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Three-dimensional imaging of viral infections

Annu Rev Virol 2014; 1: 453-473.

Risco C, Fernández de Castro I, Sanz-Sánchez L, Narayan K, Grandinetti G, Subramaniam S.

Annu Rev Virol 2014; 1: 453-473ReThree-dimensional (3D) imaging technologies are beginning to have significant impact in the field of virology, as they are helping us understand how viruses take control of cells.

In this article we review several methodologies for 3D imaging of cells and show how these technologies are contributing to the study of viral infections and the characterization of specialized structures formed in virus-infected cells. We include 3D reconstruction by transmission electron microscopy (TEM) using serial sections, electron tomography, and focused ion beam scanning electron microscopy (FIB-SEM).

We summarize from these methods selected contributions to our understanding of viral entry, replication, morphogenesis, egress and propagation, and changes in the spatial architecture of virus-infected cells. In combination with live-cell imaging, correlative microscopy, and new techniques for molecular mapping in situ, the availability of these methods for 3D imaging is expected to provide deeper insights into understanding the structural and dynamic aspects of viral infection.

Pro-oncogenic role of alternative p38 mitogen-activated protein kinases p38γ and p38δ, linking inflammation and cancer in colitis-associated colon cancer

Cancer Res. 2014 Sep 12. pii: canres.0870.2014.

Del Reino P, Alsina-Beauchamp D, Escós A, Cerezo-Guisado MI, Risco A, Aparicio N, Zur R, Fernandez-Estevez M, Collantes E, Montans J, Cuenda A.

Cancer Res. 2014;. pii: canres.0870.2014p38 mitogen-activated protein kinase (MAPK) signalling has been implicated in the regulation of processes leading to cancer development and progression. Chronic inflammation is a known risk factor for tumourigenesis, yet the precise mechanism of this association remains largely unknown. The related p38αMAPK (MAPK14) proteins p38γ (MAPK12) and p38δ (MAPK13) were recently shown to modulate the immune response, although their role in tumourigenesis remains controversial and their function in inflammation-associated cancer has not been studied.

We analysed the role of p38γ and p38δ in colon cancer associated to colitis, using the azoxymethane/dextran sodium sulphate colitis-associated colon cancer model in wild type (WT), p38γ-, p38δ- and p38γ/δ-deficient (p38γ/δ-/-) mice. We found that p38γ/δ deficiency significantly decreased tumour formation, in parallel with a decrease in proinflammatory cytokine and chemokine production. Analysis of leukocyte populations in p38γ/δ-/- mouse colon showed less macrophage and neutrophil recruitment than in WT mice. Furthermore, WT chimaeric mice with transplanted p38γ/δ-/- bone marrow (BM) had less tumours than WT mice transplanted with WT BM, whereas tumour number was significantly increased in p38γ/δ-/- chimaeric mice with WT BM compared to p38γ/δ-/- mice transplanted with p38γ/δ-/- BM.

Together, our results establish that p38γ and p38δ are central to colitis-associated colon cancer formation through regulation of haematopoietic cell response to injury, and validate p38γ and p38δ as potential targets for cancer therapy.

Characterization of a novel Zn2+-dependent intrinsic imipenemase from Pseudomonas aeruginosa

J Antimicrob Chemother. 2014; pii: dku267.

Fajardo A, Hernando-Amado S, Oliver A, Ball G, Filloux A, Martinez JL.

J Antimicrob Chemother. 2014; pii: dku267Objectives: Previous work showed that PA5542 inactivation increases Pseudomonas aeruginosa 59.20 susceptibility to carbapenems. The objective of the current study was to purify PA5542, to determine its role in carbapenem resistance and to analyse the kinetic constants of this putative new β-lactamase.

Methods: PA5542 was cloned and expressed in Escherichia coli. The enzyme was purified by affinity as a GST fusion protein and, after that, cleaved to remove the GST tag. β-Lactamase activity was measured spectrophotometrically using imipenem as substrate. Susceptibility to antibiotics was determined by Etest. Zn2+ was added when needed. The expression levels of PA5542, ampC, poxB, mexA and oprD were determined by real-time RT–PCR.

Results: Lack of PA5542 increases P. aeruginosa 59.20 susceptibility to carbapenems and its overexpression reduces E. coli susceptibility to these β-lactams. PA5542 is highly conserved in all sequenced P. aeruginosa strains. The clinical isolate 59.20 is resistant to imipenem (MIC >32 mg/L) and to meropenem (MIC 24 mg/L) and presents high-level expression of PA5542 in comparison with the wild-type strain PAO1. Spectrophotometric analyses showed that PA5542 is a Zn2+-dependent imipenemase. Analysis of the PA5542 sequence indicates that it does not belong to the classical categories of β-lactamases.

Conclusions PA5542 encodes a new Zn2+-dependent imipenemase. The presence of PA5542 in all sequenced P. aeruginosa genomes, maintaining the synteny and without adjacent gene-mobility elements, indicates that it belongs to the P. aeruginosa core genome. High PA5542 expression in 59.20 suggests it may contribute to the resistance to carbapenems of this P. aeruginosa clinical isolate.

Effect of herbicide combinations on Bt-maize rhizobacterial diversity

J Microbiol Biotechnol. 2014; doi: 10.4014/jmb.1405.05054.

Valverde JR, Marin S, Mellado RP.

J Microbiol Biotechnol. 2014; doi: 10.4014/jmb.1405.05054Reports of herbicide resistance events are proliferating worldwide leading to new cultivation strategies using combinations of pre-emergence and post-emergence herbicides. We analysed the impact during a one-year cultivation cycle of several herbicide combinations on the rhizobacterial community of glyphosate-tolerant Bt-maize and compared them to those of the untreated or glyphosate-treated soils. Samples were analysed using pyrosequencing of the V6 hypervariable region of the 16S rRNA gene.

The sequences obtained were subjected to taxonomic, taxonomy-independent and phylogeny-based diversity studies followed by a statistical analysis using principal components analysis and hierarchical clustering with jackknife statistical validation. The resilience of the microbial communities was analysed by comparing their relative composition at the end of the cultivation cycle. The bacterial communites from soil subjected to a combined treatment with mesotrione plus s-metolachlor followed by glyphosate were not statistically different from those treated with glyphosate or the untreated ones. The use of acetochlor plus terbuthylazine followed by glyphosate and the use of aclonifen plus isoxaflutole followed by mesotrione clearly affected the resilience of their corresponding bacterial communities. The treatment with pethoxamid followed by glyphosate resulted in an intermediate effect. The use of glyphosate alone seems to be the less aggressive one for bacterial communities. Should a combined treatment be needed, the combination of mesotrione and s-metolachlor shows the next best final resilience.

Our results show the relevance of comparative rhizobacterial community studies when novel combined herbicide treatments are deemed necessary to control weed growth.

Yeast mitochondrial RNAP conformational changes are regulated by interactions with the mitochondrial transcription factor

Nucleic Acids Res. 2014; pii: gku795.

Drakulic S, Wang L, Cuéllar J, Guo Q, Velázquez G, Martín-Benito J, Sousa R, Valpuesta JM.

Nucleic Acids Res. 2014; pii: gku795Mitochondrial RNA polymerases (MtRNAPs) are members of the single-subunit RNAP family, the most well-characterized member being the RNAP from T7 bacteriophage. MtRNAPs are, however, functionally distinct in that they depend on one or more transcription factors to recognize and open the promoter and initiate transcription, while the phage RNAPs are capable of performing these tasks alone. Since the transcriptional mechanisms that are conserved in phage and mitochondrial RNAPs have been so effectively characterized in the phage enzymes, outstanding structure-mechanism questions concern those aspects that are distinct in the MtRNAPs, particularly the role of the mitochondrial transcription factor(s).

To address these questions we have used both negative staining and cryo-EM to generate three-dimensional reconstructions of yeast MtRNAP initiation complexes with and without the mitochondrial transcription factor (MTF1), and of the elongation complex. Together with biochemical experiments, these data indicate that MTF1 uses multiple mechanisms to drive promoter opening, and that its interactions with the MtRNAP regulate the conformational changes undergone by the latter enzyme as it traverses the template strand.