Rethinking the common garden in invasion research

In common garden experiments, a number of genotypes are raised in a common environment in order to quantify the genetic component of phenotypic variation. Common gardens are thus ideally suited for disentangling how genetic and environmental factors contribute to the success of invasive species in their new non-native range. Although common garden experiments are increasingly employed in the study of invasive species, there has been little discussion about how these experiments should be designed for greatest utility. We argue that this has delayed progress in developing a general theory of invasion biology. We suggest a minimum optimal design (MOD) for common garden studies that target the ecological and evolutionary processes leading to phenotypic differentiation between native and invasive ranges. This involves four elements: (A) multiple, strategically sited garden locations, involving at the very least four gardens (2 in the native range and 2 in the invaded range); (B) careful consideration of the genetic design of the experiment; (C) standardization of experimental protocols across all gardens; and (D) care to ensure the biosafety of the experiment. Our understanding of the evolutionary ecology of biological invasions will be greatly enhanced by common garden studies, if and only if they are designed in a more systematic fashion, incorporating at the very least the MOD suggested here.     

The nucleotide analogue 3-deazaadenosine prevents neointima-formation after balloon injury

We have recently shown that 3-deazaadenosine (c3Ado) inhibits atherogenesis in mice. We studied whether its anti-inflammatory capacity would also affect neointima-formation after balloon injury. Sprague Dawley rats underwent balloon angioplasty. C3Ado was administered orally, starting 5 days prior to the balloon injury and continued for 2 weeks. Fourteen days after balloon injury the intima/media ratio in the c3Ado-treated group was reduced by 67% (p < 0.001) and luminal stenosis by 50% (p < 0.001). Neointimal cellular density was decreased by 25% (p < 0.001) and the induction of c-Jun and ki67 was markedly lower. The reduction of the intima/media ratio was still observed 3 months after balloon injury. Furthermore, a c3Ado-dependent inhibition of PDGF-mediated ERK-activation and proliferation could be demonstrated.Short-term administration of C3Ado inhibits neointima-formation in rats for at least 3 months after injury. The present findings implicate that c3Ado may be useful as an inhibitor of restenosis-formation after balloon angioplasty in humans.     

Familial Hemophagocytic Lymphohistiocytosis Type 5 (FHL-5) Is Caused by Mutations in Munc18-2 and Impaired Binding to Syntaxin 11

Rapid intracellular transport and secretion of cytotoxic granules through the immunological synapse requires a balanced interaction of several proteins. Disturbance of this highly regulated process underlies familial hemophagocytic lymphohistiocytosis (FHL), a genetically heterogeneous autosomal-recessive disorder characterized by a severe hyperinflammatory phenotype. Here, we have assigned FHL-5 to a 1 Mb region on chromosome 19p by using high-resolution SNP genotyping in eight unrelated FHL patients from consanguineous families. Subsequently, we found nine different mutations, either truncating or missense, in STXBP2 in twelve patients from Turkey, Saudi Arabia, and Central Europe. STXBP2 encodes syntaxin binding protein 2 (Munc18-2), involved in the regulation of vesicle transport to the plasma membrane. We have identified syntaxin 11, a SNARE protein mutated in FHL-4, as an interaction partner of STXBP2. This interaction is eliminated by the missense mutations found in our FHL-5 patients, which leads to a decreased stability of both proteins, as shown in patient lymphocytes. Activity of natural killer and cytotoxic T cells was markedly reduced or absent, as determined by CD107 degranulation. Our findings thus identify a key role for STXBP2 in lytic granule exocytosis.     

Acid-Sensitive Outwardly Rectifying Anion Channels in Human Erythrocytes

Acid-sensitive outwardly rectifying anion channels (ASOR) have been described in several mammalian cell types. The present whole-cell patch-clamp study elucidated whether those channels are expressed in erythrocytes. To this end whole-cell recordings were made in human erythrocytes from healthy donors treated with low pH and high osmotic pressure. When the pipette solution had a reduced Cl⁻ concentration, treatment of the cells with Cl⁻-containing normal and hyperosmotic (addition of sucrose and polyethelene glycol 1000 [PEG-1000] to the Ringer) media with low pH significantly increased the conductance of the cells at positive voltages. Channel activity was highest in the PEG-1000 media (95 and 300 mM PEG-1000, pH 4.5 and 4.3, respectively) where the current–voltage curves demonstrated strong outward rectification and reversed at −40 mV. Substitution of the Cl⁻-containing medium with Cl⁻-free medium resulted in a decrease of the conductance at hyperpolarizing voltages, a shift in reversal potential (to 0 mV) and loss of outward rectification. The chloride currents were inhibited by chloride channels blockers DIDS and NPPB (IC₅₀ for both was ~1 mM) but not with niflumic acid and amiloride. The observations reveal expression of ASOR in erythrocytes.     

Vancomycin and Oritavancin Have Different Modes of Action in Enterococcus faecium

The increasing frequency of Enterococcus faecium isolates with multidrug resistance is a serious clinical problem given the severely limited number of therapeutic options available to treat these infections. Oritavancin is a promising new alternative in clinical development that has potent antimicrobial activity against both staphylococcal and enterococcal vancomycin-resistant pathogens. Using solid-state NMR to detect changes in the cell-wall structure and peptidoglycan precursors of whole cells after antibiotic-induced stress, we report that vancomycin and oritavancin have different modes of action in E. faecium. Our results show the accumulation of peptidoglycan precursors after vancomycin treatment, consistent with transglycosylase inhibition, but no measurable difference in cross-linking. In contrast, after oritavancin exposure, we did not observe the accumulation of peptidoglycan precursors. Instead, the number of cross-links is significantly reduced, showing that oritavancin primarily inhibits transpeptidation. We propose that the activity of oritavancin is the result of a secondary binding interaction with the E. faecium peptidoglycan. The hypothesis is supported by results from ¹³C{¹⁹F} rotational-echo double-resonance (REDOR) experiments on whole cells enriched with l-[1-¹³C]lysine and complexed with desleucyl [¹⁹F]oritavancin. These experiments establish that an oritavancin derivative with a damaged d-Ala-d-Ala binding pocket still binds to E. faecium peptidoglycan. The ¹³C{¹⁹F} REDOR dephasing maximum indicates that the secondary binding site of oritavancin is specific to nascent and template peptidoglycan. We conclude that the inhibition of transpeptidation by oritavancin in E. faecium is the result of the large number of secondary binding sites relative to the number of primary binding sites.     

DLA-Based Strategies for Cloning Insertion Mutants: Cloning the gl4 Locus of Maize Using Mu Transposon Tagged Alleles

Digestion–ligation–amplification (DLA), a novel adaptor-mediated PCR-based method that uses a single-stranded oligo as the adaptor, was developed to overcome difficulties of amplifying unknown sequences flanking known DNA sequences in large genomes. DLA specifically overcomes the problems associated with existing methods for amplifying genomic sequences flanking Mu transposons, including high levels of nonspecific amplification. Two DLA-based strategies, MuClone and DLA-454, were developed to isolate Mu-tagged alleles. MuClone allows for the amplification of subsets of the numerous Mu transposons in the genome, using unique three-nucleotide tags at the 3′ ends of primers, simplifying the identification of flanking sequences that cosegregate with mutant phenotypes caused by Mu insertions. DLA-454, which combines DLA with 454 pyrosequencing, permits the efficient cloning of genes for which multiple independent insertion alleles are available without the need to develop segregating populations. The utility of each approach was validated by independently cloning the gl4 (glossy4) gene. Mutants of gl4 lack the normal accumulation of epicuticular waxes. The gl4 gene is a homolog of the Arabidopsis CUT1 gene, which encodes a condensing enzyme involved in the synthesis of very-long-chain fatty acids, which are precursors of epicuticular waxes.INSERTIONAL mutagenesis is widely used in functional genomics. For example, insertion mutants obtained via T-DNA in Arabidopsis (Alonso et al. 2003) and rice (Sallaud et al. 2004) and via transposons in maize (Brutnell 2002; Brutnell and Conrad 2003; May et al. 2003; McCarty et al. 2005; Settles et al. 2007), rice (Kolesnik et al. 2004; Miyao et al. 2003; Kumar et al. 2005), and Arabidopsis (Speulman et al. 1999) have been used for both forward and reverse genetics. In both situations it is necessary to identify sequences flanking the insertional mutagen. For example, the availability of sequence-indexed collections of T-DNA insertion mutants (Alonso et al. 2003) has greatly facilitated the functional analysis of Arabidopsis. Such reverse genetic resources are generated by creating large numbers of independent insertion events and then identifying and sequencing the DNA flanking the insertional mutagen. To be cost effective such flanking sequences are typically amplified using one of several available “genome-walking” strategies (Shyamala and Ames 1989; Alonso et al. 2003; O''Malley et al. 2007; Vandenbussche et al. 2008; Uren et al. 2009).Similarly, once mutant phenotypes have been identified following forward genetic screens, the challenge in cloning the affected gene is to identify the specific genic sequences that flank causative insertions. Insertional mutagensis is typically more productive if multiple copies of the insertional mutagens are present. The Mutator (Mu) transposon of maize has been widely used for forward genetics because of its high copy number and transposition activity (Benito and Walbot 1997). This high copy number can, however, complicate the identification of the specific insertion responsible for a mutant phenotype. Traditionally, identifying a gene sequence that had been tagged by an insertion involved genomic DNA blotting using multiple wild-type and mutant siblings to identify a DNA fragment that contained the insertion and that cosegregated with the mutant phenotype (James et al. 1995). However, both DNA blotting and subsequent postblotting gene isolation steps were laborious, time-consuming, and often unpredictable.Here, we report two strategies, MuClone and DLA-454, for cloning mutant alleles derived from insertional mutagenesis. Both strategies are based on an adaptation of a novel highly specific and efficient genome-walking method, digestion–ligation–amplification (DLA) that uses a single-stranded oligo as the adaptor instead of the partially double-stranded adaptors used in other methods. MuClone, a cost-efficient strategy, adds unique three-nucleotide tags to the 3′ ends of the common adaptor primer so subsets of high-copy Mu transposons can be separately amplified in a manner analogous to AFLP technology (Yunis et al. 1991). It is then possible to identify which copy of the transposon cosegregates with the mutant allele in the cosegregating population. DLA-454 combines DLA with 454 pyrosequencing to amplify and sequence multiple independent alleles of a gene to be cloned. Analysis of the resulting Mu flanking sequences (MFSs) identifies the target gene. To illustrate the applicability of the MuClone and DLA-454 strategies, each was used to independently clone the glossy4 (gl4) gene. The maize gl4 is a homolog of the Arabidopsis CUT1 gene involved in epicuticular wax accumulation.     

Identification of PpoA from Aspergillus nidulans as a Fusion Protein of a Fatty Acid Heme Dioxygenase/Peroxidase and a Cytochrome P450

The homothallic ascomycete Aspergillus nidulans serves as model organism for filamentous fungi because of its ability to propagate with both asexual and sexual life cycles, and fatty acid-derived substances regulate the balance between both cycles. These so-called psi (precocious sexual inducer) factors are produced by psi factor-producing oxygenases (Ppo enzymes). Bioinformatic analysis predicted the presence of two different heme domains in Ppo proteins: in the N-terminal region, a fatty acid heme dioxygenase/peroxidase domain is predicted, whereas in the C-terminal region, a P450 heme thiolate domain is predicted. To analyze the reaction catalyzed by Ppo enzymes, PpoA was expressed in Escherichia coli as an active enzyme. The protein was purified by 62-fold and identified as a homotetrameric ferric heme protein that metabolizes mono- as well as polyunsaturated C₁₆ and C₁₈ fatty acids at pH ∼7.25. The presence of thiolate-ligated heme was confirmed on the basis of sequence alignments and the appearance of a characteristic 450 nm CO-binding spectrum. Studies on its reaction mechanism revealed that PpoA uses different heme domains to catalyze two separate reactions. Within the heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid by abstracting a H-atom from C-8 of the fatty acid, yielding a carbon-centered radical that reacts with molecular dioxygen. In the second reaction step, 8-hydroperoxyoctadecadienoic acid is isomerized within the P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. We identify PpoA as a bifunctional P450 fusion protein that uses a previously unknown reaction mechanism for forming psi factors.The fungus Aspergillus nidulans (teleomorph Emericella nidulans) is a homothallic ascomycete that has a defined sexual and asexual developmental cycle. Therefore, it serves as a model system for the understanding of fungal development (1). Oxidized unsaturated fatty acids, so-called oxylipins, derived from endogenous fatty acids were found to influence the development of the asexual conidiophores and sexual cleistothecia (2–6). Moreover, they seem to regulate the secondary metabolism of the fungus (7). These substances were collectively named psi factors and are primarily a mixture of hydroxylated oleic (18:1^Δ9Z; x:y^Δz denotes a fatty acid with x carbons and y double bonds in position z counting from the carboxyl end), linoleic (18:2^Δ9Z,12Z), and α-linolenic (18:3^Δ9Z,12Z,15Z) acids. They are termed psiβ, psiα, and psiγ, respectively. Psi factors can be further classified by the number and positioning of hydroxy groups on the fatty acid backbone: psiB (OH at C-8, e.g. (8R)-HODE),² psiA (OH at C-5 and C-8, e.g. (5S,8R)-DiHODE), and psiC (OH at C-8 and the δ-lactone ring) (8, 9).The psi factor (8R)-HODE was first discovered in the fungus Laetisaria arvalis (10, 11); it was later also found in Gaeumannomyces graminis (12, 13), where the first enzyme, which is responsible for production of (8R)-HPODE, 7,8-LDS, was detected (13). This heme-containing enzyme is bifunctional because it oxidizes 18:2^Δ9Z,12Z in a first reaction step to (8R)-HPODE and subsequently isomerizes this intermediate compound to (7S,8S)-DiHODE (13–15).After the genome of A. nidulans was available, Keller and co-workers (6, 16, 17) found three genes that share a high homology with the sequence of 7,8-LDS, namely ppoA, ppoB, and ppoC. They showed that the deletion of these genes had a significant effect (i) on the developmental ratio between the asexual conidiospores and sexual ascospores; (ii) on the production of psi factors; and (iii) on the production of secondary metabolites, the mycotoxins (6, 7, 16, 17). Furthermore, the encoded proteins showed remarkable sequence homology to both mammalian PGHS isoforms, enzymes that are responsible for the synthesis of prostaglandins (18). Using the NCBI conserved domain search analysis tool, it turned out that ppoA amino acid residues 210–580 contain a domain similar to mammalian heme peroxidases, whereas residues 650–1050 contain a CYPX domain, similar to P450 heme thiolate enzymes (16). However, for 7,8-LDS from G. graminis, only the mammalian heme peroxidase domain is predicted. The identity of conserved catalytic domains between Ppo enzymes and mammalian PGHS ranges from 25 to 29% for PGHS-2 and from 25 to 26% for PGHS-1 (19). PpoA and 7,8-LDS show 42% amino acid identity.Oliw and co-workers (20) observed that incubation of homogenates of mycelia of A. nidulans with 18:2^Δ9Z,12Z converted the fatty acid to (8R)-HODE and (5S,8R)-DiHODE as the major products. (8R)-HPODE, (10R)-HODE, and (10R)-HPODE were detected as minor products. Incubation of mycelia of Aspergillus fumigatus with deuterium-labeled 18:2^Δ9Z,12Z revealed that the synthesis of (8R)-HPODE is accomplished via pro-S-hydrogen abstraction at C-8 and antarafacial dioxygen insertion. (5S,8R)-DiHODE is generated via an additional pro-S-hydrogen abstraction at C-5 of the substrate (20, 21).Additional studies with fungal knock-out strains led to the hypothesis that PpoA may be responsible for the synthesis of (8R)-hydroperoxides, which are partially reduced to (8R)-hydroxides (20). It was suggested that, analogous with 7,8-LDS, (8R)-hydroperoxides are then converted to 5,8-dihydroxides by PpoA. Furthermore, it was concluded that ppoC may code for linoleate (10R)-DOX (20). Analysis of Ppo enzymes from A. nidulans in studies published so far has been performed either by using knock-out mutants to demonstrate the absence of a subset of psi factors or by using crude mycelial extracts; both experimental setups have the disadvantage of observing multiple enzymatic reactions in parallel.To characterize the biochemical properties of PpoA in more detail, we cloned and expressed recombinant PpoA in Escherichia coli. After purification of the enzyme by up to 62-fold, biochemical characterization was performed. The studies revealed mechanistic as well as structural similarities to and differences from 7,8-LDS from G. graminis. Both enzymes were found to be homotetrameric ferric heme proteins that catalyze the synthesis of (8R)-HPODE. Whereas G. graminis 7,8-LDS converts the intermediate formed to (7S,8S)-DiHODE, PpoA produces 5,8-DiHODE.Using site-directed mutagenesis, we provide evidence that there are striking differences between both enzymes regarding the catalytic reaction cycle. Thus, we found that PpoA uses different domains to catalyze the two reaction steps. We suggest that the DOX reaction, yielding 8-HPODE, takes place in the N-terminal heme peroxidase domain. The isomerization of this intermediate product to the end product, 5,8-DiHODE, is accomplished, however, independently by the C-terminal P450 heme thiolate domain in an 8-hydroperoxide isomerase reaction.In addition, we are able to provide evidence that, during the catalysis, PpoA generates a carbon-centered radical presumably at C-8, like G. graminis 7,8-LDS. Furthermore, we determined the kinetic parameters for the first reaction step.     

Database on Demand – An online tool for the custom generation of FASTA‐formatted sequence databases

In mass spectrometry‐based proteomics, most conventional search engines match spectral data to sequence databases. These search databases thus play a crucial role in the identification process. While search engines can derive peptides in silico from protein sequences, this is usually limited to standard digestion algorithms. Customized search databases that provide detailed control over the search space can vastly outperform such standard strategies, especially in gel‐free proteomics experiments. Here we present Database on Demand, an easy‐to‐use web tool that can quickly produce a wide variety of customized search databases.     

flowCore: a Bioconductor package for high throughput flow cytometry

Background  

Recent advances in automation technologies have enabled the use of flow cytometry for high throughput screening, generating large complex data sets often in clinical trials or drug discovery settings. However, data management and data analysis methods have not advanced sufficiently far from the initial small-scale studies to support modeling in the presence of multiple covariates.