS and NFS isolate values (mean value±SE) were compared. Numbers followed by different letter are significantly different (P<0.05) according to Fisher's least significance difference test.</p

Solid surface assay of the South Facing Slope (SFS) and North Facing Slope (NFS) bacterial biofilm formation.

<p>The crystal violet assay was used to measure solid surface biofilm formation at 30C. Preparation and analysis were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017968#s2" target="_blank">Material and Methods</a>.</p

Human Peripheral Blood Eosinophils Express High Levels of the Purinergic Receptor P2X4

International audienceExtracellular nucleotides are important mediators of cell activation and trigger multiple responses via membrane receptors known as purinergic receptors (P2). P2X receptors are ligand-gated ion channels, activated by extracellular ATP. P2X4 is one of the most sensitive purinergic receptors, that is typically expressed by neurons, microglia, and some epithelial and endothelial cells. P2X4 mediates neuropathic pain via brain-derived neurotrophic factor and is also involved in inflammation in response to high ATP release. It is therefore involved in multiple inflammatory pathologies as well as neurodegenerative diseases. We have produced monoclonal antibodies (mAb) directed against this important human P2X4 receptor. Focusing on two mAbs, we showed that they also recognize mouse and rat P2X4. We demonstrated that these mAbs can be used in flow cytometry, immunoprecipitation and immunohistochemistry, but not in Western blot assays, indicating that they target conformational epitopes. We also characterised the expression of P2X4 receptor on mouse and human peripheral blood lymphocytes (PBL). We showed that P2X4 is expressed at the surface of several leukocyte cell types, with the highest expression level on eosinophils, making them potentially sensitive to adenosine triphosphate (ATP). P2X4 is expressed by leucocytes, in human and mouse, with a significant gender difference, males having higher surface expression levels than females. Our findings reveal that PBL express significant levels of P2X4 receptor, and suggest an important role of this receptor in leukocyte activation by ATP, particularly in P2X4high expressing eosinophils

Heating-induced transition of Potyvirus Potato Virus A coat protein into β-structure

<div><p>In our previous communication, we have reported that virions of plant Potyvirus Potato Virus A (PVA) have a peculiar structure characterized by high content of disordered regions in intravirus coat protein (CP). In this report, we describe unusual properties of the PVA CP. With the help of a number of physicochemical methods, we have observed that the PVA CP just released from the virions by heating at 60–70 °C undergoes association into oligomers and transition to β- (and even cross-β-) conformation. Transition to β-structure on heating has been recently reported for a number of viral and non-viral proteins. The PVA CP isolated by LiCl method was also transformed into cross-β-structure on heating to 60 °C. Using the algorithms for protein aggregation prediction, we found that the aggregation-prone segments should be located in the central region of a PVA CP molecule. Possibly this transition mimics some functions of PVA CP in the virus life cycle in infected plants.</p></div