Correction: Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

[This corrects the article DOI: 10.1371/journal.ppat.1008477.].

Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies.

Inside or outside? A new collection of Gateway vectors allowing plant protein subcellular localization or over-expression.

Transient expression of proteins based on agro-infiltration techniques has proven very efficient and straightforward to study the intrinsic properties of proteins. The level of protein expression has been enhanced by the use of vector plasmids containing virus-derived sequences and the cloning step has been facilitated by recombination technologies. The pEAQ-HT-DEST series of vectors fulfilling these improvements are vectors of choice. However, they lack the possibility to directly and easily fuse the protein of interest to a fluorescent tag or to address it to the secretion pathway. In the present work we describe the production of 15 pEAQ-HT-DEST1-based plasmids designed to use the Gateway® cloning technology and to generate high levels of fluorescent fusion protein by agro-infiltration, in planta. This collection of plasmids includes binary vectors allowing N-terminal or C-terminal fusion to the bright tags EGFP or TagRFP for cytoplasmic accumulation or secretion and represents therefore a valuable tool for subcellular localization or biochemical studies. A viral protein, the blue fluorescent protein TagBFP, the green fluorescent protein variant T-Sapphire and an Arabidopsis protein were transiently expressed in N. benthamiana to demonstrate the potential of these vectors.

Utility of food pellets containing 1-aminobenzotriazole for longer term in vivo inhibition of cytochrome P450 in mice.

1. The utility of 1-aminobenzotriazole (ABT), incorporated in food, has been investigated as an approach for longer term inhibition of cytochrome P450 (P450) enzymes in mice. 2. In rats, ABT inhibits gastric emptying, to investigate this potential limitation in mice we examined the effect of ABT administration on the oral absorption of NVS-CRF38. Two hour prior oral treatment with 100 mg/kg ABT inhibited the oral absorption of NVS-CRF38, Tmax was 4 hours for ABT-treated mice compared to 0.5 hours in the control group. 3. A marked inhibition of hepatic P450 activity was observed in mice fed with ABT containing food pellets for 1 month. P450 activity, as measured by the oral clearance of antipyrine, was inhibited on day 3 (88% of control), week 2 (83% of control) and week 4 (80% of control). 4. Tmax values for antipyrine were comparable between ABT-treated mice and the control group, alleviating concerns about impaired gastric function. 5. Inclusion of ABT in food provides a minimally invasive and convenient approach to achieve longer term inhibition of P450 activity in mice. This model has the potential to enable pharmacological proof-of-concept studies for research compounds which are extensively metabolised by P450 enzymes.

Arabidopsis wat1 (walls are thin1)-mediated resistance to the bacterial vascular pathogen, Ralstonia solanacearum, is accompanied by cross-regulation of salicylic acid and tryptophan metabolism.

Inactivation of Arabidopsis WAT1 (Walls Are Thin1), a gene required for secondary cell-wall deposition, conferred broad-spectrum resistance to vascular pathogens, including the bacteria Ralstonia solanacearum and Xanthomonas campestris pv. campestris, and the fungi Verticillium dahliae and Verticillium albo-atrum. Introduction of NahG, the bacterial salicylic acid (SA)-degrading salicylate hydroxylase gene, into the wat1 mutant restored full susceptibility to both R. solanacearum and X. campestris pv. campestris. Moreover, SA content was constitutively higher in wat1 roots, further supporting a role for SA in wat1-mediated resistance to vascular pathogens. By combining transcriptomic and metabolomic data, we demonstrated a general repression of indole metabolism in wat1-1 roots as shown by constitutive down-regulation of several genes encoding proteins of the indole glucosinolate biosynthetic pathway and reduced amounts of tryptophan (Trp), indole-3-acetic acid and neoglucobrassicin, the major form of indole glucosinolate in roots. Furthermore, the susceptibility of the wat1 mutant to R. solanacearum was partially restored when crossed with either the trp5 mutant, an over-accumulator of Trp, or Pro35S:AFB1-myc, in which indole-3-acetic acid signaling is constitutively activated. Our original hypothesis placed cell-wall modifications at the heart of the wat1 resistance phenotype. However, the results presented here suggest a mechanism involving root-localized metabolic channeling away from indole metabolites to SA as a central feature of wat1 resistance to R. solanacearum.

Walking with Different Insoles Changes Lower-Limb Biomechanics Globally in Patients with Medial Knee Osteoarthritis.

Using insoles to modify walking biomechanics is of keen interest for the treatment of medial-compartment knee osteoarthritis. So far, insole interventions have focused on reducing the peak of the knee adduction moment (pKAM) and have led to inconsistent clinical outcomes. This study aimed to evaluate the changes in other gait variables related to knee osteoarthritis when patients walk with different insoles to provide insights into the necessity to enlarge the biomechanical analyses to other variables. Walking trials were recorded for 10 patients in four insole conditions. Changes among conditions were computed for six gait variables, including the pKAM. The associations between the changes in pKAM and the changes in the other variables were also assessed individually. Walking with different insoles had noticeable effects on the six gait variables, with high heterogeneity among patients. For all variables, at least 36.67% of the changes were of medium-to-large effect size. The associations with the changes in pKAM varied among variables and patients. In conclusion, this study showed that varying the insole could globally influence ambulatory biomechanics and that limiting measurement to the pKAM could lead to an important loss of information. Beyond the consideration of additional gait variables, this study also encourages personalized interventions to address inter-patient variability.

Walls are thin 1 (WAT1), an Arabidopsis homolog of Medicago truncatula NODULIN21, is a tonoplast-localized protein required for secondary wall formation in fibers.

By combining Zinnia elegans in vitro tracheary element genomics with reverse genetics in Arabidopsis, we have identified a new upstream component of secondary wall formation in xylary and interfascicular fibers. Walls are thin 1 (WAT1), an Arabidopsis thaliana homolog of Medicago truncatula NODULIN 21 (MtN21), encodes a plant-specific, predicted integral membrane protein, and is a member of the plant drug/metabolite exporter (P-DME) family (transporter classification number: TC 2.A.7.3). Although WAT1 is ubiquitously expressed throughout the plant, its expression is preferentially associated with vascular tissues, including developing xylem vessels and fibers. WAT1:GFP fusion protein analysis demonstrated that WAT1 is localized to the tonoplast. Analysis of wat1 mutants revealed two cell wall-related phenotypes in stems: a defect in cell elongation, resulting in a dwarfed habit and little to no secondary cell walls in fibers. Secondary walls of vessel elements were unaffected by the mutation. The secondary wall phenotype was supported by comparative transcriptomic and metabolomic analyses of wat1 and wild-type stems, as many transcripts and metabolites involved in secondary wall formation were reduced in abundance. Unexpectedly, these experiments also revealed a modification in tryptophan (Trp) and auxin metabolism that might contribute to the wat1 phenotype. Together, our data demonstrate an essential role for the WAT1 tonoplast protein in the control of secondary cell wall formation in fibers.

Comparison of mass spectrometry data and bioinformatics predictions to assess the bona fide localization of proteins identified in cell wall proteomics studies.

Plant cell walls have complex architectures made of polysaccharides among which cellulose, hemicelluloses, pectins and cell wall proteins (CWPs). Some CWPs are anchored in the plasma membrane through a glycosylphosphatidylinositol (GPI)-anchor. The secretion pathway is the classical route to reach the extracellular space. Based on experimental data, a canonical signal peptide (SP) has been defined, and bioinformatics tools allowing the prediction of the sub-cellular localization of proteins have been designed. In the same way, the presence of GPI-anchor attachment sites can be predicted using bioinformatics programs. This article aims at comparing the bioinformatics predictions of the sub-cellular localization of proteins assumed to be CWPs to mass spectrometry (MS) data. The sub-cellular localization of a few CWPs exhibiting particular features has been checked by cell biology approaches. Although the prediction of SP length is confirmed in most cases, it is less conclusive for GPI-anchors. Three main observations were done: (i) the variability observed at the N-terminus of a few mature CWPs could play a role in the regulation of their biological activity; (ii) one protein was shown to have a double sub-cellular localization in the cell wall and the chloroplasts; and (iii) peptides were found to be located at the C-terminus of several CWPs previously identified in GPI-anchored proteomes, thus raising the issue of their actual anchoring to the plasma membrane.

Changes in ambulatory knee adduction moment with lateral wedge insoles differ with respect to the natural foot progression angle.

Lateral wedge insoles (LWI) have been proposed to reduce the knee adduction moment (KAM) during walking; a biomechanical modification notably sought in case of medial knee osteoarthritis. However, the inter-individual inconsistency in KAM changes with LWI limits their therapeutic use. Although the foot progression angle (FPA) has been frequently discussed in KAM modifications literature, there is a lack of data regarding a possible relationship between this gait measure and changes in KAM with LWI. This study aimed to test if KAM changes with LWI differ with respect to the natural FPA and to compare KAM-related variables between individuals walking with smaller and larger natural FPA. Twenty-two healthy participants (14 males, 24 ± 3 years, 22.7 ± 2.7 kg/m2) underwent gait analysis with and without LWI. They were divided into two groups based on their natural FPA, and changes in KAM 1st peak, KAM impulse, and KAM-related variables were compared between groups. KAM 1st peak and impulse decreased with LWI in the smaller natural FPA group (p ≤ 0.006), while only KAM impulse decreased in the larger natural FPA group (p < 0.001). The difference in KAM 1st peak changes was explained by a less reduced lever arm in participants walking with larger natural FPA. In conclusion, this study brought new insight into the variability in KAM response to LWI. If the findings are confirmed in patients with medial knee osteoarthritis, the FPA could become a simple measure to help identify the patients more likely to reduce their KAM with LWI.

Plant Cell Wall Proteomes: Bioinformatics and Cell Biology Tools to Assess the Bona Fide Cell Wall Localization of Proteins.

The purification of plant cell walls is challenging because they constitute an open compartment which is not limited by a membrane like the cell organelles. Different strategies have been established to limit the contamination by proteins of other compartments in cell wall proteomics studies. Non-destructive methods rely on washing intact cells with various types of solutions without disrupting the plasma membrane in order to elute cell wall proteins. In contrast, destructive protocols involve the purification of cell walls prior to the extraction of proteins with salt solutions. In both cases, proteins known to be intracellular have been identified by mass spectrometry in cell wall proteomes. The aim of this chapter is to provide tools to assess the subcellular localization of the proteins identified in cell wall proteomics studies, including: (1) bioinformatic predictions, (2) immunocytolocalization of proteins of interest on tissue sections and (3) in muro observation of proteins of interest fused to reporter fluorescent proteins by confocal microscopy. Finally, a qualitative assessment of the work can be performed and the strategy used to prepare the samples can be optimized if necessary.

Plasma Protein Binding of Highly Bound Drugs Determined With Equilibrium Gel Filtration of Nonradiolabeled Compounds and LC-MS/MS Detection.

Accurate determination of the free fraction of a drug in plasma can be challenging when it falls below 1% and even more so when below 0.1%. Equilibrium dialysis with diluted plasma has been used to determine unbound fraction below 1%, but some analytes are not amenable to this method. One robust alternative for accurately measuring very highly bound compounds is equilibrium gel filtration; however, radiolabeled compounds have been used with this technique to quantify the low analyte concentrations. This report examined results obtained using radiolabeled compounds with liquid scintillation detection and those obtained using their nonradiolabeled analogs with liquid chromatography-tandem mass spectrometry detection. The 2 methods provided comparable results over the range of 0.005%-4% free, with a slope of 1.0 and a R2 = 0.93. These results demonstrate that equilibrium gel filtration with liquid chromatography-tandem mass spectrometry detection can be used earlier in the drug discovery process to determine the unbound fraction of highly bound drugs and may help obviate the need for radiolabeled compound.

Effect of lateral wedge length on ambulatory knee kinetics.

BACKGROUND: Lateral wedge insoles (LWI) were proposed to treat medial knee osteoarthritis through reductions of the ambulatory knee adduction moment (KAM). Limited attention was however paid to the LWI length, resulting in unclear understanding of its effect on KAM reductions. The knee flexion moment (KFM) was also shown to be important in knee osteoarthritis, but little is known about the effect of LWI length on it. RESEARCH QUESTION: This study aimed to compare the KAM and KFM of healthy subjects walking with four different lengths of LWI, explicitly without LWI and with LWI below the hindfoot (HF), below the hindfoot and forefoot (HF + FF) and below the hindfoot, forefoot and hallux (HF + FF + HX) segments. METHODS: Nineteen healthy participants (63% male; 24 ±â€¯3 years old) walked in an instrumented gait lab with LWI of four different lengths. Repeated one-way ANOVAs and post-hoc t-tests were used to compare knee kinetics among LWI lengths. RESULTS: The peak value of the KAM during the first half of stance and the KAM impulse differed with respect to the LWI length (p < 0.001). A length of at least HF + FF, but not necessarily longer, was needed to decrease both KAM parameters compared to walking without LWI. The LWI length had no effect on the peak value of the KFM during the first half of stance (p = 0.86). SIGNIFICANCE: The results in this study could contribute to better selections of LWI for medial knee osteoarthritis and suggested that the length of the LWI could be a critical factor that should be considered in future research.

Photoablative inlay laser in situ keratomileusis (PAI-LASIK) in the rabbit model.

PURPOSE: To evaluate the suitability, biocompatibility, and efficacy of a proprietary hydrogel photoablative inlay (PAI) for use during laser in situ keratomileusis (LASIK). SETTING: Laboratory study, Tulane University Health Sciences Center, New Orleans, Louisiana, USA. METHODS: Eight rabbits (1 eye each) underwent the PAI-LASIK procedure; 4 eyes had a disk-shaped inlay and 4, a donut-shaped inlay. Preoperatively, the hydrogel material was ablated with a programmed correction of 5.0 diopters of hyperopia or myopia. RESULTS: The eyes were followed for 1 to 16 months. No eye showed signs of rejection or extrusion of the PAI. There was no significant difference in corneal clarity or the healing rate between eyes with donut-shaped PAIs and those with disk-shaped PAIs. One eye with a donut-shaped PAI had minimal corneal haze. The remaining inlays did not opacify or fracture during ablation. CONCLUSION: The hydrogel material can be used for the proposed PAI-LASIK procedure.

Plant Cell Wall Proteins: A Large Body of Data, but What about Runaways?

Plant cell wall proteomics has been a very dynamic field of research for about fifteen years. A full range of strategies has been proposed to increase the number of identified proteins and to characterize their post-translational modifications. The protocols are still improving to enlarge the coverage of cell wall proteomes. Comparisons between these proteomes have been done based on various working strategies or different physiological stages. In this review, two points are highlighted. The first point is related to data analysis with an overview of the cell wall proteomes already described. A large body of data is now available with the description of cell wall proteomes of seventeen plant species. CWP contents exhibit particularities in relation to the major differences in cell wall composition and structure between these plants and between plant organs. The second point is related to methodology and concerns the present limitations of the coverage of cell wall proteomes. Because of the variety of cell wall structures and of the diversity of protein/polysaccharide and protein/protein interactions in cell walls, some CWPs can be missing either because they are washed out during the purification of cell walls or because they are covalently linked to cell wall components.

Xylem sap proteomics.

Proteomic analysis of xylem sap has recently become a major field of interest to understand several biological questions related to plant development and responses to environmental clues. The xylem sap appears as a dynamic fluid undergoing changes in its proteome upon abiotic and biotic stresses. Unlike cell compartments which are amenable to purification in sufficient amount prior to proteomic analysis, the xylem sap has to be collected in particular conditions to avoid contamination by intracellular proteins and to obtain enough material. A model plant like Arabidopsis thaliana is not suitable for such an analysis because efficient harvesting of xylem sap is difficult. The analysis of the xylem sap proteome also requires specific procedures to concentrate proteins and to focus on proteins predicted to be secreted. Indeed, xylem sap proteins appear to be synthesized and secreted in the root stele or to originate from dying differentiated xylem cells. This chapter describes protocols to collect xylem sap from Brassica species and to prepare total and N-glycoprotein extracts for identification of proteins by mass spectrometry analyses and bioinformatics.

Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth.

In Arabidopsis thaliana, silencing of hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), a lignin biosynthetic gene, results in a strong reduction of plant growth. We show that, in HCT-silenced plants, lignin synthesis repression leads to the redirection of the metabolic flux into flavonoids through chalcone synthase activity. Several flavonol glycosides and acylated anthocyanin were shown to accumulate in higher amounts in silenced plants. By contrast, sinapoylmalate levels were barely affected, suggesting that the synthesis of that phenylpropanoid compound might be HCT-independent. The growth phenotype of HCT-silenced plants was shown to be controlled by light and to depend on chalcone synthase expression. Histochemical analysis of silenced stem tissues demonstrated altered tracheary elements. The level of plant growth reduction of HCT-deficient plants was correlated with the inhibition of auxin transport. Suppression of flavonoid accumulation by chalcone synthase repression in HCT-deficient plants restored normal auxin transport and wild-type plant growth. By contrast, the lignin structure of the plants simultaneously repressed for HCT and chalcone synthase remained as severely altered as in HCT-silenced plants, with a large predominance of nonmethoxylated H units. These data demonstrate that the reduced size phenotype of HCT-silenced plants is not due to the alteration of lignin synthesis but to flavonoid accumulation.

An ancient mechanism controls the development of cells with a rooting function in land plants.

Root hairs and rhizoids are cells with rooting functions in land plants. We describe two basic helix-loop-helix transcription factors that control root hair development in the sporophyte (2n) of the angiosperm Arabidopsis thaliana and rhizoid development in the gametophytes (n) of the bryophyte Physcomitrella patens. The phylogeny of land plants supports the hypothesis that early land plants were bryophyte-like and possessed a dominant gametophyte and later the sporophyte rose to dominance. If this hypothesis is correct, our data suggest that the increase in morphological complexity of the sporophyte body in the Paleozoic resulted at least in part from the recruitment of regulatory genes from gametophyte to sporophyte.

Purification, cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism.

A protein hydrolyzing hydroxycinnamoyl-CoA esters has been purified from tobacco stem extracts by a series of high pressure liquid chromatography steps. The determination of its N-terminal amino acid sequence allowed design of primers permitting the corresponding cDNA to be cloned by PCR. Sequence analysis revealed that the tobacco gene belongs to a plant acyltransferase gene family, the members of which have various functions. The tobacco cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity-purified and cleaved to yield the recombinant enzyme for use in the study of catalytic properties. The enzyme catalyzed the synthesis of shikimate and quinate esters shown recently to be substrates of the cytochrome P450 3-hydroxylase involved in phenylpropanoid biosynthesis. The enzyme has been named hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase. We show that p-coumaroyl-CoA and caffeoyl-CoA are the best acyl group donors and that the acyl group is transferred more efficiently to shikimate than to quinate. The enzyme also catalyzed the reverse reaction, i.e. the formation of caffeoyl-CoA from chlorogenate (5-O-caffeoyl quinate ester). Thus, hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase appears to control the biosynthesis and turnover of major plant phenolic compounds such as lignin and chlorogenic acid.

Silencing of hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyltransferase affects phenylpropanoid biosynthesis.

The hydroxyl group in the 3-position of the phenylpropanoid compounds is introduced at the level of coumarate shikimate/quinate esters, whose synthesis implicates an acyltransferase activity. Specific antibodies raised against the recombinant tobacco (Nicotiana tabacum) acyltransferase revealed the accumulation of the enzyme in stem vascular tissues of tobacco, in accordance with a putative role in lignification. For functional analysis, the acyltransferase gene was silenced in Arabidopsis thaliana and N. benthamiana by RNA-mediated posttranscriptional gene silencing. In Arabidopsis, gene silencing resulted in a dwarf phenotype and changes in lignin composition as indicated by histochemical staining. An in-depth study of silenced N. benthamiana plants by immunological, histochemical, and chemical methods revealed the impact of acyltransferase silencing on soluble phenylpropanoids and lignin content and composition. In particular, a decrease in syringyl units and an increase in p-hydroxyphenyl units were recorded. Enzyme immunolocalization by confocal microscopy showed a correlation between enzyme accumulation levels and lignin composition in vascular cells. These results demonstrate the function of the acyltransferase in phenylpropanoid biosynthesis.

Metabolic reprogramming in plant innate immunity: the contributions of phenylpropanoid and oxylipin pathways.

In their environment, plants interact with a multitude of living organisms and have to cope with a large variety of aggressions of biotic or abiotic origin. To survive, plants have acquired, during evolution, complex mechanisms to detect their aggressors and defend themselves. Receptors and signaling pathways that are involved in such interactions with the environment are just beginning to be uncovered. What has been known for several decades is the extraordinary variety of chemical compounds the plants are capable to synthesize, and many of these products are implicated in defense responses. The number of natural products occurring in plants may be estimated in the range of hundreds of thousands, but only a fraction have been fully characterized. Despite the great importance of these metabolites for plant and also for human health, our knowledge about their biosynthetic pathways and functions is still fragmentary. Recent progress has been made particularly for phenylpropanoid and oxylipin metabolism, which are emphasized in this review. Both pathways are involved in plant resistance at several levels: by providing building units of physical barriers against pathogen invasion, by synthesizing an array of antibiotic compounds, and by producing signals implicated in the mounting of plant resistance.