High-Resolution 1H NMR Spectroscopy of Fish Muscle,Eggs and Small Whole Fish via Hadamard-Encoded Intermolecular Multiple-Quantum Coherence

Background and Purpose

Nuclear magnetic resonance (NMR) spectroscopy has become an important technique for tissue studies. Since tissues are in semisolid-state, their high-resolution (HR) spectra cannot be obtained by conventional NMR spectroscopy. Because of this restriction, extraction and high-resolution magic angle spinning (HR MAS) are widely applied for HR NMR spectra of tissues. However, both of the methods are subject to limitations. In this study, the feasibility of HR ¹H NMR spectroscopy based on intermolecular multiple-quantum coherence (iMQC) technique is explored using fish muscle, fish eggs, and a whole fish as examples.

Materials and Methods

Intact salmon muscle tissues, intact eggs from shishamo smelt and a whole fish (Siamese algae eater) are studied by using conventional 1D one-pulse sequence, Hadamard-encoded iMQC sequence, and HR MAS.

Results

When we use the conventional 1D one-pulse sequence, hardly any useful spectral information can be obtained due to the severe field inhomogeneity. By contrast, HR NMR spectra can be obtained in a short period of time by using the Hadamard-encoded iMQC method without shimming. Most signals from fatty acids and small metabolites can be observed. Compared to HR MAS, the iMQC method is non-invasive, but the resolution and the sensitivity of resulting spectra are not as high as those of HR MAS spectra.

Conclusion

Due to the immunity to field inhomogeneity, the iMQC technique can be a proper supplement to HR MAS, and it provides an alternative for the investigation in cases with field distortions and with samples unsuitable for spinning. The acquisition time of the proposed method is greatly reduced by introduction of the Hadamard-encoded technique, in comparison with that of conventional iMQC method.     

Quantification of hepatic iron concentration in chronic viral hepatitis: usefulness of T2-weighted single-shot spin-echo echo-planar MR imaging

Objective

To investigate the usefulness of single-shot spin-echo echo-planar imaging (SSEPI) sequence for quantifying mild degree of hepatic iron stores in patients with viral hepatitis.

Methods

This retrospective study included 34 patients with chronic viral hepatitis/cirrhosis who had undergone histological investigation and magnetic resonance imaging with T2-weighted gradient-recalled echo sequence (T2-GRE) and diffusion-weighted SSEPI sequence with b-factors of 0 s/mm² (T2-EPI), 500 s/mm² (DW-EPI-500), and 1000 s/mm² (DW-EPI-1000).The correlation between the liver-to-muscle signal intensity ratio, which was generated by regions of interest placed in the liver and paraspinous muscles of each sequence image, and the hepatic iron concentration (µmol/g dry liver), which was assessed by spectrophotometry, was analyzed by linear regression using a spline model. Akaike information criterion (AIC) was used to select the optimal model.

Results

Mean ± standard deviation of the hepatic iron concentration quantified by spectrophotometry was 24.6±16.4 (range, 5.5 to 83.2) µmol/g dry liver. DW-EPI correlated more closely with hepatic iron concentration than T2-GRE (R square values: 0.75 for T2-EPI, 0.69 for DW-EPI-500, 0.62 for DW-EPI-1000, and 0.61 for T2-GRE, respectively, all P<0.0001). Using the AIC, the regression model for T2-EPI generated by spline model was optimal because of lowest cross validation error.

Conclusion

T2-EPI was sensitive to hepatic iron, and might be a more useful sequence for quantifying mild degree of hepatic iron stores in patients with chronic viral hepatitis.     

High-Resolution Echo-Planar Spectroscopic Imaging of the Human Calf

Background

This study exploits the speed benefits of echo-planar spectroscopic imaging (EPSI) to acquire lipid spectra of skeletal muscle. The main purpose was to develop a high-resolution EPSI technique for clinical MR scanner, to visualise the bulk magnetic susceptibility (BMS) shifts of extra-myocellular lipid (EMCL) spectral lines, and to investigate the feasibility of this method for the assessment of intra-myocellular (IMCL) lipids.

Methods

The study group consisted of six healthy volunteers. A two dimensional EPSI sequence with point-resolved spectroscopy (PRESS) spatial localization was implemented on a 3T clinical MR scanner. Measurements were performed by means of 64×64 spatial matrix and nominal voxel size 3×3×15 mm³. The total net measurement time was 3 min 12 sec for non-water-suppressed (1 acquisition) and 12 min 48 sec for water-suppressed scans (4 acquisitions).

Results

Spectra of the human calf had a very good signal-to-noise ratio and linewidths sufficient to differentiate IMCL resonances from EMCL. The use of a large spatial matrix reduces inter-voxel signal contamination of the strong EMCL signals. Small voxels enabled visualisation of the methylene EMCL spectral line splitting and their BMS shifts up to 0.5 ppm relative to the correspondent IMCL line. The mean soleus muscle IMCL content of our six volunteers was 0.30±0.10 vol% (range 0.18–0.46) or 3.6±1.2 mmol/kg wet weight (range: 2.1–5.4).

Conclusion

This study demonstrates that high-spatial resolution PRESS EPSI of the muscle lipids is feasible on standard clinical scanners.     

7 Tesla Magnetic Resonance Imaging to Detect Cortical Pathology in Multiple Sclerosis

Background

Neocortical lesions (NLs) are an important pathological component of multiple sclerosis (MS), but their visualization by magnetic resonance imaging (MRI) remains challenging.

Objectives

We aimed at assessing the sensitivity of multi echo gradient echo (ME-GRE) T₂ ^*-weighted MRI at 7.0 Tesla in depicting NLs compared to myelin and iron staining.

Methods

Samples from two MS patients were imaged post mortem using a whole body 7T MRI scanner with a 24-channel receive-only array. Isotropic 200 micron resolution images with varying T₂ ^* weighting were reconstructed from the ME-GRE data and converted into R₂ ^* maps. Immunohistochemical staining for myelin (proteolipid protein, PLP) and diaminobenzidine-enhanced Turnbull blue staining for iron were performed.

Results

Prospective and retrospective sensitivities of MRI for the detection of NLs were 48% and 67% respectively. We observed MRI maps detecting only a small portion of 20 subpial NLs extending over large cortical areas on PLP stainings. No MRI signal changes suggestive of iron accumulation in NLs were observed. Conversely, R₂ ^* maps indicated iron loss in NLs, which was confirmed by histological quantification.

Conclusions

High-resolution post mortem imaging using R₂ ^* and magnitude maps permits detection of focal NLs. However, disclosing extensive subpial demyelination with MRI remains challenging.     

Genome Analysis Coupled with Physiological Studies Reveals a Diverse Nitrogen Metabolism in Methylocystis sp. Strain SC2

Background

Methylocystis sp. strain SC2 can adapt to a wide range of methane concentrations. This is due to the presence of two isozymes of particulate methane monooxygenase exhibiting different methane oxidation kinetics. To gain insight into the underlying genetic information, its genome was sequenced and found to comprise a 3.77 Mb chromosome and two large plasmids.

Principal Findings

We report important features of the strain SC2 genome. Its sequence is compared with those of seven other methanotroph genomes, comprising members of the Alphaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. While the pan-genome of all eight methanotroph genomes totals 19,358 CDS, only 154 CDS are shared. The number of core genes increased with phylogenetic relatedness: 328 CDS for proteobacterial methanotrophs and 1,853 CDS for the three alphaproteobacterial Methylocystaceae members, Methylocystis sp. strain SC2 and strain Rockwell, and Methylosinus trichosporium OB3b. The comparative study was coupled with physiological experiments to verify that strain SC2 has diverse nitrogen metabolism capabilities. In correspondence to a full complement of 34 genes involved in N₂ fixation, strain SC2 was found to grow with atmospheric N₂ as the sole nitrogen source, preferably at low oxygen concentrations. Denitrification-mediated accumulation of 0.7 nmol ³⁰N₂/hr/mg dry weight of cells under anoxic conditions was detected by tracer analysis. N₂ production is related to the activities of plasmid-borne nitric oxide and nitrous oxide reductases.

Conclusions/Perspectives

Presence of a complete denitrification pathway in strain SC2, including the plasmid-encoded nosRZDFYX operon, is unique among known methanotrophs. However, the exact ecophysiological role of this pathway still needs to be elucidated. Detoxification of toxic nitrogen compounds and energy conservation under oxygen-limiting conditions are among the possible roles. Relevant features that may stimulate further research are, for example, absence of CRISPR/Cas systems in strain SC2, high number of iron acquisition systems in strain OB3b, and large number of transposases in strain Rockwell.     

Impact of RTS,S/AS02A and RTS,S/AS01B on Genotypes of P. falciparum in Adults Participating in a Malaria Vaccine Clinical Trial

Objective

RTS,S, a candidate vaccine for malaria, is a recombinant protein expressed in yeast containing part of the circumsporozoite protein (CSP) sequence of 3D7 strain of Plasmodium falciparum linked to the hepatitis B surface antigen in a hybrid protein. The RTS,S antigen is formulated with GSK Biologicals'' proprietary Adjuvant Systems AS02_A or AS01_B. A recent trial of the RTS,S/AS02_A and RTS,S/AS01_B vaccines evaluated safety, immunogenicity and impact on the development of parasitemia of the two formulations. Parasite isolates from this study were used to determine the molecular impact of RTS,S/AS02_A and RTS,S/AS01_B on the multiplicity of infection (MOI) and the csp allelic characteristics of subsequent parasitemias.

Design

The distribution of csp sequences and the MOI of the infecting strains were examined at baseline and in break-through infections from vaccinated individuals and from those receiving a non-malarial vaccine.

Setting

The study was conducted in Kombewa District, western Kenya.

Participants

Semi-immune adults from the three study arms provided isolates at baseline and during break-through infections.

Outcome

Parasite isolates used for determining MOI and divergence of csp T cell–epitopes were 191 at baseline and 87 from break-through infections.

Results

Grouping recipients of RTS,S/AS01_A and RTS,S/AS02_B together, vaccine recipients identified as parasite-positive by microscopy contained significantly fewer parasite genotypes than recipients of the rabies vaccine comparator (median in pooled RTS,S groups: 3 versus 4 in controls, P = 0.0313). When analyzed separately, parasitaemic individuals in the RTS,S/AS01_B group, but not the RTS,S/AS02_A group, were found to have significantly fewer genotypes than the comparator group. Two individual amino acids found in the vaccine construct (Q339 in Th2R and D371 in Th3R) were observed to differ in incidence between vaccine and comparator groups but in different directions; parasites harboring Q339 were less common among pooled RTS,S/AS vaccine recipients than among recipients of rabies vaccine, whereas parasites with D371 were more common among the RTS,S/AS groups.

Conclusions

It is concluded that both RTS,S/AS vaccines reduce multiplicity of infection. Our results do not support the hypothesis that RTS,S/AS vaccines elicit preferential effects against pfcsp alleles with sequence similarity to the 3D7 pfcsp sequence employed in the vaccine construct.     

Characterization of a Novel Esterase Rv0045c from Mycobacterium tuberculosis

Background

It was proposed that there are at least 250 enzymes in M. tuberculosis involved in lipid metabolism. Rv0045c was predicted to be a hydrolase by amino acid sequence similarity, although its precise biochemical characterization and function remained to be defined.

Methodology/Principal Findings

We expressed the Rv0045c protein to high levels in E. coli and purified the protein to high purity. We confirmed that the prepared protein was the Rv0045c protein by mass spectrometry analysis. Circular dichroism spectroscopy analysis showed that the protein possessed abundant β-sheet secondary structure, and confirmed that its conformation was stable in the range pH 6.0–10.0 and at temperatures ≤40°C. Enzyme activity analysis indicated that the Rv0045c protein could efficiently hydrolyze short chain p-nitrophenyl esters (C₂–C₈), and its suitable substrate was p-nitrophenyl caproate (C₆) with optimal catalytic conditions of 39°C and pH 8.0.

Conclusions/Significance

Our results demonstrated that the Rv0045c protein is a novel esterase. These experiments will be helpful in understanding ester/lipid metabolism related to M. tuberculosis.     

Fast and cost-effective mining of microsatellite markers using NGS technology: an example of a Korean water deer Hydropotes inermis argyropus

Background

Microsatellites, a special class of repetitive DNA sequence, have become one of the most popular genetic markers for population/conservation genetic studies. However, its application to endangered species has been impeded by high development costs, a lack of available sequences, and technical difficulties. The water deer Hydropotes inermis is the sole existing endangered species of the subfamily Capreolinae. Although population genetics studies are urgently required for conservation management, no species-specific microsatellite marker has been reported.

Methods

We adopted next-generation sequencing (NGS) to elucidate the microsatellite markers of Korean water deer and overcome these impediments on marker developments. We performed genotyping to determine the efficiency of this method as applied to population genetics.

Results

We obtained 98 Mbp of nucleotide information from 260,467 sequence reads. A total of 20,101 di-/tri-nucleotide repeat motifs were identified; di-repeats were 5.9-fold more common than tri-repeats. [CA]_n and [AAC]_n/[AAT]_n repeats were the most frequent di- and tri-repeats, respectively. Of the 17,206 di-repeats, 12,471 microsatellite primer pairs were derived. PCR amplification of 400 primer pairs yielded 106 amplicons and 79 polymorphic markers from 20 individual Korean water deer. Polymorphic rates of the 79 new microsatellites varied from 2 to 11 alleles per locus (H_e: 0.050–0.880; H_o: 0.000–1.000), while those of known microsatellite markers transferred from cattle to Chinese water deer ranged from 4 to 6 alleles per locus (H_e: 0.279–0.714; H_o: 0.300–0.400).

Conclusions

Polymorphic microsatellite markers from Korean water deer were successfully identified using NGS without any prior sequence information and deposited into the public database. Thus, the methods described herein represent a rapid and low-cost way to investigate the population genetics of endangered/non-model species.     

Epidemiology of Extended-Spectrum β-Lactamase Producing Escherichia coli in the Stools of Returning Japanese Travelers,and the Risk Factors for Colonization

Objective

Travel overseas has recently been considered a risk factor for colonization with drug-resistant bacteria. The purpose of this study was to establish the epidemiology and risk factors associated with the acquisition of drug-resistant bacteria by Japanese travelers.

Methods

Between October 2011 and September 2012, we screened the stools of 68 Japanese returning travelers for extended-spectrum β-lactamase (ESBL) producing Escherichia coli. All specimens were sampled for clinical reasons. Based on the results, the participants were divided into an ESBL-producing E. coli positive group (18 cases; 26%) and an ESBL-producing E. coli negative group (50 cases; 74%), and a case-control study was performed. Microbiological analyses of ESBL-producing strains, including susceptibility tests, screening tests for metallo-β-lactamase, polymerase chain reaction amplification and sequencing of bla _CTX-M genes, multilocus sequence typing, and whole genome sequencing, were also conducted.

Results

In a univariate comparison, travel to India was a risk factor (Odds Ratio 13.6, 95% Confidence Interval 3.0–75.0, p<0.0001). There were no statistical differences in the characteristics of the travel, such as backpacking, purpose of travel, interval between travel return and sampling stool, and duration of travel. Although 10 of 13 analyzed strains (77%) produced CTX-M-15, no ST131 clone was detected.

Conclusion

We must be aware of the possibilities of acquiring ESBL-producing E. coli during travel in order to prevent the spread of these bacteria not only in Japan but globally.     

National Surveillance Study on Carbapenem Non-Susceptible Klebsiella pneumoniae in Taiwan: The Emergence and Rapid Dissemination of KPC-2 Carbapenemase

Objectives

The global spread and increasing incidence of carbapenem non-susceptible Klebsiella pneumoniae (CnSKP) has made its treatment difficult, increasing the mortality. To establish nationwide data on CnSKP spread and carbapenem-resistance mechanisms, we conducted a national surveillance study in Taiwanese hospitals.

Methods

We collected 100 and 247 CnSKP isolates in 2010 and 2012, respectively. The tests performed included antibiotic susceptibility tests; detection of carbapenemase, extended-spectrum β-lactamases (ESBL), and AmpC β-lactamases genes; outer membrane porin profiles; and genetic relationship with pulsed-field gel electrophoresis and multilocus sequence type.

Results

The resistance rate of CnSKP isolates to cefazolin, cefotaxime, cefoxitin, ceftazidime, and ciprofloxacin was over 90%. Susceptibility rate to tigecycline and colistin in 2010 was 91.0% and 83.0%, respectively; in 2012, it was 91.9% and 87.9%, respectively. In 2010, carbapenemase genes were detected in only 6.0% of isolates (4 bla _IMP-8 and 2 bla _VIM-1). In 2012, carbapenemase genes were detected in 22.3% of isolates (41 bla _KPC-2, 7 bla _VIM-1, 6 bla _IMP-8, and 1 bla _NDM-1). More than 95% of isolates exhibited either OmpK35 or OmpK36 porin loss or both. Impermeability due to porin mutation coupled with AmpC β-lactamases or ESBLs were major carbapenem-resistance mechanisms. Among 41 KPC-2-producing K. pneumoniae isolates, all were ST11 with 1 major pulsotype.

Conclusions

In 2010 and 2012, the major mechanisms of CnSKP in Taiwan were the concomitance of AmpC with OmpK35/36 loss. KPC-2-KP dissemination with the same ST11 were observed in 2012. The emergence and rapid spread of KPC-2-KP is becoming an endemic problem in Taiwan. The identification of NDM-1 K. pneumoniae case is alarming.     

Recapitulation of Tumor Heterogeneity and Molecular Signatures in a 3D Brain Cancer Model with Decreased Sensitivity to Histone Deacetylase Inhibition

Introduction

Physiologically relevant pre-clinical ex vivo models recapitulating CNS tumor micro-environmental complexity will aid development of biologically-targeted agents. We present comprehensive characterization of tumor aggregates generated using the 3D Rotary Cell Culture System (RCCS).

Methods

CNS cancer cell lines were grown in conventional 2D cultures and the RCCS and comparison with a cohort of 53 pediatric high grade gliomas conducted by genome wide gene expression and microRNA arrays, coupled with immunohistochemistry, ex vivo magnetic resonance spectroscopy and drug sensitivity evaluation using the histone deacetylase inhibitor, Vorinostat.

Results

Macroscopic RCCS aggregates recapitulated the heterogeneous morphology of brain tumors with a distinct proliferating rim, necrotic core and oxygen tension gradient. Gene expression and microRNA analyses revealed significant differences with 3D expression intermediate to 2D cultures and primary brain tumors. Metabolic profiling revealed differential profiles, with an increase in tumor specific metabolites in 3D. To evaluate the potential of the RCCS as a drug testing tool, we determined the efficacy of Vorinostat against aggregates of U87 and KNS42 glioblastoma cells. Both lines demonstrated markedly reduced sensitivity when assaying in 3D culture conditions compared to classical 2D drug screen approaches.

Conclusions

Our comprehensive characterization demonstrates that 3D RCCS culture of high grade brain tumor cells has profound effects on the genetic, epigenetic and metabolic profiles of cultured cells, with these cells residing as an intermediate phenotype between that of 2D cultures and primary tumors. There is a discrepancy between 2D culture and tumor molecular profiles, and RCCS partially re-capitulates tissue specific features, allowing drug testing in a more relevant ex vivo system.     

An improved genome reference for the African cichlid,Metriaclima zebra

Background

Problems associated with using draft genome assemblies are well documented and have become more pronounced with the use of short read data for de novo genome assembly. We set out to improve the draft genome assembly of the African cichlid fish, Metriaclima zebra, using a set of Pacific Biosciences SMRT sequencing reads corresponding to 16.5× coverage of the genome. Here we characterize the improvements that these long reads allowed us to make to the state-of-the-art draft genome previously assembled from short read data.

Results

Our new assembly closed 68 % of the existing gaps and added 90.6Mbp of new non-gap sequence to the existing draft assembly of M. zebra. Comparison of the new assembly to the sequence of several bacterial artificial chromosome clones confirmed the accuracy of the new assembly. The closure of sequence gaps revealed thousands of new exons, allowing significant improvement in gene models. We corrected one known misassembly, and identified and fixed other likely misassemblies. 63.5 Mbp (70 %) of the new sequence was classified as repetitive and the new sequence allowed for the assembly of many more transposable elements.

Conclusions

Our improvements to the M. zebra draft genome suggest that a reasonable investment in long reads could greatly improve many comparable vertebrate draft genome assemblies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1930-5) contains supplementary material, which is available to authorized users.     

Silica nanoparticles aid in structural leaf coloration in the Malaysian tropical rainforest understorey herb Mapania caudata

Background and Aims

Blue-green iridescence in the tropical rainforest understorey sedge Mapania caudata creates structural coloration in its leaves through a novel photonic mechanism. Known structures in plants producing iridescent blues consist of altered cellulose layering within cell walls and in special bodies, and thylakoid membranes in specialized plastids. This study was undertaken in order to determine the origin of leaf iridescence in this plant with particular attention to nano-scale components contributing to this coloration.

Methods

Adaxial walls of leaf epidermal cells were characterized using high-pressure-frozen freeze-substituted specimens, which retain their native dimensions during observations using transmission and scanning microscopy, accompanied by energy-dispersive X-ray spectroscopy to identify the role of biogenic silica in wall-based iridescence. Biogenic silica was experimentally removed using aqueous Na₂CO₃ and optical properties were compared using spectral reflectance.

Key Results and Conclusions

Blue iridescence is produced in the adaxial epidermal cell wall, which contains helicoid lamellae. The blue iridescence from cell surfaces is left-circularly polarized. The position of the silica granules is entrained by the helicoid microfibrillar layers, and granules accumulate at a uniform position within the helicoids, contributing to the structure that produces the blue iridescence, as part of the unit cell responsible for 2 ° Bragg scatter. Removal of silica from the walls eliminated the blue colour. Addition of silica nanoparticles on existing cellulosic lamellae is a novel mechanism for adding structural colour in organisms.     

SCN5A Mutations in Brugada Syndrome Are Associated with Increased Cardiac Dimensions and Reduced Contractility

Background

The cardiac sodium channel (Na_v1.5) controls cardiac excitability. Accordingly, SCN5A mutations that result in loss-of-function of Na_v1.5 are associated with various inherited arrhythmia syndromes that revolve around reduced cardiac excitability, most notably Brugada syndrome (BrS). Experimental studies have indicated that Na_v1.5 interacts with the cytoskeleton and may also be involved in maintaining structural integrity of the heart. We aimed to determine whether clinical evidence may be obtained that Na_v1.5 is involved in maintaining cardiac structural integrity.

Methods

Using cardiac magnetic resonance (CMR) imaging, we compared right ventricular (RV) and left ventricular (LV) dimensions and ejection fractions between 40 BrS patients with SCN5A mutations (SCN5a-mut-positive) and 98 BrS patients without SCN5A mutations (SCN5a-mut-negative). We also studied 18 age/sex-matched healthy volunteers.

Results

SCN5a-mut-positive patients had significantly larger end-diastolic and end-systolic RV and LV volumes, and lower LV ejection fractions, than SCN5a-mut-negative patients or volunteers.

Conclusions

Loss-of-function SCN5A mutations are associated with dilatation and impairment in contractile function of both ventricles that can be detected by CMR analysis.     

Grifonin-1: a small HIV-1 entry inhibitor derived from the algal lectin, Griffithsin

Background

Griffithsin, a 121-residue protein isolated from a red algal Griffithsia sp., binds high mannose N-linked glycans of virus surface glycoproteins with extremely high affinity, a property that allows it to prevent the entry of primary isolates and laboratory strains of T- and M-tropic HIV-1. We used the sequence of a portion of griffithsin''s sequence as a design template to create smaller peptides with antiviral and carbohydrate-binding properties.

Methodology/Results

The new peptides derived from a trio of homologous β-sheet repeats that comprise the motifs responsible for its biological activity. Our most active antiviral peptide, grifonin-1 (GRFN-1), had an EC₅₀ of 190.8±11.0 nM in in vitro TZM-bl assays and an EC₅₀ of 546.6±66.1 nM in p24^gag antigen release assays. GRFN-1 showed considerable structural plasticity, assuming different conformations in solvents that differed in polarity and hydrophobicity. Higher concentrations of GRFN-1 formed oligomers, based on intermolecular β-sheet interactions. Like its parent protein, GRFN-1 bound viral glycoproteins gp41 and gp120 via the N-linked glycans on their surface.

Conclusion

Its substantial antiviral activity and low toxicity in vitro suggest that GRFN-1 and/or its derivatives may have therapeutic potential as topical and/or systemic agents directed against HIV-1.     

Characterization of an IncFII plasmid encoding NDM-1 from Escherichia coli ST131

Background

The current spread of the gene encoding the metallo-ß-lactamase NDM-1 in Enterobacteriaceae is linked to a variety of surrounding genetic structures and plasmid scaffolds.

Methodology

The whole sequence of plasmid pGUE-NDM carrying the bla _NDM-1 gene was determined by high-density pyrosequencing and a genomic comparative analysis with other bla _NDM-1-negative IncFII was performed.

Principal Findings

Plasmid pGUE-NDM replicating in Escherichia coli confers resistance to many antibiotic molecules including β-lactams, aminoglycosides, trimethoprim, and sulfonamides. It is 87,022 bp in-size and carries the two β-lactamase genes bla _NDM-1 and bla _OXA-1, together with three aminoglycoside resistance genes aacA4, aadA2, and aacC2. Comparative analysis of the multidrug resistance locus contained a module encompassing the bla _NDM-1 gene that is actually conserved among different structures identified in other enterobacterial isolates. This module was constituted by the bla _NDM-1 gene, a fragment of insertion sequence ISAba125 and a bleomycin resistance encoding gene.

Significance

This is the first characterized bla _NDM-1-carrying IncFII-type plasmid. Such association between the bla _NDM-1 gene and an IncFII-type plasmid backbone is extremely worrisome considering that this plasmid type is known to spread efficiently, as examplified with the worldwide dissemination of bla _CTX-M-15-borne IncFII plasmids.     

Theoretical evidence for the functional benefit of root cortical aerenchyma in soils with low phosphorus availability

Background and Aims

The formation of root cortical aerenchyma (RCA) reduces root respiration and nutrient content by converting living tissue to air volume. It was hypothesized that RCA increases soil resource acquisition by reducing the metabolic and phosphorus cost of soil exploration.

Methods

To test the quantitative logic of the hypothesis, SimRoot, a functional–structural plant model with emphasis on root architecture and nutrient acquisition, was employed. Sensitivity analyses for the effects of RCA on the initial 40 d of growth of maize (Zea mays) and common bean (Phaseolus vulgaris) were conducted in soils with varying degrees of phosphorus availability. With reference to future climates, the benefit of having RCA in high CO₂ environments was simulated.

Key Results

The model shows that RCA may increase the growth of plants faced with suboptimal phosphorus availability up to 70 % for maize and 14 % for bean after 40 d of growth. Maximum increases were obtained at low phosphorus availability (3 µm). Remobilization of phosphorus from dying cells had a larger effect on plant growth than reduced root respiration. The benefit of both these functions was additive and increased over time. Larger benefits may be expected for mature plants. Sensitivity analysis for light-use efficiency showed that the benefit of having RCA is relatively stable, suggesting that elevated CO₂ in future climates will not significantly effect the benefits of having RCA.

Conclusions

The results support the hypothesis that RCA is an adaptive trait for phosphorus acquisition by remobilizing phosphorus from the root cortex and reducing the metabolic costs of soil exploration. The benefit of having RCA in low-phosphorus soils is larger for maize than for bean, as maize is more sensitive to low phosphorus availability while it has a more ‘expensive’ root system. Genetic variation in RCA may be useful for breeding phosphorus-efficient crop cultivars, which is important for improving global food security.