Leaf rust induced volatile organic compounds signalling in willow during the infection

Plants are known to emit volatile organic compounds (VOC) in response to various biotic or abiotic stresses. Although the VOC emission in the case of insect attacks is well described, there is only little known about the impact of pathogens on plant emission. In the present study, we used a willow-leaf rust system to describe the effects of a biotrophic fungal infection on the VOC emission pattern of willow leaves. We detected that isoprene emissions from rust-infected leaves decreased threefold compared to control. The total monoterpene emissions did not change although a stress-signalling compound (Z)-β-ocimene showed an increase in infected plants on several days. The infection also increased the emission of sesquiterpenes and lipoxygenase products (LOX) by factors of 175-fold and 10-fold, respectively. The volatile emission signals showed two clear peaks during the experiment. At 6, 7 and 12 days post-infection (dpi), the relative volatile emission signal increased to about sixfold compared to uninfected plants. These time points are directly connected to rust infection since at 6 dpi the first rust pustules appeared on the leaves and at 12 dpi necrosis had developed around several pustules. We present correlations between LOX and sesquiterpene emission signals, which suggest at least two different steps in eliciting the volatile emission.     

Magnitude and variation of fat-free mass density: a cellular-level body composition modeling study

The mean density of fat-free mass (FFM) is remarkably stable at 1.10 g/cm(3) in healthy adult humans, and this stability is a cornerstone of the widely applied densitometry-based two-compartment model for estimating total body fat. At present, the usual means of exploring FFM density is by in vitro or in vivo experimental studies. The purpose of the present investigation was to develop a cellular-level body composition model that includes seven factors that determine FFM density. The model, when applied with available empirical coefficients, predicted an FFM density similar to that observed in vivo. An analysis of the seven model components indicates that the ratio of extracellular solids to total body water is a major determinant of individual variation in FFM density. The difference in FFM density across sex, race, and age groups was examined with the developed model. The present study thus provides a conceptual framework for the systematic study of FFM density in humans.     

Cbp1, a fungal virulence factor under positive selection,forms an effector complex that drives macrophage lysis

Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We observe a high rate of fixation of non-synonymous substitutions in the Cbp1 coding sequences, indicating that Cbp1 is under positive selection. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel “binocular” fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a fungal virulence factor under positive selection that localizes to the cytosol to trigger host cell lysis.     

Nucleic acid amplification tests for diagnosis of smear-negative TB in a high HIV-prevalence setting: a prospective cohort study

Background

Nucleic acid amplification tests are sensitive for identifying Mycobacterium tuberculosis in populations with positive sputum smears for acid-fast bacilli, but less sensitive in sputum-smear-negative populations. Few studies have evaluated the clinical impact of these tests in low-income countries with high burdens of TB and HIV.

Methods

We prospectively enrolled 211 consecutive adults with cough ≥2 weeks and negative sputum smears at Mulago Hospital in Kampala, Uganda. We tested a single early-morning sputum specimen for Mycobacterium tuberculosis DNA using two nucleic acid amplification tests: a novel in-house polymerase chain reaction targeting the mycobacterial secA1 gene, and the commercial Amplified® Mycobacterium tuberculosis Direct (MTD) test (Gen-Probe Inc, San Diego, CA). We calculated the diagnostic accuracy of these index tests in reference to a primary microbiologic gold standard (positive mycobacterial culture of sputum or bronchoalveolar lavage fluid), and measured their likely clinical impact on additional tuberculosis cases detected among those not prescribed initial TB treatment.

Results

Of 211 patients enrolled, 170 (81%) were HIV-seropositive, with median CD4+ T-cell count 78 cells/µL (interquartile range 29-203). Among HIV-seropositive patients, 94 (55%) reported taking co-trimoxazole prophylaxis and 29 (17%) reported taking antiretroviral therapy. Seventy-five patients (36%) had culture-confirmed TB. Sensitivity of MTD was 39% (95% CI 28–51) and that of secA1 was 24% (95% CI 15–35). Both tests had specificities of 95% (95% CI 90–98). The MTD test correctly identified 18 (24%) TB patients not treated at discharge and led to a 72% relative increase in the smear-negative case detection rate.

Conclusions

The secA1 and MTD nucleic acid amplification tests had moderate sensitivity and high specificity for TB in a predominantly HIV-seropositive population with negative sputum smears. Although newer, more sensitive nucleic acid assays may enhance detection of Mycobacterium tuberculosis in sputum, even currently available tests can provide substantial clinical impact in smear-negative populations.     

Identification of immunologically related proteins in sieve-tube exudate collected from monocotyledonous and dicotyledonous plants

The mature, functional sieve-tube system in higher plants is dependent upon protein import from the companion cells to maintain a functional long-distance transport system. Soluble proteins present within the sieve-tube lumen were investigated by analysis of sieve-tube exudates which revealed the presence of distinct sets of polypeptides in seven monocotyledonous and dicotyledonous plant species. Antibodies directed against sieve-tube exudate proteins from Ricinus communis L. demonstrated the presence of shared antigens in the phloem sap collected from Triticum aestivum L., Oryza sativa L., Yucca filamentosa L., Cucurbita maxima Duch., Robinia pseudoacacia L. and Tilia platyphyllos L. Specific antibodies were employed to identify major polypeptides. Molecular chaperones related to Rubisco-subunit-binding protein and cyclophilin, as well as ubiquitin and the redox proteins, thioredoxin h and glutaredoxin, were detected in the sieve-tube exudate of all species examined. Actin and profilin, a modulator of actin polymerization, were also present in all analyzed phloem exudates. However, some proteins were highly species-specific, e.g. cystatin, a protease-inhibitor was present in R. communis but was not detected in exudates from other species, and orthologs of the well-known squash phloem lectin, phloem protein 2, were only identified in the sieve-tube exudate of R. communis and R. pseudoacacia. These findings are discussed in terms of the likely roles played by phloem proteins in the maintenance and function of the enucleate sieve-tube system of higher plants. Received: 12 February 1998 / Accepted: 16 March 1998     

Factors Likely to Affect the Long-term Results of Ventricular Stimulation After Myocardial Infarction

Background

The results of programmed ventricular stimulation (PVS) may change after myocardial infarction (MI). The objective was to study the factors that could predict the results of a second PVS.

Methods

Left ventricular ejection fraction (LVEF) and QRS duration were determined and PVS performed within 3 to 14 years of one another (mean 7.5±5) in 50 patients studied systematically between 1 and 3 months after acute MI.

Results

QRS duration increased from 120±23 ms to 132±29 (p 0.04). LVEF did not decrease significantly (36±12 % vs 37±13 %). Ventricular tachycardia with cycle length (CL) > 220ms (VT) was induced in 11 patients at PVS 1, who had inducible VT with a CL > 220 ms (8) or < 220 ms (ventricular flutter, VFl) (3) at PVS 2. VFl or fibrillation (VF) was induced in 14 patients at PVS 1 and remained inducible in 5; 5 patients had inducible VT and 4 had a negative 2nd PVS. 2. 25 patients had initially negative PVS; 7 had secondarily inducible VT, 4 a VFl/VF, 14 a negative PVS. Changes of PVS were related to initially increasing QRS duration and secondarily changes in LVEF and revascularization but not to the number of extrastimuli required to induce VFl.

Conclusions

In patients without induced VT at first study, changes of PVS are possible during the life. Patients with initially long QRS duration and those who developed decreased LVEF are more at risk to have inducible monomorphic VT at 2nd study, than other patients.     

Bacterial product cantastim derived from Pseudomonas aeruginosa induces migration and maturation of dendritic cells

Dendritic cells (DCs) play a pivotal role in linking innate and adaptive immunity. Migration to the lymph nodes and maturation of DCs are crucial steps in the initiation of specific immune responses. The bacterial product CANTASTIM (CS) is a purified extract of Pseudomonas aeruginosa that induces non-specific protection against bacterial infection, enhances macrophage effector functions and modulates cytokines production. In this study, we used a mouse skin explant culture model and human monocyte-derived DCs to study the effect of CS on the migration and maturation of DCs, respectively. We noticed a significant increase in the number of DCs which migrated from the skin explants when CS was added to the culture medium. Also, CS was able to induce the expression of maturation-associated marker CD83 on human monocyte-derived DCs. DC-based tumor vaccines represent a promising approach for cancer immunotherapy and the migration rate and maturation state of DCs are important parameters for their clinical effectiveness. CS may be an attractive candidate to be tested for the production of DC-based vaccine.