Crystal structure of Haemophilus influenzae NadR protein. A bifunctional enzyme endowed with NMN adenyltransferase and ribosylnicotinimide kinase activities

Haemophilus influenzae NadR protein (hiNadR) has been shown to be a bifunctional enzyme possessing both NMN adenylytransferase (NMNAT; EC ) and ribosylnicotinamide kinase (RNK; EC ) activities. Its function is essential for the growth and survival of H. influenzae and thus may present a new highly specific anti-infectious drug target. We have solved the crystal structure of hiNadR complexed with NAD using the selenomethionine MAD phasing method. The structure reveals the presence of two distinct domains. The N-terminal domain that hosts the NMNAT activity is closely related to archaeal NMNAT, whereas the C-terminal domain, which has been experimentally demonstrated to possess ribosylnicotinamide kinase activity, is structurally similar to yeast thymidylate kinase and several other P-loop-containing kinases. There appears to be no cross-talk between the two active sites. The bound NAD at the active site of the NMNAT domain reveals several critical interactions between NAD and the protein. There is also a second non-active-site NAD molecule associated with the C-terminal RNK domain that adopts a highly folded conformation with the nicotinamide ring stacking over the adenine base. Whereas the RNK domain of the hiNadR structure presented here is the first structural characterization of a ribosylnicotinamide kinase from any organism, the NMNAT domain of hiNadR defines yet another member of the pyridine nucleotide adenylyltransferase family.     

A Rapid Simultaneous Saccharification and Fermentation (SSF) Technique to Determine Ethanol Yields

We have developed a relatively simple simultaneous saccharification and fermentation (SSF) technique to determine the ethanol production potential for large sets of biomass samples. The technique is based on soaking approximately 0.5 grams of a biomass sample in aqueous ammonia at room temperature and at atmospheric pressure for 24 h, then fermenting with Saccharomyces cerevisiae D₅A for 24 h using Spezyme CP, for enzymatic hydrolysis of structural polysaccharides. We have tested the technique on a set of corn stover samples representing much of the genetic variability in the commercial corn hybrid population. The samples were weighed into modified Ankom filter bags (F57) before soaking to avoid biomass loss during the process. Fermentation samples were analyzed for ethanol after 24 h by HPLC. Percentages of theoretical maximum ethanol yields of the samples ranged between 44.9 and 73%. We observed that percentages of theoretical maximum ethanol yields were highly correlated (r ²?=?0.90) with acid detergent lignin concentration while a low correlation was observed between cellulose concentration and ethanol yield. Near infrared spectra of corn stover samples were also examined. The coefficient of determination (r ²) from regression of predicted versus measured percent theoretical maximum ethanol yield was 0.96. This result suggests that using NIRS is a promising method for predicting ethanol yield, but larger calibration sets are necessary for obtaining improved accuracy for larger sample populations. We conclude that the developed SSF technique could be applied to large numbers of biomass samples to rapidly estimate ethanol yields and to compare different biomass samples in terms of ethanol yields.     

Cytoskeletal responses during early development of the downy mildew of grapevine (Plasmopara viticola)

A host-free system was established to induce the early development of the obligate biotrophic pathogen Plasmopara viticola, the downy mildew of grapevine. This system was used to study cytoskeletal responses during encystation and germ tube formation. During these processes, both the actin and the tubulin cytoskeleton show a stage-specific pattern of distribution. Elimination of the cytoskeleton by the actin drug latrunculin B and the microtubule drug ethyl-N-phenyl-carbamate did not affect the release of mobile zoospores from the sporangia, nor the encystation process, but efficiently inhibited the formation of a germ tube. The data are discussed with respect to a role of both actin and microtubules for the establishment of the cell polarity guiding the emergence and the growth of the germ tube.     

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture.

Methodology/Principal Findings

A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (C_q values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination.

Conclusions/Significance

C_q values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.     

Eicosanoid Profiling in an Orthotopic Model of Lung Cancer Progression by Mass Spectrometry Demonstrates Selective Production of Leukotrienes by Inflammatory Cells of the Microenvironment

Eicosanoids are bioactive lipid mediators derived from arachidonic acid¹ (AA), which is released by cytosolic phospholipase A₂ (cPLA₂). AA is metabolized through three major pathways, cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450, to produce a family of eicosanoids, which individually have been shown to have pro- or anti-tumorigenic activities in cancer. However, cancer progression likely depends on complex changes in multiple eicosanoids produced by cancer cells and by tumor microenvironment and a systematic examination of the spectrum of eicosanoids in cancer has not been performed. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitate eicosanoids produced during lung tumor progression in an orthotopic immunocompetent mouse model of lung cancer, in which Lewis lung carcinoma (LLC) cells are injected into lungs of syngeneic mice. The presence of tumor increased products of both the cyclooxygenase and the lipoxygenase pathways in a time-dependent fashion. Comparing tumors grown in cPLA₂ knockout vs wild-type mice, we demonstrated that prostaglandins (PGE₂, PGD₂ and PGF_2a) were produced by both cancer cells and the tumor microenvironment (TME), but leukotriene (LTB₄, LTC₄, LTD₄, LTE₄) production required cPLA₂ expression in the TME. Using flow cytometry, we recovered tumor-associated neutrophils and 2 types of tumor-associated macrophages from tumor-bearing lungs and we defined their distinct eicosanoid profiles by LC/MS/MS. The combination of flow cytometry and LC/MS/MS unravels the complexity of eicosanoid production in lung cancer and provides a rationale to develop therapeutic strategies that target select cell populations to inhibit specific classes of eicosanoids.     

High-Efficiency Thermal Asymmetric Interlaced PCR (hiTAIL-PCR) for Determination of a Highly Degenerated Prophage WO Genome in a Wolbachia Strain Infecting a Fig Wasp Species

Temperate bacteriophage WO is a model system for studying tripartite interactions among viruses, bacteria, and eukaryotes, especially investigations of the genomic stability of obligate intracellular bacteria. Few WO genomes exist because of the difficulty in isolating viral DNA from eukaryotic hosts, and most reports are by-products of Wolbachia sequencing. Only one partial genome of a WO phage has been determined directly from isolated particles. We determine the complete genome sequence of prophage WO (WOSol) in Wolbachia strain wSol, which infects the fig wasp Ceratosolen solmsi (Hymenoptera: Chalcidoidea), by high-efficiency thermal asymmetric interlaced PCR. The genome of WOSol is highly degenerated and disrupted by a large region (14,267 bp) from Wolbachia. Consistent with previous molecular studies of multiple WO genomes, the genome of WOSol appears to have evolved by single nucleotide mutations and recombinations.     

Evolution in a Changing Environment

We propose a simple model for genetic adaptation to a changing environment, describing a fitness landscape characterized by two maxima. One is associated with “specialist” individuals that are adapted to the environment; this maximum moves over time as the environment changes. The other maximum is static, and represents “generalist” individuals not affected by environmental changes. The rest of the landscape is occupied by “maladapted” individuals. Our analysis considers the evolution of these three subpopulations. Our main result is that, in presence of a sufficiently stable environmental feature, as in the case of an unchanging aspect of a physical habitat, specialists can dominate the population. By contrast, rapidly changing environmental features, such as language or cultural habits, are a moving target for the genes; here, generalists dominate, because the best evolutionary strategy is to adopt neutral alleles not specialized for any specific environment. The model we propose is based on simple assumptions about evolutionary dynamics and describes all possible scenarios in a non-trivial phase diagram. The approach provides a general framework to address such fundamental issues as the Baldwin effect, the biological basis for language, or the ecological consequences of a rapid climate change.     

A Trio of Viral Proteins Tunes Aphid-Plant Interactions in Arabidopsis thaliana

Background

Virus-induced deterrence to aphid feeding is believed to promote plant virus transmission by encouraging migration of virus-bearing insects away from infected plants. We investigated the effects of infection by an aphid-transmitted virus, cucumber mosaic virus (CMV), on the interaction of Arabidopsis thaliana, one of the natural hosts for CMV, with Myzus persicae (common names: ‘peach-potato aphid’, ‘green peach aphid’).

Methodology/Principal Findings

Infection of Arabidopsis (ecotype Col-0) with CMV strain Fny (Fny-CMV) induced biosynthesis of the aphid feeding-deterrent 4-methoxy-indol-3-yl-methylglucosinolate (4MI3M). 4MI3M inhibited phloem ingestion by aphids and consequently discouraged aphid settling. The CMV 2b protein is a suppressor of antiviral RNA silencing, which has previously been implicated in altering plant-aphid interactions. Its presence in infected hosts enhances the accumulation of CMV and the other four viral proteins. Another viral gene product, the 2a protein (an RNA-dependent RNA polymerase), triggers defensive signaling, leading to increased 4MI3M accumulation. The 2b protein can inhibit ARGONAUTE1 (AGO1), a host factor that both positively-regulates 4MI3M biosynthesis and negatively-regulates accumulation of substance(s) toxic to aphids. However, the 1a replicase protein moderated 2b-mediated inhibition of AGO1, ensuring that aphids were deterred from feeding but not poisoned. The LS strain of CMV did not induce feeding deterrence in Arabidopsis ecotype Col-0.

Conclusions/Significance

Inhibition of AGO1 by the 2b protein could act as a booby trap since this will trigger antibiosis against aphids. However, for Fny-CMV the interplay of three viral proteins (1a, 2a and 2b) appears to balance the need of the virus to inhibit antiviral silencing, while inducing a mild resistance (antixenosis) that is thought to promote transmission. The strain-specific effects of CMV on Arabidopsis-aphid interactions, and differences between the effects of Fny-CMV on this plant and those seen previously in tobacco (inhibition of resistance to aphids) may have important epidemiological consequences.     

TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita

Patients with dyskeratosis congenita (DC), a heterogeneous inherited bone marrow failure syndrome, have abnormalities in telomere biology, including very short telomeres and germline mutations in DKC1, TERC, TERT, or NOP10, but approximately 60% of DC patients lack an identifiable mutation. With the very short telomere phenotype and a highly penetrant, rare disease model, a linkage scan was performed on a family with autosomal-dominant DC and no mutations in DKCI, TERC, or TERT. Evidence favoring linkage was found at 2p24 and 14q11.2, and this led to the identification of TINF2 (14q11.2) mutations, K280E, in the proband and her five affected relatives and TINF2 R282H in three additional unrelated DC probands, including one with Revesz syndrome; a fifth DC proband had a R282S mutation. TINF2 mutations were not present in unaffected relatives, DC probands with mutations in DKC1, TERC, or TERT or 298 control subjects. We demonstrate that a fifth gene, TINF2, is mutated in classical DC and, for the first time, in Revesz syndrome. This represents the first shelterin complex mutation linked to human disease and confirms the role of very short telomeres as a diagnostic test for DC.