A conifer genome spruces up plant phylogenomics

The Norway spruce genome provides key insights into the evolution of plant genomes, leading to testable new hypotheses about conifer, gymnosperm, and vascular plant evolution.In the past year a burst of plant genome sequences have been published, providing enhanced phylogenetic coverage of green plants (Figure ​(Figure1)1) and inclusion of new agricultural, ecological, and evolutionary models. Collectively, these sequences are revealing some extraordinary structural and evolutionary attributes in plant genomes. Perhaps most surprising is the exceptionally high frequency of whole-genome duplication (WGD): nearly every genome that has been analyzed has borne the signature of one or more WGDs, with particularly notable events having occurred in the common ancestors of seed plants, of angiosperms, and of core eudicots (the latter ''WGD'' represents two WGDs in close succession) [1,2]. Given this tendency for plant genomes to duplicate and then return to an essentially diploid genetic system (an example is the cotton genomes, which have accumulated the effects of perhaps 15 WGDs [3]), the conservation of genomes in terms of gene number, chromosomal organization, and gene content is astonishing. From the publication of the first plant genome, Arabidopsis thaliana [4], the number of inferred genes has been between 25,000 and 30,000, with many gene families shared across all land plants, although the number of members and patterns of expansion and contraction vary. Furthermore, conserved synteny has been detected across the genomes of diverse angiosperms, despite WGDs, diploidization, and millions of years of evolution.Open in a separate windowFigure 1Simplified phylogeny of land plants, showing major clades and their component lineages. Asterisks indicate species (or lineage) for which whole-genome sequence (or sequences) is (are) available. Increases and decreases in genome size are shown by arrows.Despite the proliferation of genome sequences available for angiosperms, genome-level data for both ferns (and their relatives, collectively termed monilophytes; Figure ​Figure1)1) and gymnosperms have been conspicuously lacking - until recently, with the publication of the genome sequence of the gymnosperm Norway spruce (Picea abies) [5]. The large genome sizes for both monilophytes and gymnosperms have discouraged attempts at genome sequencing and assembly, whereas the smaller genome size of angiosperms has resulted in more genome sequences being available (Table ​(Table1)1) [6]. Because of this limited phylogenetic sample, our understanding of the timing and phylogenetic positions of WGDs, the core number of plant genes, possible conserved syntenic regions, and patterns of expansion and contraction of gene families across both tracheophytes (vascular plants) and across all land plants is imperfect. This sampling problem is particularly acute in analyses of the genes and genomes of seed plants; many hundreds of genes are present in angiosperms that are not present in mosses or lycophytes, but whether these genes arose in the common ancestor of seed plants or of angiosperms cannot be determined without a gymnosperm genome sequence. The Norway spruce genome therefore offers tremendous power, not only for understanding the structure and evolution of conifer genomes, but also as a reference for interpreting gene and genome evolution in angiosperms.

Table 1

Genome sizes in land plants

Association of Campylobacter jejuni Cj0859c Gene (fspA) Variants with Different C. jejuni Multilocus Sequence Types

Cj0859c variants fspA1 and fspA2 from 669 human, poultry, and bovine Campylobacter jejuni strains were associated with certain hosts and multilocus sequence typing (MLST) types. Among the human and poultry strains, fspA1 was significantly (P < 0.001) more common than fspA2. FspA2 amino acid sequences were the most diverse and were often truncated.Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide and responsible for more than 90% of Campylobacter infections (7). Case-control studies have identified consumption or handling of raw and undercooked poultry meat, drinking unpasteurized milk, and swimming in natural water sources as risk factors for acquiring domestic campylobacteriosis in Finland (7, 9). Multilocus sequence typing (MLST) has been employed to study the molecular epidemiology of Campylobacter (4) and can contribute to virulotyping when combined with known virulence factors (5). FspA proteins are small, acidic, flagellum-secreted nonflagellar proteins of C. jejuni that are encoded by Cj0859c, which is expressed by a σ²⁸ promoter (8). Both FspA1 and FspA2 were shown to be immunogenic in mice and protected against disease after challenge with a homologous strain (1). However, FspA1 also protected against illness after challenge with a heterologous strain, whereas FspA2 failed to do the same at a significant level. Neither FspA1 nor FspA2 protected against colonization (1). On the other hand, FspA2 has been shown to induce apoptosis in INT407 cells, a feature not exhibited by FspA1 (8). Therefore, our aim was to study the distributions of fspA1 and fspA2 among MLST types of Finnish human, chicken, and bovine strains.In total, 367 human isolates, 183 chicken isolates, and 119 bovine isolates (n = 669) were included in the analyses (3). PCR primers for Cj0859c were used as described previously (8). Primer pgo6.13 (5′-TTGTTGCAGTTCCAGCATCGGT-3′) was designed to sequence fspA1. Fisher''s exact test or a chi-square test was used to assess the associations between sequence types (STs) and Cj0859c. The SignalP 3.0 server was used for prediction of signal peptides (2).The fspA1 and fspA2 variants were found in 62.6% and 37.4% of the strains, respectively. In 0.3% of the strains, neither isoform was found. Among the human and chicken strains, fspA1 was significantly more common, whereas fspA2 was significantly more frequent among the bovine isolates (Table ​(Table1).1). Among the MLST clonal complexes (CCs), fspA1 was associated with the ST-22, ST-45, ST-283, and ST-677 CCs and fspA2 was associated with the ST-21, ST-52, ST-61, ST-206, ST-692, and ST-1332 CCs and ST-58, ST-475, and ST-4001. Although strong CC associations of fspA1 and fspA2 were found, the ST-48 complex showed a heterogeneous distribution of fspA1 and fspA2. Most isolates carried fspA2, and ST-475 was associated with fspA2. On the contrary, ST-48 commonly carried fspA1 (Table ​(Table1).1). In our previous studies, ST-48 was found in human isolates only (6), while ST-475 was found in both human and bovine isolates (3, 6). The strict host associations and striking difference between fspA variants in human ST-48 isolates and human/bovine ST-475 isolates suggest that fspA could be important in host adaptation.

TABLE 1.

Percent distributions of fspA1 and fspA2 variants among 669 human, poultry, and bovine Campylobacter jejuni strains and their associations with hosts, STs, and CCs

Sheep with scrapie and mastitis transmit infectious prions through the milk

Prions are misfolded proteins that are infectious and naturally transmitted, causing a fatal neurological disease in humans and animals. Prion shedding routes have been shown to be modified by inflammation in excretory organs, such as the kidney. Here, we show that sheep with scrapie and lentiviral mastitis secrete prions into the milk and infect nearly 90% of naïve suckling lambs. Thus, lentiviruses may enhance prion transmission, conceivably sustaining prion infections in flocks for generations. This study also indicates a risk of prion spread to sheep and potentially to other animals through dietary exposure to pooled sheep milk or milk products.Prion diseases have emerged globally as a significant threat to human and animal health. Recently, human-to-human spread of prions is believed to have occurred through blood transfusions (9, 12, 16), underscoring the importance of understanding possible transmission routes. PrP^Sc, a misfolded, aggregated form of the normal prion protein, PrP^C, commonly accumulates in the follicles of lymphoid tissues, prior to entering the central nervous system (2, 11, 14). Inflammation can cause lymphoid follicles to form in other organs, such as liver and kidney, which leads to prion invasion of organs that are not typically prion permissive (1). In mice, prion infection in the inflamed kidney has the untoward consequence of prion excretion in urine (13). This finding, together with our report of sheep with PrP^Sc in the inflamed mammary gland (8), has raised concerns of prion secretion into milk.Here, we investigated whether PrP^Sc in the inflamed mammary gland leads to prion secretion in milk and infection of naïve lambs through suckling. Prion infectivity has been detected in the milk of sheep expressing a prion gene (Prnp) coding for VRQ/VRQ or VRQ/ARQ at polymorphic codons 136, 154, and 171 (3, 4). However, whether (i) sheep-to-lamb transmission of prions in milk leads to clinical prion disease or (ii) sheep with the common ARQ/ARQ Prnp genotype can infect lambs through milk is unknown. We induced a chronic lentiviral mastitis and inoculated ARQ/ARQ Sarda breed sheep with infectious prions. After 14 months, we bred the sheep and collected the milk. To avoid cross-contamination of newborn lambs, we fed the milk to imported known-naïve lambs and then monitored the lambs for signs of prion infection (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.Sheep infected with prions and maedi-visna virus (MVV) develop lymphofollicular mastitis with PrP^Sc. (A) Experimental scheme. Sheep were inoculated with culture medium or MVV and were then orally exposed to scrapie prions and bred. Milk was collected near the time point that neurologic signs of scrapie developed and was fed to naïve lambs. The ratio of lambs with detectable PrP^Sc to lambs fed the indicated milk is shown for each experiment. (B) PrP immunohistochemistry assay of brain and tonsil from milk source sheep shows staining for PrP^Sc in the brainstem, particularly in the vagal nucleus (indicated by asterisks) and in the tonsillar follicles of scrapie-infected sheep (arrows). (C) Mammary gland (MG) of milk source sheep shows lymphoid follicles (arrowheads) with associated PrP^Sc (arrows) adjacent to milk ducts (md) in the MVV-inoculated sheep, whereas the medium-inoculated sheep had a histologically normal MG with no detectable PrP^Sc. Insets show a high magnification of follicles containing PrP^Sc. Scale bar = 100 μm; scale bar in inset = 25 μm. (D) Western blot analysis shows PrP^Sc detection in MG of sheep inoculated with MVV/scrapie agents but not in sheep inoculated with scrapie prions only. The sheep identification number is indicated for each lane. PK, proteinase K digested; pos. br, positive brain control; neg. br, negative brain control.To induce a chronic lymphofollicular mastitis, we exposed 7- to 10-day-old lambs (groups of 10) by intratracheal and intravenous routes to a common sheep lentivirus known as maedi-visna virus (MVV) or to cell culture medium only. To do this, lambs were inoculated with 3.5 ml intravenously and 0.5 ml intratracheally of MVV in culture supernatant containing 1.5 × 10⁶ tissue culture infectious doses/ml of the “rapid/high” MVV strain 85/34 (5, 15). Twenty days later, all lambs were orally inoculated with 25 ml of 10% scrapie-infected brain homogenate from a pool of naturally infected Sarda sheep.We sequenced the entire Prnp gene and found that all lambs expressed the ARQ/ARQ Prnp genotype, indicating that the sheep should be susceptible to scrapie. As negative controls, 2 lambs of Prnp genotype ARR/ARR and ARQ/ARQ were mock inoculated with cell culture medium and healthy brain homogenate. All lambs originated from scrapie-free flocks that had been monitored for clinical scrapie cases for at least 3 years.All inoculated sheep were naturally bred to rams at 15 months postinoculation (p.i.) and produced lambs at 20 months p.i. Sheep developed early signs of scrapie just after the lambs were born. Milk from each sheep was manually collected and frozen daily.Eight of 10 MVV-and-scrapie (denoted MVV/scrapie)-inoculated sheep and 9 of 10 scrapie-inoculated sheep showed clinical signs of scrapie, with mean incubation periods of 22 ± 1.4 and 23 ± 1.5 months postinoculation, respectively, and were euthanized. There was no significant difference in incubation period between the groups (Student''s t test, P = 0.5), indicating that inflammation associated with the MVV infection does not accelerate prion disease. This finding is consistent with the results of previous studies that showed that chronic pancreatitis or nephritis did not affect the scrapie incubation period (1). Scrapie infection was confirmed postmortem by the detection of PrP^Sc in brain and lymphoid tissues by Western blot and immunohistochemistry assays (Fig. ​(Fig.1B).1B). Interestingly, scrapie did not develop in 3 sheep with a Prnp gene encoding a rare polymorphism at codon 176 (K), consistent with recent reports describing scrapie resistance for this genotype (10).Antibodies to MVV were detected in serum of all the MVV-inoculated sheep by indirect enzyme-linked immunosorbent assay (ELISA) (Elitest kit; Hyphen BioMed). Five of 8 MVV/scrapie-infected sheep (63%) showed a lymphofollicular mastitis (Fig. ​(Fig.1C),1C), and 3 had a diffuse interacinar lymphoid infiltrate. Of the 5 sheep with lymphofollicular mastitis, 4 had PrP^Sc deposits detectable by immunohistochemistry and Western blot assays (Fig. 1C and D), whereas no sheep with diffuse lymphoid infiltrates had detectable PrP^Sc. Surprisingly, 2 of 9 sheep inoculated only with scrapie also had lymphofollicular mastitis and anti-MVV antibodies, one of which had visible PrP^Sc deposits. MVV is a common pathogen in Europe, and it is possible that these sheep were infected from the dam. The remaining 7 scrapie-inoculated sheep had histologically normal mammary glands (Fig. ​(Fig.1C)1C) and no detectable PrP^Sc (Fig. ​(Fig.1D)1D) or anti-MVV antibodies.We selected the stored milk from the 4 MVV/scrapie-infected sheep with PrP^Sc in the mammary glands and from the 7 scrapie-infected sheep with histologically normal mammary glands. Milk samples from the early, middle, and late stages of lactation were pooled for each group. We imported naïve Cheviot lambs (n = 9) from flocks that originated from scrapie-free New Zealand and had been bred and housed under strict biosecurity containment in the United Kingdom to ensure that the lambs had not been exposed to scrapie. The Sarda lambs (n = 4) originated from a scrapie-free flock in Sardinia. We then fed pooled milk from MVV/scrapie-infected sheep to each of 8 naïve ARQ/ARQ lambs and from scrapie-infected sheep to 3 naïve ARQ/ARQ lambs ad libitum. Each lamb ingested a total volume of 1 to 2 liters over a total period of 3 days (Table ​(Table1).1). Two lambs were orally inoculated with brain homogenate pooled from the scrapie-infected milk donors as positive controls. Groups of lambs were housed in separate stalls and subjected to isolation conditions.

TABLE 1.

Genotype, breed, and PrP^Sc detection in lambs fed milk from MVV/scrapie- or scrapie-infected sheep

Widespread Distribution of Cell Defense against d-Aminoacyl-tRNAs

Several l-aminoacyl-tRNA synthetases can transfer a d-amino acid onto their cognate tRNA(s). This harmful reaction is counteracted by the enzyme d-aminoacyl-tRNA deacylase. Two distinct deacylases were already identified in bacteria (DTD1) and in archaea (DTD2), respectively. Evidence was given that DTD1 homologs also exist in nearly all eukaryotes, whereas DTD2 homologs occur in plants. On the other hand, several bacteria, including most cyanobacteria, lack genes encoding a DTD1 homolog. Here we show that Synechocystis sp. PCC6803 produces a third type of deacylase (DTD3). Inactivation of the corresponding gene (dtd3) renders the growth of Synechocystis sp. hypersensitive to the presence of d-tyrosine. Based on the available genomes, DTD3-like proteins are predicted to occur in all cyanobacteria. Moreover, one or several dtd3-like genes can be recognized in all cellular types, arguing in favor of the nearubiquity of an enzymatic function involved in the defense of translational systems against invasion by d-amino acids.Although they are detected in various living organisms (reviewed in Ref. 1), d-amino acids are thought not to be incorporated into proteins, because of the stereospecificity of aminoacyl-tRNA synthetases and of the translational machinery, including EF-Tu and the ribosome (2). However, the discrimination between l- and d-amino acids by aminoacyl-tRNA synthetases is not equal to 100%. Significant d-aminoacylation of their cognate tRNAs by Escherichia coli tyrosyl-, tryptophanyl-, aspartyl-, lysyl-, and histidyl-tRNA synthetases has been characterized in vitro (3–9). Recently, using a bacterium, transfer of d-tyrosine onto tRNA^Tyr was shown to occur in vivo (10).With such misacylation reactions, the resulting d-aminoacyl-tRNAs form a pool of metabolically inactive molecules, at best. At worst, d-aminoacylated tRNAs infiltrate the protein synthesis machinery. Although the latter harmful possibility has not yet been firmly established, several cells were shown to possess a d-tyrosyl-tRNA deacylase, or DTD, that should help them counteract the accumulation of d-aminoacyl-tRNAs. This enzyme shows a broad specificity, being able to remove various d-aminoacyl moieties from the 3′-end of a tRNA (4–6, 11). Such a function makes the deacylase a member of the family of enzymes capable of editing in trans mis-aminoacylated tRNAs. This family includes several homologs of aminoacyl-tRNA synthetase editing domains (12), as well as peptidyl-tRNA hydrolase (13, 14).Two distinct deacylases have already been discovered. The first one, called DTD1, is predicted to occur in most bacteria and eukaryotes (see d-amino acids, including d-tyrosine (6). In fact, in an E. coli Δdtd strain grown in the presence of 2.4 mm d-tyrosine, as much as 40% of the cellular tRNA^Tyr pool becomes esterified with d-tyrosine (10).

TABLE 1

Distribution of DTD1 and DTD2 homologs in various phylogenetic groupsHomologs of DTD1 and DTD2 were searched for using a genomic Blast analysis against complete genomes in the NCBI Database (www.ncbi.nlm.nih.gov). Values in the table are number of species. For instance, E. coli is counted only once in γ-proteobacteria despite the fact that several E. coli strains have been sequenced.

Efflux of hydraulically lifted water from mycorrhizal fungal hyphae during imposed drought

Apart from improving plant and soil water status during drought, it has been suggested that hydraulic lift (HL) could enhance plant nutrient capture through the flow of mineral nutrients directly from the soil to plant roots, or by maintaining the functioning of mycorrhizal fungi. We evaluated the extent to which the diel cycle of water availability created by HL covaries with the efflux of HL water from the tips of extramatrical (external) mycorrhizal hyphae, and the possible effects on biogeochemical processes. Phenotypic mycorrhizal fungal variables, such as total and live hyphal lengths, were positively correlated with HL efflux from hyphae, soil water potential (dawn), and plant response variables (foliar ¹⁵N). The efflux of HL water from hyphae was also correlated with bacterial abundance and soil enzyme activity (P), and the moistening of soil organic matter. Such findings indicate that the efflux of HL water from the external mycorrhizal mycelia may be a complementary explanation for plant nutrient acquisition and survival during drought.Key words: hydraulic lift, nitrogen, phosphorus, microbial abundance, mycorrhizal hyphae, QuercusIn environments that experience seasonal or extended drought, plant productivity, resource partitioning, and competition are limited by the availability of water and mineral nutrients. One mechanism that is important to whole plant water balance in these environments is hydraulic lift (HL), a passive process driven by gradients in water potential among soils layers. Soil water is transported upwards from deep moist soils and released into the nutrient-rich upper soil layers by root systems accessing both deep and shallow soil layers.¹ HL water may improve the lifespan and activity of fine roots in a wide variety of plant life forms.²Hydraulic lift may also have a second ecological function in facilitating plant nutrient acquisition.² It been hypothesized that HL water could enhance the supply of nutrients to roots through mass flow or diffusion,³ or trigger episodes of soil biotic activity such as microbe-mediated nutrient transformations^4,5 that are analogous to the increased inflow of nitrogen (N) into roots and flushes of carbon (C) and N mineralization respectively that follow precipitation events.^4,6 However, few data currently exist with which to test these possibilities.Hydraulically lifted water also sustains mycorrhizal fungi,^7,8 a mutualism that enhances the acquisition of water and mineral nutrients in many terrestrial plant species. Mycorrhizal fungal hyphae provide comprehensive exploration and rapid access to small-scale or temporary nutrient flushes that may not be available to plant roots.⁹ This resource flow has often been assumed to be a unidirectional flux whereby resources are moved from source (soil) into the sink (plant) by the fungal hyphae. However, there is now evidence to suggest that the physiological plasticity of the peripheral extramatrical hyphae, and in particular the hyphal tips, permits the exudation, and subsequent reabsorption, of water and solutes.^10,11 Laboratory experiments using pure cultures have demonstrated that water may be exuded from the hyphal tips, especially in fungal species with hydrophobic hyphae, along with a variety of organic molecules, such as free amino acids.^10–13 At the same time, water, mobile minerals, amino acids and other low-molecular weight metabolites may be selectively and actively reabsorbed by mycorrhizal fungal hyphae.¹¹ However, quantitative data on the environmental impact of hyphal exudation and reabsorption is still largely lacking.We ask: could the diel cycle of water availability created by HL produce a water efflux from hyphal tips and if so, would this be sufficient to impact biogeochemical processes? Is there also an opposite rhythm driven by plant transpiration so that any resultant soil solution is pulled towards hyphal tips and consequently, the host plant? By imposing drought on seedlings of Quercus agrifolia Nee (coast live oak; Fagaceae) grown in mesocosms (Fig. 1), we identified a composite of feedbacks that could influence nutrient capture with HL (Fig. 2). Our analyses provide support for the key predictions of the HL-nutrient cycling scenario including the efflux of HL water from the extramatrical hyphae (Fig. 3), moistening of soil organic matter (Figs. 3 and ​and4),4), and the maintenance of soil microbial activity and nutrient capture (N, P; Open in a separate windowFigure 1Quercus mesocosms demonstrating the plant, root, and hyphal compartments. Details of soil conditions, plant inoculation protocol, mycorrhizal fungi and dye injection methods are detailed in previous work (ref. 7) Point 1 (tap root compartment) denotes the region in which fluorescent tracer dyes were injected into the mesocosm at dusk to track the path of HL water. Point 2 (hyphal chamber) denotes spots adjacent to or distant from the mesh screen into which a small volume (200 µl) of fluorescent and ¹⁵N tracers (99% as ¹⁵NH₄¹⁵NO₃) were injected at dawn to measure water and nutrient uptake by the external hyphae.Open in a separate windowFigure 2Path analysis of the influence of different soil and mycorrhizal factors on nutrient capture with HL, and resultant model showing the significant path coefficients among variables in the Q. agrifolia mesocosms. Lines with a single arrow denote possible cause-effect relationships. The partial correlation coefficients adjacent to each line indicate the strength of the association between the individual factors. Thick lines are statistically significant (p < 0.05) whereas thin lines indicate no significant relationship between parameters (p > 0.05) and only significant coefficients are given (p < 0.05).Open in a separate windowFigure 3Fluorescently-labeled structures recovered from the hyphal chamber of Quercus microcosms following 80 days of soil drying and with nocturnal hydraulic lift. Yellow-green fluorescence indicates samples labeled with Lucifer yellow CH (LYCH), blue fluorescence denotes samples labeled with Cascade blue (CB) hydrazide. (A) CB-labeled leaf litter from the soil and (B) soil particle; (C) LYCH-labeled root fragment in the soil mixture with adherent extramatrical hyphae; (D) LYCH tracer dye fluorescence in labeled extramatrical hyphae and in efflux (arrow) from the hyphal tip onto organic matter; (E and F) external hyphae filled with LYCH (influx; arrow) and (G) background fluorescence in non-labeled extramatrical hyphae.Open in a separate windowFigure 4Measurements of hyphal efflux and influx based on the quantitative analysis of LYCH fluorescence intensity in soil solution. Fluorescent intensity values were converted to LYCH concentration using a standard curve generated for the dye since fluorescent intensity correlates with the number of fluorescent molecules in solution. Influx is the uptake of LYCH by hyphae as driven by plant transpiration demands (day), and measured efflux is the passive loss of LYCH from hyphae into the surrounding soil during HL (night). Vertical bars indicate the standard error of the means.

Table 1

Summary of soil, microbial, mycorrhizal and plant parameters in plant or hyphal compartments

Mechanisms and Evidence of Genital Coevolution: The Roles of Natural Selection,Mate Choice,and Sexual Conflict

Genital coevolution between the sexes is expected to be common because of the direct interaction between male and female genitalia during copulation. Here we review the diverse mechanisms of genital coevolution that include natural selection, female mate choice, male–male competition, and how their interactions generate sexual conflict that can lead to sexually antagonistic coevolution. Natural selection on genital morphology will result in size coevolution to allow for copulation to be mechanically possible, even as other features of genitalia may reflect the action of other mechanisms of selection. Genital coevolution is explicitly predicted by at least three mechanisms of genital evolution: lock and key to prevent hybridization, female choice, and sexual conflict. Although some good examples exist in support of each of these mechanisms, more data on quantitative female genital variation and studies of functional morphology during copulation are needed to understand more general patterns. A combination of different approaches is required to continue to advance our understanding of genital coevolution. Knowledge of the ecology and behavior of the studied species combined with functional morphology, quantitative morphological tools, experimental manipulation, and experimental evolution have been provided in the best-studied species, all of which are invertebrates. Therefore, attention to vertebrates in any of these areas is badly needed.Of all the evolutionary interactions between the sexes, the mechanical interaction of genitalia during copulation in species with internal fertilization is perhaps the most direct. For this reason alone, coevolution between genital morphologies of males and females is expected. Morphological and genetic components of male and female genitalia have been shown to covary in many taxa (Sota and Kubota 1998; Ilango and Lane 2000; Arnqvist and Rowe 2002; Brennan et al. 2007; Rönn et al. 2007; Kuntner et al. 2009; Tatarnic and Cassis 2010; Cayetano et al. 2011; Evans et al. 2011, 2013; Simmons and García-González 2011; Yassin and Orgogozo 2013; and see examples in

Perioperative Opioid Counseling Reduces Opioid Use Following Primary Total Joint Arthroplasty

BackgroundPreoperative counseling may reduce postoperative opioid requirements; however, there is a paucity of randomized controlled trials (RCTs) demonstrating efficacy. The purpose of this study was to perform an interventional, telehealth-based RCT evaluating the effect of peri-operative counseling on quantity and duration of opioid consumption following primary total joint arthroplasty (TJA).MethodsParticipants were randomized into three groups: 1. Control group, no perioperative counseling; 2. Intervention group, preoperative educational video; 3. Intervention group, preoperative educational video and postoperative acceptance and commitment therapy (ACT). Opioid consumption was evaluated daily for 14 days and at 6 weeks postoperatively. Best-case and worse-case intention to treat analyses were performed to account for non-responses. Bonferroni corrections were applied.Results183 participants were analyzed (63 in Group 1, 55 in Group 2, and 65 in Group 3). At 2 weeks postoperatively, there was no difference in opioid consumption between Groups 1, 2, and 3 (p>0.05 for all). At 6 weeks postoperatively, Groups 2 and 3 had consumed significantly less opioids than Group 1 (p=0.04, p<0.001) (VariableGroupp-value1. Control2. Video OnlyVideo + ACTSex (n, % female)39 (62%)32 (58%)40 (62%)0.90Surgery (n, % THA)26 (41%)21 (38%)31 (47%)0.56Age (mean ± SD; years)59 ± 1159 ± 1158 ± 9Overall: 0.83
1v2: 0.98
2v3: 0.65
2v3: 0.56Prolonged Opioid Use > 60 mo. (n, %)000-Opioid Use Within 3 mo. of Index Surgery (n, %)0 (14%)4 (7%)5 (8%)0.34Open in a separate windowSD – standard deviation.Table 2.Quantity of Opioid Consumption at 2 Weeks Postoperatively, Best-Case Scenario

Variation in Adult Plant Phenotypes and Partitioning among Seed and Stem-Borne Roots across Brachypodium distachyon Accessions to Exploit in Breeding Cereals for Well-Watered and Drought Environments

Seedling roots enable plant establishment. Their small phenotypes are measured routinely. Adult root systems are relevant to yield and efficiency, but phenotyping is challenging. Root length exceeds the volume of most pots. Field studies measure partial adult root systems through coring or use seedling roots as adult surrogates. Here, we phenotyped 79 diverse lines of the small grass model Brachypodium distachyon to adults in 50-cm-long tubes of soil with irrigation; a subset of 16 lines was droughted. Variation was large (total biomass, ×8; total root length [TRL], ×10; and root mass ratio, ×6), repeatable, and attributable to genetic factors (heritabilities ranged from approximately 50% for root growth to 82% for partitioning phenotypes). Lines were dissected into seed-borne tissues (stem and primary seminal axile roots) and stem-borne tissues (tillers and coleoptile and leaf node axile roots) plus branch roots. All lines developed one seminal root that varied, with branch roots, from 31% to 90% of TRL in the well-watered condition. With drought, 100% of TRL was seminal, regardless of line because nodal roots were almost always inhibited in drying topsoil. Irrigation stimulated nodal roots depending on genotype. Shoot size and tillers correlated positively with roots with irrigation, but partitioning depended on genotype and was plastic with drought. Adult root systems of B. distachyon have genetic variation to exploit to increase cereal yields through genes associated with partitioning among roots and their responsiveness to irrigation. Whole-plant phenotypes could enhance gain for droughted environments because root and shoot traits are coselected.Adult plant root systems are relevant to the size and efficiency of seed yield. They supply water and nutrients for the plant to acquire biomass, which is positively correlated to the harvest index (allocation to seed grain), and the stages of flowering and grain development. Modeling in wheat (Triticum aestivum) suggested that an extra 10 mm of water absorbed by such adult root systems during grain filling resulted in an increase of approximately 500 kg grain ha⁻¹ (Manschadi et al., 2006). This was 25% above the average annual yield of wheat in rain-fed environments of Australia. This number was remarkably close to experimental data obtained in the field in Australia (Kirkegaard et al., 2007). Together, these modeling and field experiments have shown that adult root systems are critical for water absorption and grain yield in cereals, such as wheat, emphasizing the importance of characterizing adult root systems to identify phenotypes for productivity improvements.Most root phenotypes, however, have been described for seedling roots. Seedling roots are essential for plant establishment, and hence, the plant’s potential to set seed. For technical reasons, seedlings are more often screened than adult plants because of the ease of handling smaller plants and the high throughput. Seedling-stage phenotyping may also improve overall reproducibility of results because often, growth media are soil free. Seedling soil-free root phenotyping conditions are well suited to dissecting fine and sensitive mechanisms, such as lateral root initiation (Casimiro et al., 2003; Péret et al., 2009a, 2009b). A number of genes underlying root processes have been identified or characterized using seedlings, notably with the dicotyledonous models Arabidopsis (Arabidopsis thaliana; Mouchel et al., 2004; Fitz Gerald et al., 2006; Yokawa et al., 2013) and Medicago truncatula (Laffont et al., 2010) and the cereals maize (Zea mays; Hochholdinger et al., 2001) and rice (Oryza sativa; Inukai et al., 2005; Kitomi et al., 2008).Extrapolation from seedling to adult root systems presents major questions (Hochholdinger and Zimmermann, 2008; Chochois et al., 2012; Rich and Watt, 2013). Are phenotypes in seedling roots present in adult roots given developmental events associated with aging? Is expression of phenotypes correlated in seedling and adult roots if time compounds effects of growth rates and growth conditions on roots? Watt et al. (2013) showed in wheat seedlings that root traits in the laboratory and field correlated positively but that neither correlated with adult root traits in the field. Factors between seedling and adult roots seemed to be differences in developmental stage and the time that growing roots experience the environment.Seedling and adult root differences may be larger in grasses than dicotyledons. Grass root systems have two developmental components: seed-borne (seminal) roots, of which a number emerge at germination and continue to grow and branch throughout the plant life, and stem-borne (nodal or adventitious) roots, which emerge from around the three-leaf stage and continue to emerge, grow, and branch throughout the plant life. Phenotypes and traits of adult root systems of grasses, which include the major cereal crops wheat, rice, and maize, are difficult to predict in seedling screens and ideally identified from adult root systems first (Gamuyao et al., 2012).Phenotyping of adult roots is possible in the field using trenches (Maeght et al., 2013) or coring (Wasson et al., 2014). A portion of the root system is captured with these methods. Alternatively, entire adult root systems can be contained within pots dug into the ground before sowing. These need to be large; field wheat roots, for example, can reach depths greater than 1.5 m depending on genotype and environment. This method prevents root-root interactions that occur under normal field sowing of a plant canopy and is also a compromise.A solution to the problem of phenotyping adult cereal root systems is a model for monocotyledon grasses: Brachypodium distachyon. B. distachyon is a small-stature grass with a small genome that is fully sequenced (Vogel et al., 2010). It has molecular tools equivalent to those available in Arabidopsis (Draper et al., 2001; Brkljacic et al., 2011; Mur et al., 2011). The root system of B. distachyon reference line Bd21 is more similar to wheat than other model and crop grasses (Watt et al., 2009). It has a seed-borne primary seminal root (PSR) that emerges from the embryo at seed germination and multiple stem-borne coleoptile node axile roots (CNRs) and leaf node axile roots (LNRs), also known as crown roots or adventitious roots, that emerge at about three leaves through to grain development. Branch roots emerge from all root types. There are no known anatomical differences between root types of wheat and B. distachyon (Watt et al., 2009). In a recent study, we report postflowering root growth in B. distachyon line Bd21-3, showing that this model can be used to answer questions relevant to the adult root systems of grasses (Chochois et al., 2012).In this study, we used B. distachyon to identify adult plant phenotypes related to the partitioning among seed-borne and stem-borne shoots and roots for the genetic improvement of well-watered and droughted cereals (Fig. 1; Krassovsky, 1926; Navara et al., 1994), nitrogen, phosphorus (Tennant, 1976; Brady et al., 1995), oxygen (Wiengweera and Greenway, 2004), soil hardness (Acuna et al., 2007), and microorganisms (Sivasithamparam et al., 1978). Of note is the study by Krassovsky (1926), which was the first, to our knowledge, to show differences in function related to water. Krassovsky (1926) showed that seminal roots of wheat absorbed almost 2 times the water as nodal roots per unit dry weight but that nodal roots absorbed a more diluted nutrient solution than seminal roots. Krassovsky (1926) also showed by removing seminal or nodal roots as they emerged that “seminal roots serve the main stem, while nodal roots serve the tillers” (Krassovsky, 1926). Volkmar (1997) showed, more recently, in wheat that nodal and seminal roots may sense and respond to drought differently. In millet (Pennisetum glaucum) and sorghum (Sorghum bicolor), Rostamza et al. (2013) found that millet was able to grow nodal roots in a dryer soil than sorghum, possibly because of shoot and root vigor.Open in a separate windowFigure 1.B. distachyon plant scanned at the fourth leaf stage, with the root and shoot phenotypes studied indicated. Supplemental Table S1.

A Review of Principal Studies on the Development and Treatment of Epithelial Ovarian Cancer in the Laying Hen Gallus gallus

Often referred to as the silent killer, ovarian cancer is the most lethal gynecologic malignancy. This disease rarely shows any physical symptoms until late stages and no known biomarkers are available for early detection. Because ovarian cancer is rarely detected early, the physiology behind the initiation, progression, treatment, and prevention of this disease remains largely unclear. Over the past 2 decades, the laying hen has emerged as a model that naturally develops epithelial ovarian cancer that is both pathologically and histologically similar to that of the human form of the disease. Different molecular signatures found in human ovarian cancer have also been identified in chicken ovarian cancer including increased CA125 and elevated E-cadherin expression, among others. Chemoprevention studies conducted in this model have shown that decreased ovulation and inflammation are associated with decreased incidence of ovarian cancer development. The purpose of this article is to review the major studies performed in laying hen model of ovarian cancer and discuss how these studies shape our current understanding of the pathophysiology, prevention and treatment of epithelial ovarian cancer.

Ovarian cancer is the leading cause of death among female gynecologic malignancies, with a 47% 5 y relative survival rate.¹⁵⁴ Early detection of the disease is necessary for decreasing the high mortality rate. However, early detection is difficult due to the lack of known specific biomarkers and clinically detectable symptoms until the tumor reaches at an advanced stage. The disease has multiple subtypes. Epithelial ovarian cancer (EOC) is the most common type of ovarian cancer, accounting for about 90% of all reported cases.^127,164 EOC is commonly subdivided into 5 histotypes: high-grade serous (HGSOC), low-grade serous, mucinous, endometroid (EC), and clear cell. The histotypes differ in terms of tumor cell morphology, severity, systemic effect, and response to treatment. Among the different subtypes, HGSOC accounts for about 70% of cases of EOC observed in women. HGSOC has a higher mitotic index and is a more aggressive form of cancer with a worse prognosis. HGSOC and low-grade serous histotypes exhibit distinctly different presentations of the disease^82,166 and demand different treatment modalities. EC (10% to 20%), mucinous (5% to 20%), and clear cell (3% to 10%) histotypes are less common forms of the disease. The subtypes of EOC also differ in terms of 5 y survival rates of patients; that is, HGSOC (20% to 35%), EC (40% to 63%), mucinous (40% to 69%), and clear cell (35% to 50%).^20,76,148Developing a representative animal model for EOC has been challenging due to the histologic and pathologic differences among different subtypes of EOC. While developing a reliable animal model is challenging due to the vast complexity and limited understanding of the origin of the disease, laying hens naturally develop EOC that is histopathologically very similar to the human form of the disease (Figure 1).¹⁵ All the different human ovarian cancer histotypes have been observed in laying hen ovarian cancer (Figure 2). In addition, the presentation of the disease in chickens is remarkably similar to the human form of the disease, with early-stage ovarian cancer in laying hens having similar precursor lesions as occur in women.¹⁵ The laying hen develops ovarian cancer spontaneously, allowing analysis of early events and investigation into the natural course of the disease, as tumors can be examined as they progress from normal to late-stage ovarian carcinoma. The gross appearance of these stages is shown in Figure 3.Open in a separate windowFigure 1.Gross pathologic presentation of chicken compared with human ovarian cancer. The remarkably similar presentation in hens (A,B) and women (C,D) at the gross anatomic level with profuse abdominal ascites and peritoneal dissemination of metastasis. A) Ascites in abdominal cavity chicken with advanced ovarian cancer (photo credit: DB Hales); (B) Chicken ovarian cancer with extensive peritoneal dissemination of metastasis (photo credit: DB Hales); (C) Distended abdomen from ascites fluid accumulation in woman with ovarian cancer (http://www.pathguy.com/bryanlee/ovca.html) (D) Human ovarian cancer with extensive peritoneal dissemination of metastasis (http://www.pathguy.com/bryanlee/ovca.html).Open in a separate windowFigure 2.Gross anatomic appearance of different stages of ovarian cancer in the chicken The progression from the normal hen ovary to late-stage metastatic ovarian cancer. (A) Normal chicken ovary showing hierarchal clutch of developing follicles and postovulatory follicle; (B) Stage 1 ovarian cancer, confined to ovary with vascularized follicles; (C) Stage 2/3 ovarian cancer, metastasis locally to peritoneal cavity with ascites; (D) Stage 4 ovarian cancer, late stage with metastasis to lung and liver with extensive ascites (photo credits: DB Hales).Open in a separate windowFigure 3.Histologic subtypes in chicken compared with human ovarian cancers. H and E staining of formalin fixed paraffin embedded tissues from hens with ovarian cancer (A through D) and women (E through G). (A) Chicken clear cell carcinoma; (B) Chicken endometrioid carcinoma; (C) Chicken mucinous adenocarcinoma; (D) Chicken serous papillary adenocarcinoma (photo credits: DB Hales). (E) Human clear cell carcinoma; (F) Human endometrioid carcinoma; (G) Human mucinous cystadenocarcinoma; (H) Human serous adenocarcinoma (https://www.womenshealthsection.com).Over the past 2 decades, the laying hen has emerged as a valuable experimental model for EOC, in addition to other in vivo models such as Patient-Derived Xenografts (PDX) and Genetically Engineered Mouse Models (GEMMs). Comparison of the hen model with other animal models has been reviewed elsewhere.⁷² Modern-day laying hens, such as the white leghorn, have been selected from their ancestor red jungle fowl⁵⁷ for decreased broodiness and persistent ovulation, resulting in approximately one egg per day, if proper nutrition and light-dark cycles are maintained. Daily rupture and consequent repair of the ovarian surface epithelia (OSE) due to the persistent ovulation promotes potential error during rapid DNA replication. This increases the probability of oncogenic mutations, ultimately leading to neoplasia.¹³⁷ Inflammation resulting from continuous ovulation also promotes the natural development of EOC.⁸¹ By the age of 2.5 to 3 y, laying hens have undergone a similar number of ovulations as a perimenopausal woman. The risk of ovarian cancer in white leghorn hens in this time (4%) is similar to the lifetime risk of ovarian cancer in women (0.35% to 8.8%).¹²⁵ By the age of 4 to 6 y, the risk of ovarian cancer in hens rises to 30% to 60%.⁵⁴ The incidence of ovarian carcinoma in the hens, however, depends on the age, genetic strain,⁸⁰ and the egg-laying frequency of the specific breed.⁵⁴ The common white leghorn hen has routinely been employed in chicken ovarian cancer studies. On average, hens are exposed to 17 h of light per day, with lights turned on at 0500 h and turned off at 2200 h. The laying hen model of EOC does present some considerable challenges. Despite its great utility for research, the model is still used mainly by agricultural poultry scientists and a small number of ovarian cancer researchers.Comprehensive and proper vivarium support is required to conduct large-scale cancer prevention studies. Only a few facilities are available for biomedical chicken research, including University of Illinois Urbana-Champaign, Cornell University, Penn State University, NC State, Auburn University, and MS State University. Another difficulty is a lack of available antibodies specific for chicken antigens. Because of the structural dissimilarities between most human proteins and murine antigens to their chicken counterparts, cross-reactivity of available antibodies is also limited. The entire chicken genome was sequenced in 2004;⁷⁸ however, the chromosomal locus of many key genes, such as p53, are still unknown. Overall, humans and chickens share about 60% of genetic commonality, whereas humans and rats share about 88% of their genes. Specific pathway-mutated strains of chickens are not yet available, limiting the ability to study key pathways in carcinogenesis and prevention of cancer using this model. Although all 5 different subtypes of ovarian cancer are present in hens, their most predominant subtype is different from women. Close to 70% of women diagnosed with ovarian cancer have serous EOC, while the predominant subtype reported in hens is endometrioid.¹⁵ However, these comparisons are complicated because observations of cancer in hens consist of both early and late stages of the disease, wherein women, most of the data is from late stage and aggressive ovarian carcinoma.The spontaneous onset of ovarian cancer and the histologic and pathologic similarities to the human form of the disease make laying hens an excellent model for continued research on EOC. To date, a large number of studies have been performed on laying hens. Here we have divided the current studies into 2 groups— (A) studies that have described the molecular presentation of EOC to be similar to that in women; (AuthorYearSignificanceKey molecular targetsCitationHaritani and colleagues.1984Investigating ovarian tumors for key gene signaturesOvalbumin 71 Rodriguez-Burford and colleagues.2001Investigating expressions of clinically important prognostic markers in cancerous hensCA125, cytokeratin AE1/AE3, pan cytokeratin, Lewis Y, CEA, Tag 72, PCNA, EGFR, erbB-2, p27, TGF{α}, Ki-67, MUC1, and MUC2 135 Giles and colleagues.2004, 2006Investigating ovarian tumors for key gene signaturesOvalbumin, PR, PCNA, Vimentin62, 63Jackson and colleagues.2007CA125 expression in hen ovarian tumorsCA125 79 Stammer and colleagues.2008SELENBP1 downregulation in hen ovarian tumorsSELENBP1 149 Hales and colleagues.2008Cyclooxygenase expressions in hen ovarian tumorsCOX1, COX2, PGE2 67 Urick and colleagues.2008-2009VEGF expression in cultured ascites cells from hen ovarian tumorsVEGF160, 161Ansenberger and colleagues.2009Elevation of E-cadherin in hen ovarian tumorsE-cad 6 Hakim and colleagues.2009Investigating oncogenic mutations in hen ovarian tumorsp53, K-ras, H-ras 66 Zhuge and colleagues.2009CYP1B1 levels in chicken ovarian tumorsCYP1B1 175 Seo and colleagues.2010Upregulation of Claudin-10 in hen ovarian tumorsClaudin-10 145 Trevino and colleagues.2010Investigating ovarian tumors for key gene signaturesOvalbumin, Pax2, SerpinB3, OVM, LTF, RD 157 Choi and colleagues.2011Upregulation of MMP-3 in hen ovarian tumor stromaMMP-3 28 Barua and colleagues.2012Upregulation of DR6 in hen ovarian tumorsDR6 16 Lee and colleagues.2012-2014Upregulation of DNA methylation in hen ovarian tumorsDNMT1, DNMT3A, DNMT3B,
SPP1, SERPINB11, SERPINB1394, 101, 103, 104Lim and colleagues.2013-2014Key genes upregulated in endometrioid hen tumorsAvBD-11, CTNNB1, Wnt4102, 11, 100Bradaric and colleagues.2013Investigating immune cells in hen ovarian tumors 23 Ma and colleagues.2014Identifying unique proteins from proteomic profilingF2 thrombin, ITIH2 106 Hales and colleagues.2014Key genes upregulated in hen ovarian tumorsPAX2, MSX2, FOXA2, EN1 68 Parada and colleagues,2017Unique ganglioside expressed in hen ovarian tumorsNeuGcGM3 124 Open in a separate windowTable 2.Ovarian cancer prevention studies using laying hen model

Intra- and inter-generic transfer of pathogenicity island-encoded virulence genes by cos phages

Bacteriophage-mediated horizontal gene transfer is one of the primary driving forces of bacterial evolution. The pac-type phages are generally thought to facilitate most of the phage-mediated gene transfer between closely related bacteria, including that of mobile genetic elements-encoded virulence genes. In this study, we report that staphylococcal cos-type phages transferred the Staphylococcus aureus pathogenicity island SaPIbov5 to non-aureus staphylococcal species and also to different genera. Our results describe the first intra- and intergeneric transfer of a pathogenicity island by a cos phage, and highlight a gene transfer mechanism that may have important implications for pathogen evolution.Classically, transducing phages use the pac site-headful system for DNA packaging. Packaging is initiated on concatemeric post-replicative DNA by terminase cleavage at the sequence-specific pac site, a genome slightly longer than unit length is packaged, and packaging is completed by non-sequence-specific cleavage (reviewed in Rao and Feiss, 2008). Generalized transduction results from the initiation of packaging at pac site homologs in host chromosomal or plasmid DNA, and typically represents ∼1% of the total number of phage particles. In the alternative cos site mechanism packaging is also initiated on concatemeric post-replicative DNA by terminase cleavage at a sequence-specific (cos) site. Here, however, packaging is completed by terminase cleavage at the next cos site, generating a precise monomer with the cohesive termini used for subsequent circularization (Rao and Feiss, 2008). Although cos site homologs may exist in host DNA, it is exceedingly rare that two such sites would be appropriately spaced. Consequently, cos phages, of which lambda is the prototype, do not engage in generalized transduction. For this reason, cos-site phages have been preferred for possible phage therapy, since they would not introduce adventitious host DNA into target organisms.The Staphylococcus aureus pathogenicity islands (SaPIs) are the best-characterized members of the phage-inducible chromosomal island family of mobile genetic elements (MGEs; Novick et al., 2010). SaPIs are ∼15 kb mobile elements that encode virulence factors and are parasitic on specific temperate (helper) phages. Helper phage proteins are required to lift their repression (Tormo-Más et al., 2010, 2013), thereby initiating their excision, circularization and replication. Phage-induced lysis releases vast numbers of infectious SaPI particles, resulting in high frequencies of transfer. Most SaPI helper phages identified to date are pac phages, and many well-studied SaPIs are packaged by the headful mechanism (Ruzin et al., 2001; Ubeda et al., 2007). Recently, we have reported that some SaPIs, of which the prototype is SaPIbov5 (Viana et al., 2010), carry phage cos sequences in their genomes, and can be efficiently packaged and transferred by cos phages to S. aureus strains at high frequencies (Quiles-Puchalt et al., 2014). Here we show that this transfer extends to non-aureus staphylococci and to Listeria monocytogenes.Since the pac phages transfer SaPIs to non-aureus staphylococci and to the Gram-positive pathogen Listeria monocytogenes (Maiques et al., 2007; Chen and Novick, 2009), we reasoned that cos phages might also be capable of intra- and intergeneric transfer. We tested this with SaPIbov5, into which we had previously inserted a tetracycline resistance (tetM) marker to enable selection, and with lysogens of two helper cos phages, φ12 and φSLT, carrying SaPIbov5 (strains JP11010 and JP11194, respectively; Supplementary Table 1). The prophages in these strains were induced with mitomycin C, and the resulting lysates were adjusted to 1 μg ml⁻¹ DNase I and RNase A, filter sterilized (0.2 μm pore), and tested for SaPI transfer with tetracycline selection, as previously described (Ubeda et al., 2008). To test for trans-specific or trans-generic transduction, coagulase-negative staphylococci species and L. monocytogenes strains were used as recipients for SaPIbov5 transfer, respectively, as previously described (Maiques et al., 2007; Chen and Novick, 2009). As shown in Figure 1 and Supplementary Table 2). In contrast, deletion of the SaPIbov5 cos site (strains JP11229 and JP11230) did not affect SaPI replication (Supplementary Figure 1), but completely eliminated SaPIbov5 transfer (Supplementary Table 2). The TerS protein is essential for φ12 and SaPIbov5 DNA packaging, but not for phage-mediated lysis (Quiles-Puchalt et al., 2014). As expected, this mutation abolished SaPIbov5 transfer (Open in a separate windowFigure 1(a) Map of SaPIbov5. Arrows represent the localization and orientation of ORFs greater than 50 amino acids in length. Rectangles represent the position of the ori (in purple) or cos (in red) sites. Positions of different primers described in the text are shown. (b) Amplimers generated for detection of SaPIbov5 in the different recipient strains. Supplementary Table 2 lists the sequence of the different primers used. The element was detected in S. epidermidis JP829 (Se-1), S. epidermidis JP830 (Se-2), L. monocytogenes SK1351 (Lm-1), L. monocytogenes EGDe (Lm-2), S. xylosus C2a (Sx) and S. aureus JP4226 (Sa).

Table 1

Intra- and intergeneric SaPIbov5 transfer^a

Detection and Quantification of the Coral Pathogen Vibrio coralliilyticus by Real-Time PCR with TaqMan Fluorescent Probes

A real-time quantitative PCR-based detection assay targeting the dnaJ gene (encoding heat shock protein 40) of the coral pathogen Vibrio coralliilyticus was developed. The assay is sensitive, detecting as little as 1 CFU per ml in seawater and 10⁴ CFU per cm² of coral tissue. Moreover, inhibition by DNA and cells derived from bacteria other than V. coralliilyticus was minimal. This assay represents a novel approach to coral disease diagnosis that will advance the field of coral disease research.Vibrio coralliilyticus has recently emerged as a coral pathogen of concern on reefs throughout the Indo-Pacific. It was first implicated as the etiological agent responsible for bleaching and tissue lysis of the coral Pocillopora damicornis on Zanzibar reefs (2). More recently, V. coralliilyticus has been identified as the causative agent of white syndrome (WS) outbreaks on several Pacific reefs (14). WS is a collective term describing coral diseases characterized by a spreading band of tissue loss exposing white skeleton on Indo-Pacific scleractinian corals (16). V. coralliilyticus is an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease (13) and therefore provides an ideal model for the development of diagnostic assays to detect coral disease. Current coral disease diagnostic methods, which are based primarily upon field-based observations of macroscopic disease signs, often detect disease only at the latest stages of infection, when control measures are least effective. The development of diagnostic tools targeting pathogens underlying coral disease pathologies may provide early indications of infection, aid the identification of disease vectors and reservoirs, and assist managers in developing strategies to prevent the spread of coral disease outbreaks. In this paper, we describe the development and validation of a TaqMan-based real-time quantitative PCR (qPCR) assay that targets a segment of the V. coralliilyticus heat shock protein 40-encoding gene (dnaJ).Nucleotide sequences of the dnaJ gene were retrieved from relevant Vibrio species, including V. coralliilyticus (LMG 20984), using the National Center for Biotechnology Information''s (NCBI) Entrez Nucleotide Database search tool (http://www.ncbi.nlm.nih.gov/). Gene sequences of strains not available in public databases (V. coralliilyticus strains LMG 21348, LMG 21349, LMG 21350, LMG 10953, LMG 20538, LMG 23696, LMG 23691, LMG 23693, LMG 23692, and LMG 23694) were obtained through extraction of total DNA using a Promega Wizard Prep DNA Purification Kit (Promega, Sydney, Australia), PCR amplification, and sequencing using primers and thermal cycling parameters described by Nhung et al. (8). A 128-bp region (nucleotides 363 to 490) containing high concentrations of single nucleotide polymorphisms (SNPs), which were conserved within V. coralliilyticus strains but differed from non-V. coralliilyticus strains, was identified, and oligonucleotide primers Vc_dnaJ_F1 (5′-CGG TTC GYG GTG TTT CAA AA-3′) and Vc_dnaJ_R1 (5′-AAC CTG ACC ATG ACC GTG ACA-3′) and a TaqMan probe, Vc_dnaJ_TMP (5′-6-FAM-CAG TGG CGC GAA G-MGBNFQ-3′; 6-FAM is 6-carboxyfluorescein and MGBNFQ is molecular groove binding nonfluorescent quencher), were designed to target this region. The qPCR assay was optimized and validated using DNA extracted from V. coralliilyticus isolates, nontarget Vibrio species, and other bacterial species grown in marine broth (MB) (Table ​(Table1),1), under the following optimal conditions: 1× TaqMan buffer A, 0.5 U of AmpliTaq Gold DNA polymerase, 200 μM deoxynucleotide triphosphates (with 400 μM dUTP replacing deoxythymidine triphosphate), 0.2 U of AmpErase uracil N-glycosylase (UNG), 3 mM MgCl₂, 0.6 μM each primer, 0.2 μM fluorophore-labeled TaqMan, 1 μl of template, and sterile MilliQ water for a total reaction volume to 20 μl. All assays were conducted on a RotoGene 300 (Corbett Research, Sydney, Australia) real-time analyzer with the following cycling parameters: 50°C for 120 s (UNG activation) and 95°C for 10 min (AmpliTaq Gold DNA polymerase activation), followed by 40 cycles of 95°C for 15 s (denaturation) and 60°C for 60 s (annealing/extension). During the annealing/extension phase of each thermal cycle, fluorescence was measured in the FAM channel (470-nm excitation and 510-nm detection).

TABLE 1.

Species, strain, and threshold cycle for all bacterial strains tested^a

Novel Carbohydrate-Binding Module Identified in a Ruminal Metagenomic Endoglucanase

Endoglucanase C5614-1 comprises a catalytic module (CM) and an X module (XM). The XM showed no significant homology with known carbohydrate-binding modules (CBMs). Recombinant full-length endoglucanase could bind Avicel, whereas the CM could not. The XM could bind various polysaccharides. The results demonstrated that the XM was a new CBM.Most cellulases are modular proteins that comprise two or more discrete modules, such as catalytic modules (CMs) and carbohydrate-binding modules (CBMs), each of which can function independently (9). CBMs are classified into 59 families based on their amino acid similarity in the CAZY database (http://www.cazy.org/fam/acc_CBM.html). The main functions of CBMs are to recognize and bind polysaccharides and to increase the hydrolytic activities of the enzymes against insoluble and soluble substrates (3). Endoglucanase C5614-1 (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"ACA61140","term_id":"169640132","term_text":"ACA61140"}}ACA61140), which was identified from the metagenome of the contents of buffalo rumen (5), is a modular enzyme comprising an N-terminal signal peptide (amino acids [aa] 1 to 20), a CM belonging to the glycosyl hydrolase family 5 (aa 40 to 334), and a C-terminal X module (XM) of unknown function (aa 335 to 537) (Fig. ​(Fig.1A).1A). No linker region rich in Ser/Pro/Thr was found between the CM and the XM. In this study, we aimed to ascertain the function of the XM in the endoglucanase C5614-1.Open in a separate windowFIG. 1.Endoglucanase C5614-1 and its derivatives. (A) Modular organization of C5614-1 and its truncated derivatives. Abbreviations: SP, signal peptide; GHF5, GHF5 catalytic module; X, the X module (XM). (B) SDS-PAGE of purified recombinant proteins. Protein samples were analyzed on a 10% gel. Lane 1, protein molecular mass standard (molecular masses are shown on the left); lane 2, rC5614-1; lane 3, rGHF5; lane 4, rX.The XM showed no significant homology to known CBMs. The XM shared 25% to 33% identities and 41% to 45% similarities with about 200 amino acids at the C terminus of a xylanase (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAC36862","term_id":"143974","term_text":"AAC36862"}}AAC36862) from the ruminal bacterium Prevotella ruminicola, uncultured ruminal microbial cellulases ({"type":"entrez-protein","attrs":{"text":"ABX76045","term_id":"161898163","term_text":"ABX76045"}}ABX76045, {"type":"entrez-protein","attrs":{"text":"ACA61132","term_id":"169640121","term_text":"ACA61132"}}ACA61132, {"type":"entrez-protein","attrs":{"text":"ACA61135","term_id":"169640125","term_text":"ACA61135"}}ACA61135, {"type":"entrez-protein","attrs":{"text":"ACA61137","term_id":"169640128","term_text":"ACA61137"}}ACA61137, and {"type":"entrez-protein","attrs":{"text":"ABB46200","term_id":"78522584","term_text":"ABB46200"}}ABB46200), and an uncultured bacterial bifunctional mannanase-xyloglucanase ({"type":"entrez-protein","attrs":{"text":"ADA62505","term_id":"281335881","term_text":"ADA62505"}}ADA62505). None of these homologous polypeptides was confirmed to show carbohydrate-binding activity. An alignment of XM with these homologous sequences using ClustalW (http://www.ebi.ac.uk/Tools/clustalw) is shown in Fig. ​Fig.2.2. Seven conserved aromatic amino acid residues were found in the respective sequences. Aromatic amino acid residues in CBMs play critical roles in recognizing and binding polysaccharides (4).Open in a separate windowFIG. 2.Multiple sequence alignment of the XM (203 aa) in endoglucanase C5614-1 (aa 335 to 537), with its homologous sequences. The identical and similar amino acid residues are indicated by asterisks and dots, respectively, below the alignment. The conserved aromatic amino acid residues are indicated by arrows. GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAC36862","term_id":"143974","term_text":"AAC36862"}}AAC36862, Prevotella ruminicola xylanase (aa 376 to 584); {"type":"entrez-protein","attrs":{"text":"ABB46200","term_id":"78522584","term_text":"ABB46200"}}ABB46200, ruminal uncultured bacterium endoglycosidase precursor protein (aa 719 to 917); {"type":"entrez-protein","attrs":{"text":"ABX76045","term_id":"161898163","term_text":"ABX76045"}}ABX76045, ruminal uncultured microorganism endo-1,4-beta-d-glucanase (aa 342 to 552); {"type":"entrez-protein","attrs":{"text":"ADA62505","term_id":"281335881","term_text":"ADA62505"}}ADA62505, ruminal uncultured bacterium bifunctional mannanase-xyloglucanase (aa 721 to 919); {"type":"entrez-protein","attrs":{"text":"ACA61132","term_id":"169640121","term_text":"ACA61132"}}ACA61132, ruminal uncultured microorganism cellulase C29-2 (aa 341 to 553); {"type":"entrez-protein","attrs":{"text":"ACA61135","term_id":"169640125","term_text":"ACA61135"}}ACA61135, ruminal uncultured microorganism cellulase C35-2 (aa 337 to 552); {"type":"entrez-protein","attrs":{"text":"ACA61137","term_id":"169640128","term_text":"ACA61137"}}ACA61137, ruminal uncultured microorganism cellulase C67-1 (aa 335 to 546). The amino acid numbers in each of the parentheses above define the range of the homologous region in each sequence.A PCR-based approach was used to produce constructs expressing C5614-1 derivatives (Fig. ​(Fig.1A).1A). Plasmid C5614 (5), carrying the endoglucanase gene C5614-1, was used as a PCR template. The primer pairs used to amplify the portions of C5614-1 encoding C5614-1 amino acids 20 to 537 (recombinant C5614-1 [rC5614-1], comprising the CM and the XM), 20 to 348 (rGHF5, containing the CM only), and 349 to 537 (rX, containing the XM only) are, respectively, as follows: C5614-1F (5′CAGCCATGGAGGCACAAGATTTTGAGACTGCTACCGAA-3′) and C5614-1R (5′-GACCTCGAGTTGTGCTATGTATTTTTTGCCGTTCTGG-3′), C5614-1F and C5614-1CM (5′-GGGCTCGAGGGTTAATGTCTCAGCCAGGTCAGGCTG-3′), and C5614-1X (5′-GGGCCATGGCCAAAGCCTATCATGGCAGCGCGTTC-3′) and C5614-1R. The underlined sequences in the primers are NcoI and XhoI restriction sites.The digested PCR products were ligated into the same digested expression vector, pET-30a(+) (Novagen, Madison, WI). The recombinant plasmids were transformed into Escherichia coli Rosetta(DE3)pLysS (Novagen), and the cloned fragments were expressed as proteins with 6×His tags at both the N and C termini. The recombinant C5614-1 derivatives were purified by affinity chromatography with Cobalt immobilized metal chromatography resin (Clontech, Palo Alto, CA), according to the user manual. Each of the purified proteins produced a single band on an SDS-PAGE gel (8), and their molecular sizes were in agreement with those of the deduced polypeptides (Fig. ​(Fig.1B1B).The hydrolytic activities of rC5614-1 and rGHF5 toward carboxymethyl cellulose (CMC) were determined essentially as described by Duan et al. (5). The pH profiles of the enzymatic reactions of rC5614-1 and rGHF5 were similar, and both showed maximum activities at pH 5.0 (data not shown). However, rC5614-1 and rGHF5 showed different temperature profiles (Fig. ​(Fig.3A).3A). The rGHF5 showed narrower pH stability and lower temperature stability profiles than those of rC5614-1 (Fig. 3B and C). These results indicated that the XM is required for the stability of rC5614-1.Open in a separate windowFIG. 3.Effects of pH and temperature on the activities and stability of rC5614-1 and rGHF5. (A) Influence of temperature on the activities of rC5614-1 and rGHF5. Cellulase activity was measured at pH 5.0 in citrate-phosphate buffer at the indicated temperatures for 10 min. Values are expressed as percentages of maximal activity at 50°C and 35°C for rC5614-1 and rGHF5, respectively. (B) Influence of pH on rC5614-1 and rGHF5 stability. Purified enzyme was first incubated in citrate-phosphate buffer (pH 2.5 to 7.0), 0.1 M Tris-HCl buffer (pH 7.5 to 8.5), or 0.1 M glycine-NaOH buffer (pH 9.0 to 12) at 4°C for 24 h, and activity was measured under optimal conditions for 10 min. Values are expressed as percentages of maximal activity when the sample was incubated under pH 5.5 and pH 6.0 for rC5614-1 and rGHF5, respectively. (C) Thermal stability of recombinant rC5614-1 and rGHF5. Purified enzymes were first incubated at the indicated temperatures for 1 h; activity was then measured under optimal conditions for 10 min. Values are expressed as percentages of untreated-sample activity.The hydrolytic activities of rC5614-1 toward particle substrates, including birch wood xylan, lichenan, and acid-swollen cellulose (ASC), were about three times greater than those of rGHF5; however, rC5614-1 and rGHF5 showed similar hydrolytic activities toward soluble substrates (Table ​(Table1).1). This result indicated that the presence of the XM enhanced the hydrolytic activity of the enzyme toward insoluble substrates but not toward soluble substrates. Similar phenomena were reported for Irpex lacteus exocellulase I (7), Clostridium thermocellum Xyn10C (1), and Clostridium stercorarium Xyn10B (2).

TABLE 1.

Specific activities of rC5614-1 and rGHF5 toward various substrates