Resistance gene homologues in Theobroma cacao as useful genetic markers

Resistance gene homologue (RGH) sequences have been developed into useful genetic markers for marker-assisted selection (MAS) of disease resistant Theobroma cacao. A plasmid library of amplified fragments was created from seven different cultivars of cacao. Over 600 cloned recombinant amplicons were evaluated. From these, 74 unique RGHs were identified that could be placed into 11 categories based on sequence analysis. Primers specific to each category were designed. The primers specific for a single RGH category amplified fragments of equal length from the seven different cultivars used to create the library. However, these fragments exhibited single-strand conformational polymorphism (SSCP), which allowed us to map six of the RGH categories in an F(2) population of T. cacao. RGHs 1, 4 and 5 were in the same linkage group, with RGH 4 and 5 separated by less than 4 cM. As SSCP can be efficiently performed on our automated sequencer, we have developed a convenient and rapid high throughput assay for RGH alleles.     

Comparison of ribotyping and repetitive extragenic palindromic-PCR for identification of fecal Escherichia coli from humans and animals

This report compares the performances of two popular genotypic methods used for tracking the sources of fecal pollution in water, ribotyping and repetitive extragenic palindromic-PCR (rep-PCR). The rep-PCR was more accurate, reproducible, and efficient in associating DNA fingerprints of fecal Escherichia coli with human and animal hosts of origin.     

Objective threshold selection procedure (OTS) for segmentation of scanning laser confocal microscope images

The determination of volumes and interface areas from confocal laser scanning microscopy (CLSM) images requires the identification of component objects by segmentation. An automated method for the determination of segmentation thresholds for CLSM imaging of biofilms was developed. The procedure, named objective threshold selection (OTS), is a three-dimensional development of the approach introduced by the popular robust automatic threshold selection (RATS) method. OTS is based on the statistical properties of local gray-values and gradients in the image. By characterizing the dependence between a volumetric feature and the intensity threshold used for image segmentation, the former can be determined with an arbitrary confidence level, with no need for user intervention. The identification of an objective segmentation procedure renders the possibility for the full automation of volume and interfacial area measurement. Images from two distinct biofilm systems, acquired using different experimental techniques and instrumental setups were segmented by OTS to determine biofilm volume and interfacial area. The reliability of measurements for each case was analyzed to identify optimal procedure for image acquisition. The automated OTS method was shown to reproduce values obtained manually by an experienced operator.     

Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of the archaeon Thermococcus litoralis

The members of the ABC transporter family transport a wide variety of molecules into or out of cells and cellular compartments. Apart from a translocation pore, each member possesses two similar nucleoside triphosphate-binding subunits or domains in order to couple the energy-providing reaction with transport. In the maltose transporter of several Gram-negative bacteria and the archaeon Thermo coccus litoralis, the nucleoside triphosphate-binding subunit contains a C-terminal regulatory domain. A dimer of the subunit is attached cytoplasmically to the translocation pore. Here we report the crystal structure of this dimer showing two bound pyrophosphate molecules at 1.9 A resolution. The dimer forms by association of the ATPase domains, with the two regulatory domains attached at opposite poles. Significant deviation from 2-fold symmetry is seen at the interface of the dimer and in the regions corresponding to those residues known to be in contact with the translocation pore. The structure and its relationship to function are discussed in the light of known mutations from the homologous Escherichia coli and Salmonella typhimurium proteins.     

Retrovirally transduced mouse dendritic cells require CD4+ T cell help to elicit antitumor immunity: implications for the clinical use of dendritic cells

Presentation of MHC class I-restricted peptides by dendritic cells (DCs) can elicit vigorous antigen-specific CTL responses in vivo. It is well established, however, that T cell help can augment CTL function, raising the question of how best to present tumor-associated MHC class I epitopes to induce effective tumor immunity. To this end, we have examined the role of MHC class II peptide-complexes present on the immunizing DCs in a murine melanoma model. To present MHC class I- and II-restricted Ags reliably on the same cell, we retrovirally transduced bone marrow-derived DCs with the model Ag OVA encoding well-defined class I- and II-restricted epitopes. The importance of CD4+ T cells activated by the immunizing DCs in this model is demonstrated by the following findings: 1) transduced DCs presenting class I and class II epitopes are more efficient than class I peptide-pulsed DCs; 2) MHC class II-deficient DCs fail to induce tumor protection; 3) CD4+ T cell depletion abolishes induction of tumor protection; and 4) DCs presenting bovine serum Ags are more effective in establishing tumor immunity than DCs cultured in syngeneic serum. When MHC class II-deficient DCs were directly activated via their CD40 receptor, we indeed observed a moderate elevation of OVA-specific CTL activity. However, this increase in CTL activity was not sufficient to induce in vivo tumor rejection. Thus, our results demonstrate the potency of genetically modified DCs that express both MHC class I and II epitopes, but caution against the use of DCs presenting only the former.     

Evaluation of fluorescently labeled lectins for noninvasive localization of extracellular polymeric substances in Sphingomonas biofilms

Three strains of Sphingomonas were grown as biofilms and tested for binding of five fluorescently labeled lectins (Con A-type IV-TRITC or -Cy5, Pha-E-TRITC, PNA-TRITC, UEA 1-TRITC, and WGA-Texas red). Only ConA and WGA were significantly bound by the biofilms. Binding of the five lectins to artificial biofilms made of the commercially available Sphingomonas extracellular polysaccharides was similar to binding to living biofilms. Staining of the living and artificial biofilms by ConA might be explained as binding of the lectin to the terminal mannosyl and terminal glucosyl residues in the polysaccharides secreted by Sphingomonas as well as to the terminal mannosyl residue in glycosphingolipids. Staining of the biofilms by WGA could only be explained as binding to the Sphingomonas glycosphingolipid membrane, binding to the cell wall, or nonspecific binding. Glycoconjugation of ConA and WGA with the target sugars glucose and N-acetylglucosamine, respectively, was used as a method for evaluation of the specificity of the lectins towards Sphingomonas biofilms and Sphingomonas polysaccharides. Our results show that the binding of lectins to biofilms does not necessarily prove the presence of specific target sugars in the extracellular polymeric substances (EPS) in biofilms. The lectins may bind to non-EPS targets or adhere nonspecifically to components of the biofilm matrix.     

Localization of iron-reducing activity in paddy soilby profile studies

Profiles of iron speciations (porewaterFe(II) and Fe(III), solid-phase Fe(II) andFe(III)) have been studied to localize both ironreduction and oxidation in flooded paddy soil. Sulfateand nitrate were determined to analyze interactions ofredox reactions involved in the iron cycle with thoseof the sulfur and nitrogen cycle. The development ofthe iron(II) and iron(III) profiles was observed inmicroscale over a time period of 11 weeks. After 11weeks the profiles were stable and showed lowestconcentrations of solid-phase iron(II) on the soilsurface with increasing concentrations to a soil depthof 10 mm ( 100 µmol/cm³). Profilesof iron(III) showed a maximum of iron(III) at a depthof 2 to 4 mm ( 100--200 µmol/cm³).Porewater iron(II) concentrations were three orders ofmagnitude lower than extracted iron(II) and indicatedthat most iron(II) was adsorbed to the solid-phase orimmobilized as siderite and vivianite. Diffusive lossof iron from the soil was indicated by iron recovery(0.3 µmol gdw^–1) in the flooding water after12 weeks. The organic content of the soil influencedthe concentrations of solid-phase iron(II) in deepersoil layers (> 6 mm); higher Fe(II) concentrationsin soil with limiting amounts of electron donors mayindicate lower consumption of CO₂ by methanogenicbacteria and therefore a higher sideriteprecipitation. Soil planted with rice showed similariron(II) profiles of fresh paddy soil cores. However,maximal iron(III) concentrations ( 350µmol/cm³) were present in planted soil at adepth of 1 to 2.5 mm where oxygen is provided by a matof fine roots. Sulfate and nitrate concentrations inthe porewater were highest on the soil surface (10µM NO₃ ^–, 40 µM SO₄ ^2–) anddecreased with depth. Similar profiles were detectedfor malate, acetate, lactate, and propionate, theconcentrations decreased gradually from the surface toa depth of 4 mm. Profiles of oxygen showed highestconcentrations at the surface due to photosyntheticproduction and a depletion of oxygen below 3 mm depth.Methane production rates measured from soil layersincubated separately in closed vessels were zero atthe soil surface and increased with depth. In soildepths below 4 mm where iron(III) concentrationsdecreased higher methane production rates werefound.     

Insertion of telomere repeat sequence decreases plasmid DNA condensation by cobalt (III) hexaammine.

Telomere repeat sequence (TRS) DNA is found at the termini of most eukaryotic chromosomes. The sequences are highly repetitive and G-rich (e.g., [C(1-3)A/TG(1-3)]n for the yeast Saccharomyces cerevisiae) and are packaged into nonnucleosomal protein-DNA structures in vivo. We have used total intensity light scattering and electron microscopy to monitor the effects of yeast TRS inserts on in vitro DNA condensation by cobalt (III) hexaammine. Insertion of 72 bp of TRS into a 3.3-kb plasmid depresses condensation as seen by light scattering and results in a 22% decrease in condensate thickness as measured by electron microscopy. Analysis of toroidal condensate dimensions suggests that the growth stages of condensation are inhibited by the presence of a TRS insert. The depression in total light scattering intensity is greater when the plasmid is linearized with the TRS at an end (39-49%) than when linearized with the TRS in the interior (18-22%). Circular dichroism of a 95-bp fragment containing the TRS insert gives a spectrum that is intermediate between the A-form and B-form, and the anomalous condensation behavior of the TRS suggests a noncanonical DNA structure. We speculate that under conditions in which the plasmid DNA condenses, the telomeric insert assumes a helical geometry that is similar to the A-form and is incompatible with packing into the otherwise B-form lattice of the condensate interior.     

Parasitism and epibiosis in native and non-native gammarids in freshwater in Ireland

The introduced amphipod crustaceans Gammarus pulex and G. tigrinus are displacing the native G. ducheni celticus in a number of freshwater sites in Northern Ireland. We investigated parasite and epibiont infection in populations in the Rivet Lagan and Lough Neagh where both native and invading species occur. Prevalence of the four parasites and epibionts observed was higher in the native G. d. celticus than in the invading amphipods at both field sites. In Lough Neagh. G. d. celticus individuals suffered higher burdens of the rotifer Embata parasitica and the protozoan Epistylis in comparison with the invading species. These patterns may reflect host specificity by the parasites or may result from different susceptibilities of the native and invading host species. We consider the influence of parasitism on host invasions and resulting species distributions.     

Discovery and Targeted Proteomics on Cutaneous Biopsies Infected by Borrelia to Investigate Lyme Disease

Lyme disease is the most important vector-borne disease in the Northern hemisphere and represents a major public health challenge with insufficient means of reliable diagnosis. Skin is rarely investigated in proteomics but constitutes in the case of Lyme disease the key interface where the pathogens can enter, persist, and multiply. Therefore, we investigated proteomics on skin samples to detect Borrelia proteins directly in cutaneous biopsies in a robust and specific way. We first set up a discovery gel prefractionation-LC-MS/MS approach on a murine model infected by Borrelia burgdorferi sensu stricto that allowed the identification of 25 Borrelia proteins among more than 1300 mouse proteins. Then we developed a targeted gel prefractionation-LC-selected reaction monitoring (SRM) assay to detect 9/33 Borrelia proteins/peptides in mouse skin tissue samples using heavy labeled synthetic peptides. We successfully transferred this assay from the mouse model to human skin biopsies (naturally infected by Borrelia), and we were able to detect two Borrelia proteins: OspC and flagellin. Considering the extreme variability of OspC, we developed an extended SRM assay to target a large set of variants. This assay afforded the detection of nine peptides belonging to either OspC or flagellin in human skin biopsies. We further shortened the sample preparation and showed that Borrelia is detectable in mouse and human skin biopsies by directly using a liquid digestion followed by LC-SRM analysis without any prefractionation. This study thus shows that a targeted SRM approach is a promising tool for the early direct diagnosis of Lyme disease with high sensitivity (<10 fmol of OspC/mg of human skin biopsy).Lyme borreliosis is an arthropod-borne disease transmitted by hard ticks (Ixodes spp.). The causative agents are bacteria belonging to the Borrelia burgdorferi sensu lato group. In the United States, more than 30,000 cases have been reported to the Centers for Disease Control and Prevention in 2012. There, the unique pathogenic species of Borrelia is B. burgdorferi sensu stricto (s.s.). In Europe, between 65,000 and 85,000 cases are reported depending on the epidemiological study (1, 2), and the three most prevalent pathogenic species of Borrelia are Borrelia afzelii, Borrelia garinii, and B. burgdorferi s.s. The disease in both Europe and the United States is first characterized in most patients by an inflammatory skin lesion, erythema migrans (EM),¹ which is the most frequent manifestation of the disease. Dissemination to other sites occurs secondarily and can involve among others articulation, nervous system, heart, and skin at other sites (3, 4). The diagnosis can be a real challenge because of the proteiform clinical manifestations. When an EM is present, which is the case for 80% of patients (3), early diagnosis is facilitated. However, EM presentation can be clinically atypical, making the recognition of this manifestation of Lyme borreliosis difficult (5). Later on, when Borrelia has disseminated to the target organs, biological diagnosis is based either on the direct detection of the pathogen in different patient body fluids and biopsies by means of culture and/or PCR or on the indirect demonstration of presence of Borrelia by detection of anti-pathogen-directed IgM and IgG antibodies (enzyme-linked immunosorbent assay (ELISA) and Western blot) (6).Concerning the direct detection of Borrelia, culture of the bacteria has allowed the spirochete isolation since the 80s in different specific Barbour-Stoenner-Kelly-based media by using skin biopsies or biological fluids such as blood or cerebrospinal fluid (7, 8). However, Borrelia culture is not routinely used as a diagnostic test because the bacterial growth takes several weeks and does not yield timely results. Indeed, it requires the use of the specific and expensive Barbour-Stoenner-Kelly medium and a dark field microscope to detect, frequently after at least 2 weeks of incubation, the presence of Borrelia in tissues or biological fluids. When performed from patients with EM, only 40–80% of the cultures are positive (6). In addition, the success of culture varies greatly according to the Borrelia species. PCR is quicker and generally more sensitive than culture with a range of 36–88%, although the success of bacterial detection varies with the gene selected for the assay (6). PCR is efficient for Borrelia detection in synovial liquid (60–85% of the cases) in the case of arthritis (9, 10) but less sensitive in cases of neuroborreliosis in cerebrospinal fluid (<20–40% of the cases) (9, 11). Moreover, PCR detects DNA and not proteins and therefore prevents the detection of active infection. As far as the skin biopsies are concerned, the sensitivity of detection is variable in cases of EM or acrodermatitis chronica atrophicans (12). Conversely, indirect detection using serological tests is not adapted to the early diagnosis as it relies on antibodies only detectable after at least 4–6 weeks after the infectious tick bite. These tests also suffer from lack of specificity (13). New diagnostic approaches are therefore required. Selected reaction monitoring (SRM) has been recognized as an efficient mass spectrometry-based technique for the biomarker verification and validation in several biological fluids (blood, plasma, and urine) (14 –18). The demonstrated specificity, selectivity, and high sensitivity (low attomole range) of the technique (19) makes it promising for the development of an SRM-based method for early diagnosis of Lyme disease. To our knowledge, this strategy has only rarely been used on skin tissue (20). It would allow the direct and rapid detection of Borrelia proteins in the skin, demonstrating the presence of an active infection very early after the tick transmission.In the present study, we set up a workflow to develop a robust and sensitive SRM assay to detect Borrelia in human skin samples (Fig. 1). First, we looked for Borrelia proteins in infected mouse skin samples by using a classical shotgun/discovery strategy. This experiment afforded a list of bacterial proteins that are expressed in vivo in the skin of an infected mammalian host. Then, we selected protein targets and optimized a Ge-LC-SRM assay to specifically detect and quantify these proteins in mouse skin samples. We demonstrated the transferability of the SRM assay for the detection of the targeted proteins in human skin samples naturally infected with Borrelia. Finally, we improved the experimental protocol to avoid gel prefractionation.Open in a separate windowFig. 1.Summary of the experimental workflow. Experimentally infected mouse skin biopsies were analyzed by a shotgun Ge-LC-MS/MS strategy to identify Borrelia target proteins. Then we developed targeted LC-SRM assays with or without gel prefractionation. Finally, these targeted methods were transferred on tick-infected human skin samples.