Consistency in the habitat degree of invasion for three invasive plant species across Mediterranean islands

Habitats are known to vary in their vulnerability to invasion by alien plants and different species often colonise distinct habitats. To assess the consistency in the degree of invasion of particular habitats, this study examined the frequency of occurrence and local abundance of three invasive plant taxa: Ailanthus altissima, Carpobrotus spp., and Oxalis pes-caprae across different habitat types on four representative Mediterranean islands. We conducted systematic field surveys recording the presence-absence and cover of these taxa on the islands of Mallorca, Corsica, Sardinia and Crete. Drawing on the results of 5,285 sample points, the frequency of occurrence of the three invaders tends to be higher than expected in urban, ruderal and roadside habitats. In contrast, scrub habitats rarely contain any of the three invaders, indicating that they may be more resistant to invasion. The degree of invasion, determined by the local abundance of an invasive plant in any one habitat, varies according to the identity of the invader and the island. However, based on average abundance, Oxalis pes-caprae exhibits the highest degree of invasion, and Carpobrotus spp. the least. There is no indication that any one of the four islands is more prone to either higher frequencies or abundances of the three invaders. These patterns suggest that anthropogenic changes in Mediterranean islands will increase the vulnerability of certain habitats to invasion and increase the distribution of these three invasive taxa at any of the four islands.     

A high-throughput <Emphasis Type="Italic">de novo</Emphasis> sequencing approach for shotgun proteomics using high-resolution tandem mass spectrometry

Background  

High-resolution tandem mass spectra can now be readily acquired with hybrid instruments, such as LTQ-Orbitrap and LTQ-FT, in high-throughput shotgun proteomics workflows. The improved spectral quality enables more accurate de novo sequencing for identification of post-translational modifications and amino acid polymorphisms.     

Identification of qRT-PCR reference genes for analysis of opioid gene expression in a hibernator

Previous work has suggested that central and peripheral opioid signaling are involved in regulating torpor behavior and tissue protection associated with the hibernation phenotype. We used quantitative real-time PCR (qRT-PCR) to measure mRNA levels of opioid peptide precursors and receptors in the brain and heart of summer ground squirrels (Ictidomys tridecemlineatus) and winter hibernating squirrels in the torpid or interbout arousal states. The use of appropriate reference genes for normalization of qRT-PCR gene expression data can have profound effects on the analysis and interpretation of results. This may be particularly important when experimental subjects, such as hibernating animals, undergo significant morphological and/or functional changes during the study. Therefore, an additional goal of this study was to identify stable reference genes for use in qRT-PCR studies of the 13-lined ground squirrel. Expression levels of 10 potential reference genes were measured in the small intestine, liver, brain, and heart, and the optimal combinations of the most stable reference genes were identified by the GeNorm Excel applet. Based on this analysis, we provide recommendations for reference genes to use in each tissue that would be suitable for comparative studies among different activity states. When appropriate normalization of mRNA levels was used, there were no changes in opioid-related genes in heart among the three activity states; in brain, DOR expression was highest during torpor, lowest in interbout arousal and intermediate in summer. The results support the idea that changes in DOR expression may regulate the level of neuronal activity in brain during the annual hibernation cycle and may contribute to hibernation-associated tissue protection.     

Manipulating Each MreB of Bdellovibrio bacteriovorus Gives Diverse Morphological and Predatory Phenotypes

We studied the two mreB genes, encoding actinlike cytoskeletal elements, in the predatory bacterium Bdellovibrio bacteriovorus. This bacterium enters and replicates within other Gram-negative bacteria by attack-phase Bdellovibrio squeezing through prey outer membrane, residing and growing filamentously in the prey periplasm forming an infective “bdelloplast,” and septating after 4 h, once the prey contents are consumed. This lifestyle brings challenges to the Bdellovibrio cytoskeleton. Both mreB genes were essential for viable predatory growth, but C-terminal green fluorescent protein tagging each separately with monomeric teal-fluorescent protein (mTFP) gave two strains with phenotypic changes at different stages in predatory growth and development. MreB1-mTFP cells arrested growth early in bdelloplast formation, despite successful degradation of prey nucleoid. A large population of stalled bdelloplasts formed in predatory cultures and predation proceeded very slowly. A small proportion of bdelloplasts lysed after several days, liberating MreB1-mTFP attack-phase cells of wild-type morphology; this process was aided by subinhibitory concentrations of an MreB-specific inhibitor, A22. MreB2-mTFP, in contrast, was predatory at an almost wild-type rate but yielded attack-phase cells with diverse morphologies, including spherical, elongated, and branched, the first time such phenotypes have been described. Wild-type predatory rates were seen for all but spherical morphotypes, and septation of elongated morphotypes was achieved by the addition of A22.The predatory bacterium Bdellovibrio bacteriovorus shows novel filamentous growth within the periplasm of the Gram-negative prey bacterium on which it feeds. This study focuses on the cytoskeletal protein MreB and the role that two homologues of it play in B. bacteriovorus predatory or host-dependent (HD) growth. The HD B. bacteriovorus life cycle can be split into two phases: an attack phase and a growth phase (Fig. ​(Fig.1).1). The attack-phase B. bacteriovorus is a small free-swimming, highly motile cell within which replication has been arrested and which does not take up organic nutrients from the environment or grow extensively (24, 26). Once an attack-phase cell has collided with a suitable prey bacterium, B. bacteriovorus opens and squeezes through a small hole, formed in the outer membrane, using type IV pili to pull itself inside (7, 10). The B. bacteriovorus reseals the hole upon entering the periplasm. Once inside, the prey is killed rapidly within 15 min, and the prey cell wall is partially digested (35), forming a rounded structure called the bdelloplast (see Fig. ​Fig.11 and 4Ac and d). The HD B. bacteriovorus cell then enters the second, growth phase, part of the life cycle, (Fig. ​(Fig.1),1), whereby it grows filamentously while simultaneously coordinating the digestion and transportation of monomers from the prey cytoplasm. The mature growth-phase cell is multiploid and elongates typically 3 to 10 times the length of an attack-phase cell, its length being a reflection of the nutritional resources available in the prey (20). Once resources within the bdelloplast are depleted, the mature filament septates sequentially from one pole to form multiple progeny. These lyse the exhausted bdelloplast, mature into attack-phase cells, and repress growth once again (Fig. ​(Fig.1).1). B. bacteriovorus can be cultured slowly, without prey, as host-independent (HI) cells growing upon peptone-rich medium (30). In these conditions they grow pleiomorphically as mainly long filamentous or serpentine cells, from which some small attack-phase cells septate (30).Open in a separate windowFIG. 1.Schematic host-dependent (HD) predatory cycle for B. bacteriovorus on E. coli prey, showing the different phases of growth and inferred demands on the B. bacteriovorus cell cytoskeleton. References where the roles of the cytoskeleton in cell development have been proven for other bacteria are provided in parentheses. The status of the prey genome is both drawn from an earlier study (26) and confirmed by our work in the present study (see Fig. ​Fig.44).The predatory lifestyle of B. bacteriovorus presents a number of novel developmental challenges to the B. bacteriovorus cell and its cytoskeleton. It is not known how the attack-phase cells deform, allowing the B. bacteriovorus to squeeze through a pore it makes in the prey outer membrane that is narrower than the width of an attack-phase cell, as was imaged by Burnham et al. in the 1960s and more recently by Evans et al. (7, 10). It is also not known how the growth-phase filamentous cell within the bdelloplast is generated and remains resistant to division until terminal sequential septation begins, despite having multiple potential sites for septation along its length while elongating.The processes of cell elongation in rod-shaped bacteria are coordinated by an internal MreB cell cytoskeleton (9). MreB is a eukaryotic actin homologue and has been well studied in Escherichia coli, Bacillus subtilis, and Caulobacter crescentus (38). MreB monomers polymerize on ATP binding, forming helical structures in vivo that appear to associate with the cytoplasmic side of the bacterial cytoplasmic membrane (11, 18, 31). Bacterial two-hybrid experiments in Escherichia coli suggest that MreB forms a transmembrane complex with the two proteins MreC and MreD, each of which have been shown to form helical structures in vivo (21). A complex of MreBCD, together with the RodA protein, influences the shape of the peptidoglycan cell wall and thus the shape of the cell by positioning the peptidoglycan biosynthetic machinery so that its action is directionally specific (9, 19, 37). The MreB filament has also been shown to have roles in chromosome segregation, septation, and cell polarity (13, 14, 22, 37).Depletion of the MreB protein levels in E. coli and B. subtilis led to cells taking on a spherical morphology and eventual loss of viability since new peptidoglycan is not synthesized evenly along the cell wall (9, 36). Uneven incorporation of new peptidoglycan is potentially driven by the tubulin homologue FtsZ (4, 36). A similar phenotype is achieved by addition of the MreB inhibitor A22 that causes the reversible loss of MreB filament localization in vivo (14, 17). A22 was discovered in a chemical library being screened for the ability to generate anucleate minicells from E. coli (16, 17). It has been used extensively by others to examine MreB function in bacteria of many different genera. The addition of A22 to E. coli cells at 3.13 μg/ml leads to the breakdown of MreB filaments, spheroplasting and the generation of minicells (16). In B. subtilis, A22 at concentrations in excess of 100 μg/ml is required to generate spheroplasts; in C. crescentus 6-h incubations of A22 at 10 μg/ml are needed before any change to the cell shape can be observed—in this case cells take on a characteristic “lemon shape” (14, 16). Isolation and sequencing of A22-resistant mutants of C. crescentus, as well as biochemical evidence from purified MreB from Thermotoga maritima, revealed that A22 binds in the nucleotide binding pocket of MreB (3, 14). In vitro light scattering assays of MreB filamentation showed that A22 acted as a competitive inhibitor of ATP binding and was able in inhibit the formation of MreB filaments, presumably by sequestering and inactivating MreB monomers preventing their recycling (3). This study also demonstrated that in vitro A22 can have a role in stabilizing ADP-bound MreB (3).In the present study we investigated the functions of the two MreB homologues found in B. bacteriovorus, testing the role each has in predation and cell morphology by a combination of genetic approaches and A22 treatment. A reduction in function in both MreB proteins, achieved by C-terminal teal fluorescent protein (TFP) tagging, suggests that the MreB1 (Bd0211) protein was required in the growth-phase cell in bdelloplasts, whereas the MreB2 (Bd1737) protein is required later in the predatory process, for maintaining and allowing successful resolution of the growth-phase filament into short attack-phase vibroid cells.     

The First Bite— Profiling the Predatosome in the Bacterial Pathogen Bdellovibrio

Bdellovibrio bacteriovorus is a Gram-negative bacterium that is a pathogen of other Gram-negative bacteria, including many bacteria which are pathogens of humans, animals and plants. As such Bdellovibrio has potential as a biocontrol agent, or living antibiotic. B. bacteriovorus HD100 has a large genome and it is not yet known which of it encodes the molecular machinery and genetic control of predatory processes. We have tried to fill this knowledge-gap using mixtures of predator and prey mRNAs to monitor changes in Bdellovibrio gene expression at a timepoint of early-stage prey infection and prey killing in comparison to control cultures of predator and prey alone and also in comparison to Bdellovibrio growing axenically (in a prey-or host independent “HI” manner) on artificial media containing peptone and tryptone. From this we have highlighted genes of the early predatosome with predicted roles in prey killing and digestion and have gained insights into possible regulatory mechanisms as Bdellovibrio enter and establish within the prey bdelloplast. Approximately seven percent of all Bdellovibrio genes were significantly up-regulated at 30 minutes of infection- but not in HI growth- implicating the role of these genes in prey digestion. Five percent were down-regulated significantly, implicating their role in free-swimming, attack-phase physiology. This study gives the first post- genomic insight into the predatory process and reveals some of the important genes that Bdellovibrio expresses inside the prey bacterium during the initial attack.     

Association of chromosome 9p21 SNPs with cardiovascular phenotypes in morbid obesity using electronic health record data

Genomic medicine research requires substantial time and resources to obtain phenotype data. The electronic health record offers potential efficiencies in addressing these temporal and economic challenges, but few studies have explored the feasibility of using such data for genetics research. The main objective of this study was to determine the association of two genetic variants located on chromosome 9p21 conferring susceptibility to coronary heart disease and type 2 diabetes with a variety of clinical phenotypes derived from the electronic health record in a population of morbidly obese patients. Data on more than 100 clinical measures including diagnoses, laboratory values, and medications were extracted from the electronic health records of a total of 709 morbidly obese (body mass index (BMI) >/= 40 kg/m(2)) patients. Two common single nucleotide polymorphisms located at chromosome 9p21 recently linked to coronary heart disease and type 2 diabetes (McPherson et al. Science 316:1488-1491, 2007; Saxena et al. Science 316:1331-1336, 2007; Scott et al. Science 316:1341-1345, 2007) were genotyped to assess statistical association with clinical phenotypes. Neither the type 2 diabetes variant nor the coronary heart disease variant was related to any expected clinical phenotype, although high-risk type 2 diabetes/coronary heart disease compound genotypes were associated with several coronary heart disease phenotypes. Electronic health records may be efficient sources of data for validation studies of genetic associations.     

Venezuelan Equine Encephalitis Virus in Iquitos,Peru: Urban Transmission of a Sylvatic Strain

Enzootic strains of Venezuelan equine encephalitis virus (VEEV) have been isolated from febrile patients in the Peruvian Amazon Basin at low but consistent levels since the early 1990s. Through a clinic-based febrile surveillance program, we detected an outbreak of VEEV infections in Iquitos, Peru, in the first half of 2006. The majority of these patients resided within urban areas of Iquitos, with no report of recent travel outside the city. To characterize the risk factors for VEEV infection within the city, an antibody prevalence study was carried out in a geographically stratified sample of urban areas of Iquitos. Additionally, entomological surveys were conducted to determine if previously incriminated vectors of enzootic VEEV were present within the city. We found that greater than 23% of Iquitos residents carried neutralizing antibodies against VEEV, with significant associations between increased antibody prevalence and age, occupation, mosquito net use, and overnight travel. Furthermore, potential vector mosquitoes were widely distributed across the city. Our results suggest that while VEEV infection is more common in rural areas, transmission also occurs within urban areas of Iquitos, and that further studies are warranted to identify the precise vectors and reservoirs involved in urban VEEV transmission.