Iron Availability within the Leaf Vasculature Determines the Magnitude of Iron Deficiency Responses in Source and Sink Tissues in Arabidopsis.

Iron (Fe) uptake and translocation in plants are fine-tuned by complex mechanisms that are not yet fully understood. In Arabidopsis thaliana, local regulation of Fe homeostasis at the root level has been extensively studied and is better understood than the systemic shoot-to-root regulation. While the root system is solely a sink tissue that depends on photosynthates translocated from source tissues, the shoot system is a more complex tissue, where sink and source tissues occur synchronously. In this study, and to gain better insight into the Fe deficiency responses in leaves, we overexpressed Zinc/Iron-regulated transporter-like Protein (ZIP5), an Fe/Zn transporter, in phloem-loading cells (proSUC2::AtZIP5) and determined the timing of Fe deficiency responses in sink (young leaves and roots) and source tissues (leaves). Transgenic lines overexpressing ZIP5 in companion cells displayed increased sensitivity to Fe deficiency in root growth assays. Moreover, young leaves and roots (sink tissues) displayed either delayed or dampened transcriptional responses to Fe deficiency compared to wild-type (WT) plants. We also took advantage of the Arabidopsis mutant nas4x-1 to explore Fe transcriptional responses in the opposite scenario, where Fe is retained in the vasculature but in an unavailable and precipitated form. In contrast to proSUC2::AtZIP5 plants, nas4x-1 young leaves and roots displayed a robust and constitutive Fe deficiency response, while mature leaves showed a delayed and dampened Fe deficiency response compared to WT plants. Altogether, our data provide evidence suggesting that Fe sensing within leaves can also occur locally in a leaf-specific manner.

Cadmium interference with iron sensing reveals transcriptional programs sensitive and insensitive to reactive oxygen species.

Iron (Fe) is an essential micronutrient whose uptake is tightly regulated to prevent either deficiency or toxicity. Cadmium (Cd) is a non-essential element that induces both Fe deficiency and toxicity; however, the mechanisms behind these Fe/Cd-induced responses are still elusive. Here we explored Cd- and Fe-associated responses in wild-type Arabidopsis and in a mutant that overaccumulates Fe (opt3-2). Gene expression profiling revealed a large overlap between transcripts induced by Fe deficiency and Cd exposure. Interestingly, the use of opt3-2 allowed us to identify additional gene clusters originally induced by Cd in the wild type but repressed in the opt3-2 background. Based on the high levels of H2O2 found in opt3-2, we propose a model where reactive oxygen species prevent the induction of genes that are induced in the wild type by either Fe deficiency or Cd. Interestingly, a defined cluster of Fe-responsive genes was found to be insensitive to this negative feedback, suggesting that their induction by Cd is more likely to be the result of an impaired Fe sensing. Overall, our data suggest that Fe deficiency responses are governed by multiple inputs and that a hierarchical regulation of Fe homeostasis prevents the induction of specific networks when Fe and H2O2 levels are elevated.

Protection of Cattle Elicited Using a Bivalent Lumpy Skin Disease Virus-Vectored Recombinant Rift Valley Fever Vaccine.

Lumpy skin disease and Rift Valley fever are two high-priority livestock diseases which have the potential to spread into previously free regions through animal movement and/or vectors, as well as intentional release by bioterrorists. Since the distribution range of both diseases is similar in Africa, it makes sense to use a bivalent vaccine to control them. This may lead to the more consistent and sustainable use of vaccination against Rift Valley fever through a more cost-effective vaccine. In this study, a recombinant lumpy skin disease virus was constructed in which the thymidine kinase gene was used as the insertion site for the Gn and Gc protective glycoprotein genes of Rift Valley fever virus using homologous recombination. Selection markers, the enhanced green fluorescent protein and Escherichia coli guanidine phosphoribosyl transferase (gpt), were used for selection of recombinant virus and in a manner enabling a second recombination event to occur upon removal of the gpt selection-pressure allowing the removal of both marker genes in the final product. This recombinant virus, LSD-RVF.mf, was selected to homogeneity, characterized and evaluated in cattle as a vaccine to show protection against both lumpy skin disease and Rift Valley fever in cattle. The results demonstrate that the LSD-RVF.mf is safe, immunogenic and can protect cattle against both diseases.

Simulated evolution assembles more realistic food webs with more functionally similar species than invasion.

While natural communities are assembled by both ecological and evolutionary processes, ecological assembly processes have been studied much more and are rarely compared with evolutionary assembly processes. We address these disparities here by comparing community food webs assembled by simulating introductions of species from regional pools of species and from speciation events. Compared to introductions of trophically dissimilar species assumed to be more typical of invasions, introducing species trophically similar to native species assumed to be more typical of sympatric or parapatric speciation events caused fewer extinctions and assembled more empirically realistic networks by introducing more persistent species with higher trophic generality, vulnerability, and enduring similarity to native species. Such events also increased niche overlap and the persistence of both native and introduced species. Contrary to much competition theory, these findings suggest that evolutionary and other processes that more tightly pack ecological niches contribute more to ecosystem structure and function than previously thought.

Copper uptake mechanism of Arabidopsis thaliana high-affinity COPT transporters.

Copper (Cu) is an essential plant micronutrient. Under scarcity, Cu2+ is reduced to Cu+ and taken up through specific high-affinity transporters (COPTs). In Arabidopsis, the COPT family consists of six members, either located at the plasma membrane (COPT1, COPT2, and COPT6) or in internal membranes (COPT3 and COPT5). Cu uptake by COPT proteins has been mainly assessed through complementation studies in corresponding yeast mutants, but the mechanism of this transport has not been elucidated. To test whether Cu is incorporated by an electrogenic mechanism, electrophysiological changes induced by Cu addition were studied in Arabidopsis thaliana. Mutant (T-DNA insertion mutants, copt2-1 and copt5-2) and overexpressing lines (COPT1OE and COPT5OE) with altered expression of COPT transporters were compared to wild-type plants. No significant changes of the membrane potential (Em) were detected, regardless of genotype or Cu concentration supplied. In contrast, membrane depolarization was detected in response to iron supply in both wild-type and in mutant or transgenic plants. Similar results were obtained for trans-plant potentials (TPP). GFP fusions of the plasma membrane COPT2 and the internal COPT5 transporters were expressed in Xenopus laevis oocytes to potentiate Cu uptake signals, and the cRNA-injected oocytes were tested for electrical currents upon Cu addition using two-electrode voltage clamp. Results with oocytes confirmed those obtained in plants. Cu accumulation in injected oocytes was measured by ICP-OES, and a significant increase in Cu content with respect to controls occurred in oocytes expressing COPT2:GFP. The possible mechanisms driving this transport are discussed in this manuscript.

Changes in iron availability in Arabidopsis are rapidly sensed in the leaf vasculature and impaired sensing leads to opposite transcriptional programs in leaves and roots.

The OLIGOPEPTIDE TRANSPORTER 3 (OPT3) has recently been identified as a component of the systemic network mediating iron (Fe) deficiency responses in Arabidopsis. Reduced expression of OPT3 induces an over accumulation of Fe in roots and leaves, due in part by an elevated expression of the IRON-REGULATED TRANSPORTER 1. Here we show however, that opt3 leaves display a transcriptional program consistent with an Fe overload, suggesting that Fe excess is properly sensed in opt3 leaves and that the OPT3-mediated shoot-to-root signaling is critical to prevent a systemic Fe overload. We also took advantage of the tissue-specific localization of OPT3, together with other Fe-responsive genes, to determine the timing and location of early transcriptional events during Fe limitation and resupply. Our results show that the leaf vasculature responds more rapidly than roots to both Fe deprivation and resupply, suggesting that the leaf vasculature is within the first tissues that sense and respond to changes in Fe availability. Our data highlight the importance of the leaf vasculature in Fe homeostasis by sensing changes in apoplastic levels of Fe coming through the xylem and relaying this information back to roots via the phloem to regulate Fe uptake at the root level.

Whole genome sequencing of Shigella sonnei through PulseNet Latin America and Caribbean: advancing global surveillance of foodborne illnesses.

OBJECTIVES: Shigella sonnei is a globally important diarrhoeal pathogen tracked through the surveillance network PulseNet Latin America and Caribbean (PNLA&C), which participates in PulseNet International. PNLA&C laboratories use common molecular techniques to track pathogens causing foodborne illness. We aimed to demonstrate the possibility and advantages of transitioning to whole genome sequencing (WGS) for surveillance within existing networks across a continent where S. sonnei is endemic. METHODS: We applied WGS to representative archive isolates of S. sonnei (n = 323) from laboratories in nine PNLA&C countries to generate a regional phylogenomic reference for S. sonnei and put this in the global context. We used this reference to contextualise 16 S. sonnei from three Argentinian outbreaks, using locally generated sequence data. Assembled genome sequences were used to predict antimicrobial resistance (AMR) phenotypes and identify AMR determinants. RESULTS: S. sonnei isolates clustered in five Latin American sublineages in the global phylogeny, with many (46%, 149 of 323) belonging to previously undescribed sublineages. Predicted multidrug resistance was common (77%, 249 of 323), and clinically relevant differences in AMR were found among sublineages. The regional overview showed that Argentinian outbreak isolates belonged to distinct sublineages and had different epidemiologic origins. CONCLUSIONS: Latin America contains novel genetic diversity of S. sonnei that is relevant on a global scale and commonly exhibits multidrug resistance. Retrospective passive surveillance with WGS has utility for informing treatment, identifying regionally epidemic sublineages and providing a framework for interpretation of prospective, locally sequenced outbreaks.

Exceptional river gorge formation from unexceptional floods.

An understanding of rates and mechanisms of incision and knickpoint retreat in bedrock rivers is fundamental to perceptions of landscape response to external drivers, yet only sparse field data are available. Here we present eye witness accounts and quantitative surveys of rapid, amphitheatre-headed gorge formation in unweathered granite from the overtopping of a rock-cut dam spillway by small-moderate floods (∼100-1,500 m(3) s(-1)). The amount of erosion demonstrates no relationship with flood magnitude or bedload availability. Instead, structural pattern of the bedrock through faults and joints appears to be the primary control on landscape change. These discontinuities facilitate rapid erosion (>270 m headward retreat; ∼100 m incision; and ∼160 m widening over 6 years) principally through fluvial plucking and block topple. The example demonstrates the potential for extremely rapid transient bedrock erosion even when rocks are mechanically strong and flood discharges are moderate. These observations are relevant to perceived models of gorge formation and knickpoint retreat.

Antimicrobial Resistance Profiles and Diversity in Salmonella from Humans and Cattle, 2004-2011.

Analysis of long-term anti-microbial resistance (AMR) data is useful to understand source and transmission dynamics of AMR. We analysed 5124 human clinical isolates from Washington State Department of Health, 391 cattle clinical isolates from the Washington Animal Disease Diagnostic Laboratory and 1864 non-clinical isolates from foodborne disease research on dairies in the Pacific Northwest. Isolates were assigned profiles based on phenotypic resistance to 11 anti-microbials belonging to eight classes. Salmonella Typhimurium (ST), Salmonella Newport (SN) and Salmonella Montevideo (SM) were the most common serovars in both humans and cattle. Multinomial logistic regression showed ST and SN from cattle had greater probability of resistance to multiple classes of anti-microbials than ST and SN from humans (P < 0.0001). While these findings could be consistent with the belief that cattle are a source of resistant ST and SN for people, occurrence of profiles unique to cattle and not observed in temporally related human isolates indicates these profiles are circulating in cattle only. We used various measures to assess AMR diversity, conditional on the weighting of rare versus abundant profiles. AMR profile richness was greater in the common serovars from humans, although both source data sets were dominated by relatively few profiles. The greater profile richness in human Salmonella may be due to greater diversity of sources entering the human population compared to cattle or due to continuous evolution in the human environment. Also, AMR diversity was greater in clinical compared to non-clinical cattle Salmonella, and this could be due to anti-microbial selection pressure in diseased cattle that received treatment. The use of bootstrapping techniques showed that although there were shared profiles between humans and cattle, the expected and observed number of profiles was different, suggesting Salmonella and associated resistance from humans and cattle may not be wholly derived from a common population.

A multi-country outbreak of Salmonella Newport gastroenteritis in Europe associated with watermelon from Brazil, confirmed by whole genome sequencing: October 2011 to January 2012.

In November 2011, the presence of Salmonella Newport in a ready-to-eat watermelon slice was confirmed as part of a local food survey in England. In late December 2011, cases of S. Newport were reported in England, Wales, Northern Ireland, Scotland, Ireland and Germany. During the outbreak, 63 confirmed cases of S. Newport were reported across all six countries with isolates indistinguishable by pulsed-field gel electrophoresis from the watermelon isolate.A subset of outbreak isolates were whole-genome sequenced and were identical to, or one single nucleotide polymorphism different from the watermelon isolate.In total, 46 confirmed cases were interviewed of which 27 reported watermelon consumption. Further investigations confirmed the outbreak was linked to the consumption of watermelon imported from Brazil.Although numerous Salmonella outbreaks associated with melons have been reported in the United States and elsewhere, this is the first of its kind in Europe.Expansion of the melon import market from Brazil represents a potential threat for future outbreaks. Whole genome sequencing is rapidly becoming more accessible and can provide a compelling level of evidence of linkage between human cases and sources of infection,to support public health interventions in global food markets.

Moving toward a precise nutrition: preferential loading of seeds with essential nutrients over non-essential toxic elements.

Plants and seeds are the main source of essential nutrients for humans and livestock. Many advances have recently been made in understanding the molecular mechanisms by which plants take up and accumulate micronutrients such as iron, zinc, copper and manganese. Some of these mechanisms, however, also facilitate the accumulation of non-essential toxic elements such as cadmium (Cd) and arsenic (As). In humans, Cd and As intake has been associated with multiple disorders including kidney failure, diabetes, cancer and mental health issues. Recent studies have shown that some transporters can discriminate between essential metals and non-essential elements. Furthermore, sequestration of non-essential elements in roots has been described in several plant species as a key process limiting the translocation of non-essential elements to aboveground edible tissues, including seeds. Increasing the concentration of bioavailable micronutrients (biofortification) in grains while lowering the accumulation of non-essential elements will likely require the concerted action of several transporters. This review discusses the most recent advances on mineral nutrition that could be used to preferentially enrich seeds with micronutrients and also illustrates how precision breeding and transport engineering could be used to enhance the nutritional value of crops by re-routing essential and non-essential elements to separate sink tissues (roots and seeds).

Distinguishable epidemics of multidrug-resistant Salmonella Typhimurium DT104 in different hosts.

The global epidemic of multidrug-resistant Salmonella Typhimurium DT104 provides an important example, both in terms of the agent and its resistance, of a widely disseminated zoonotic pathogen. Here, with an unprecedented national collection of isolates collected contemporaneously from humans and animals and including a sample of internationally derived isolates, we have used whole-genome sequencing to dissect the phylogenetic associations of the bacterium and its antimicrobial resistance genes through the course of an epidemic. Contrary to current tenets supporting a single homogeneous epidemic, we demonstrate that the bacterium and its resistance genes were largely maintained within animal and human populations separately and that there was limited transmission, in either direction. We also show considerable variation in the resistance profiles, in contrast to the largely stable bacterial core genome, which emphasizes the critical importance of integrated genotypic data sets in understanding the ecology of bacterial zoonoses and antimicrobial resistance.

Serum 25 hydroxy-vitamin D does not exhibit an acute phase reaction after acute myocardial infarction.

BACKGROUND: There is growing epidemiological evidence linking serum 25 hydroxy-vitamin D (25(OH)D) concentrations to outcome in cardiovascular and other diseases. We have studied patients with acute myocardial infarction (AMI) to determine if they exhibit an acute phase reaction affecting 25(OH)D. METHODS: Patients (n=32) with first AMI who had been treated with primary percutaneous coronary intervention within 12 h of symptom onset had venous blood samples taken two days, one week, one month and three months after presentation. Samples were analysed for troponin I, C-reactive protein (CRP) and 25(OH)D. RESULTS: All patients had significant rises in troponin confirming the myocardial damage and CRP, both of which resolved by 28 days. In contrast, 25(OH)D remained unchanged throughout the 90-day observation period with a median concentration of 46 nmol/L. CONCLUSION: Serum 25(OH)D does not change after AMI and is likely to be a reliable marker of vitamin D status in patients with cardiovascular disease.

From phenotype to genotype: a Bayesian solution.

The study of biological systems commonly depends on inferring the state of a 'hidden' variable, such as an underlying genotype, from that of an 'observed' variable, such as an expressed phenotype. However, this cannot be achieved using traditional quantitative methods when more than one genetic mechanism exists for a single observable phenotype. Using a novel latent class Bayesian model, it is possible to infer the prevalence of different genetic elements in a population given a sample of phenotypes. As an exemplar, data comprising phenotypic resistance to six antimicrobials obtained from passive surveillance of Salmonella Typhimurium DT104 are analysed to infer the prevalence of individual resistance genes, as well as the prevalence of a genomic island known as SGI1 and its variants. Three competing models are fitted to the data and distinguished between using posterior predictive p-values to assess their ability to predict the observed number of unique phenotypes. The results suggest that several SGI1 variants circulate in a few fixed forms through the population from which our data were derived. The methods presented could be applied to other types of phenotypic data, and represent a useful and generic mechanism of inferring the genetic population structure of organisms.

High glucose induced endothelial cell growth inhibition is associated with an increase in TGFbeta1 secretion and inhibition of Ras prenylation via suppression of the mevalonate pathway.

OBJECTIVE: Ras proteins are known to affect cellular growth and function. The influence of the prenylation status of Ras on the observed changes in endothelial cell growth under high glucose conditions has not previously been examined. METHODS: Human umbilical vein endothelial cells were exposed to normal or high glucose conditions for 72 h. They were then examined for proliferative and hypertrophic effects, transforming growth factor beta(1) (TGFbeta(1)) release, and phosphorylated p38 expression. The importance of prenylation was explored by the addition of mevalonate, isoprenoids or farnesyltransferase inhibitors to control the high glucose media and by measuring changes induced by high glucose and exogenous TGFbeta(1) in Ras prenylation and farnesyltransferase activity. Kidneys from diabetic rats treated with atorvastatin were also compared to specimens from untreated animals and the expression of the Ras effector p-Akt examined. RESULTS: High glucose conditions caused a reduction in cell number. This was reversed in the presence of mevalonate or farnesylpyrophosphate (FPP), suggesting that the cell growth abnormalities observed are due to high glucose induced inhibition of the mevalonate pathway and subsequent prenylation of proteins. Endothelial cells exposed to high glucose increased their secretion of TGFbeta(1) and the phosphorylation of p38 both of which were reversed by concurrent exposure to FPP. A reduction in farnesyltransferase activity was observed after exposure to both high glucose and TGFbeta(1). Exposure to a farnesyltransferase inhibitor in control conditions mimicked the growth response observed with high glucose exposure and prenylated Ras was reduced by exposure to both high glucose and TGFbeta(1). Finally, interruption of the mevalonate pathway with a statin reduced the expression of p-Akt in diabetic rat kidneys. CONCLUSION: This study demonstrates that high glucose induced significant alterations in endothelial cell growth by inhibition of the mevalonate pathway, which subsequently mediates the increase in TGFbeta(1) and inhibition of Ras prenylation.

Factors associated with cross-contamination of hides of Scottish cattle by Escherichia coli O157.

The putative source of hide contamination for 236 cattle in Scotland followed from the farm through to slaughter was determined using phage and verocytotoxin type data. The majority of cattle (84%) were found to have subtypes of Escherichia coli O157 on their hide that had not been found previously in any animal from the farm of origin, strongly suggesting that contamination occurred once animals had left the farm of origin. Using logistic regression analysis, several variables and factors were found to be strongly associated (P < 0.01) with cross-contamination of cattle hides at the univariate level; commercial transport to slaughter, transport with other animals, use of a crush, line automation, and increasing slaughterhouse throughput were all risk factors, while feeding hay in lairage, processing an animal earlier in a slaughter cohort, and cleaning the landing area poststunning were protective. In the multivariable model, with the slaughterhouse and the farm group included as random effects, factors associated with the cross-contamination of cattle hides were identified. Transport to the slaughterhouse by a commercial hauler had a borderline-significant association with increased odds of an animal having a cross-contaminated hide (odds ratio [OR] [95% confidence interval (CI)] = 5.7 [0.99, 33.0]; P = 0.05). At the slaughterhouse, providing hay to cattle waiting in lairage (OR [95% CI] = 0.04 [<0.01, 1.04]; P = 0.05) and cleaning the landing area (OR [95% CI] = 0.03 [<0.01, 1.15,]; P = 0.06) also had a borderline-significant association with decreased odds of an animal having a cross-contaminated hide. Although the prevalence of carcass contamination remains very low, targeted intervention at the preslaughter stage may have the potential to reduce further the risk to public health.

Risk factors for hide contamination of Scottish cattle at slaughter with Escherichia coli O157.

In the slaughter processing of cattle, contaminated hides have been identified as one of the major sources of Escherichia coli O157 carcase contamination. Logistic regression analysis was applied to data collected in a large scale study in Scotland involving 222 cattle forming 34 groups sent for slaughter from 30 farms to 10 slaughterhouses. Aspects of individual animal characteristics, farm management practices and slaughterhouse features were examined to identify potential risk factors for hide contamination at harvest. Two models were developed, the first in which slaughterhouse was modelled as a fixed effect, and a second model where slaughterhouse and farm groups were modelled as random effects. In the first model, there was a significantly increased risk of a carcase testing positive for E. coli O157 on the hide if either the hide of the carcase immediately before or after it on the line was contaminated (OR 3.6; 95% CI: 1.4-9.9). If both adjacent carcases had contaminated hides, the odds ratio for the study carcase having a contaminated hide rose to 11.5 (95% CI: 4.4-32.5). If animals were held in lairage, receiving hay as feed appeared to have a protective effect on hide contamination. Transportation to the slaughterhouse by haulier, as opposed to transport by the farmer, was associated with a 5.4 increase in the odds of E. coli O157 contamination. The use of a crush in the lairage, often employed when reading ear tags, was also found to significantly increase the odds of hide contamination with E. coli O157. In the second model, the inclusion of slaughterhouse and farm group as random effects resulted in two of the previously identified factors being associated with hide contamination. If at least one of the adjacent carcases on the line had a contaminated hide, the associated odds ratio was 6.6 (95% CI: 2.8-15.9), which rose to 22.7 (95% CI: 9.3-55.5) if both adjacent hides were contaminated. Receiving hay in lairage was found to be important to the model, although not significant in itself (OR 0.005; 95% CI: 1.2e(-6)-20.7). These results suggest that modifiable risk factors for hide contamination exist. However, in order best to reduce the prevalence of hide contamination at slaughter, individual slaughterhouse risk assessment and intervention strategies are appropriate.

A time-resolved fluorescence immunoassay for the determination of a novel respiratory therapeutic agent, AR-C68397XX (Viozan) in human plasma.

A dissociation-enhanced lathanide fluorescence immunoassay (DELFIA) method has been developed for the determination of AR-C68397XX, a novel respiratory therapeutic agent, in human plasma. The method is a 'direct' immunoassay and provides an alternative to the solid phase extraction RIA described in a previous publication, which employs the same specific antiserum. The DELFIA method is suitable for the determination of the analyte at pg ml(-1) concentrations. The non-isotopic label was prepared by complexation of a DTPA derivative of AR-C68397XX with free europium cation (Eu3+). Plasma samples were diluted at least 5-fold prior to analysis to eliminate matrix interference. The calibration range is 10-2000 pg ml(-1), and the LOQ of the method is 50 pg ml(-1) using 50 microl of diluted human plasma sample.