A five-gene qPCR signature can classify type 2 asthma comparably to microscopy of induced sputum from severe asthma patients.

Asthma is a heterogenous disease characterized by airway inflammation and variable expiratory airflow limitation resulting in variable respiratory symptoms. Characterization of airway inflammation is important to choose the optimal treatment for severe asthma patients eligible for biological treatment. However, counting cells in induced sputum samples are a time-consuming process, highly dependent on personal skills. Replacing eosinophil and neutrophil cell counting with qPCR for transcripts of selected mast cell, and basophil genes may provide more reproducible results. Aims: The objective of this study was to compare qPCR with microscopy in asthma endotyping. Methods: A qPCR method measuring five mast cell/basophil genes was applied on induced sputum samples from 30 severe asthma patients and compared with microscopy. Target gene Ct-values (CPA3, GATA2, HDC, MS4A2, TPSAB1/TPSB2) were referenced to household ß-actin Ct values as a measure of relative mRNA abundance of the target in each sample. Target/ß-actin-ratios in eosinophilic and non-eosinophilic groups determined by microscopy with an eosinophil threshold of 3% in 400 cells were compared using Mann-Whitney U Test. Spearman´s correlations were used to test for correlation between targets vs. FENO and targets vs. blood eosinophil counts. Results: The study demonstrated a statistical difference in relative mRNA abundance for four mast cell/basophil specific genes. CPA3, GATA2, HDC and MS4A2 were elevated in eosinophilic asthma versus non-eosinophilic asthma patients. The study found that GATA2, CPA3, MS4A2 and TPSAB1/TPSB2 transcripts are positively correlated with FENO. Neither the five mast cell genes nor the five-gene signature correlated with blood eosinophils. The five-gene signature with a target/ß-actin-ratio cut-off ≥2 generated sensitivity = 87%, specificity = 94%, NPV = 88% and PPV = 92% compared to microscopy. Conclusion: This study confirms the contribution of mast cells in the pathogenesis of EA and suggests that mast cell mRNA markers could be one of the biomarkers used to identify EA.

Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans.

IMPORTANCE: Intracellular calcium signaling plays an important role in the resistance and adaptation to stresses encountered by fungal pathogens within the host. This study reports the optimization of the GCaMP fluorescent calcium reporter for live-cell imaging of dynamic calcium responses in single cells of the pathogen, Candida albicans, for the first time. Exposure to membrane, osmotic or oxidative stress generated both specific changes in single cell intracellular calcium spiking and longer calcium transients across the population. Repeated treatments showed that calcium dynamics become unaffected by some stresses but not others, consistent with known cell adaptation mechanisms. By expressing GCaMP in mutant strains and tracking the viability of individual cells over time, the relative contributions of key signaling pathways to calcium flux, stress adaptation, and cell death were demonstrated. This reporter, therefore, permits the study of calcium dynamics, homeostasis, and signaling in C. albicans at a previously unattainable level of detail.

Network traits predict ecological strategies in fungi.

Colonization of terrestrial environments by filamentous fungi relies on their ability to form networks that can forage for and connect resource patches. Despite the importance of these networks, ecologists rarely consider network features as functional traits because their measurement and interpretation are conceptually and methodologically difficult. To address these challenges, we have developed a pipeline to translate images of fungal mycelia, from both micro- and macro-scales, to weighted network graphs that capture ecologically relevant fungal behaviour. We focus on four properties that we hypothesize determine how fungi forage for resources, specifically: connectivity; relative construction cost; transport efficiency; and robustness against attack by fungivores. Constrained ordination and Pareto front analysis of these traits revealed that foraging strategies can be distinguished predominantly along a gradient of connectivity for micro- and macro-scale mycelial networks that is reminiscent of the qualitative 'phalanx' and 'guerilla' descriptors previously proposed in the literature. At one extreme are species with many inter-connections that increase the paths for multidirectional transport and robustness to damage, but with a high construction cost; at the other extreme are species with an opposite phenotype. Thus, we propose this approach represents a significant advance in quantifying ecological strategies for fungi using network information.

Comparative Effectiveness Analyses of Salvage Prostatectomy and Salvage Radiotherapy Outcomes Following Focal or Whole-Gland Ablative Therapy (High-Intensity Focused Ultrasound, Cryotherapy or Electroporation) for Localised Prostate Cancer.

AIMS: Ablative therapy, such as focal therapy, cryotherapy or electroporation, aims to treat clinically significant prostate cancer with reduced treatment-related toxicity. Up to a third of patients may require further local salvage treatment after ablative therapy failure. Limited descriptive, but no comparative, evidence exists between different salvage treatment outcomes. The aim of this study was to compare oncological and functional outcomes after salvage robot-assisted radical prostatectomy (SRARP) and salvage radiotherapy (SRT). MATERIALS AND METHODS: Data were collected prospectively and retrospectively on 100 consecutive SRARP cases and 100 consecutive SRT cases after ablative therapy failure in a high-volume tertiary centre. RESULTS: High-risk patients were over-represented in the SRARP group (66.0%) compared with the SRT group (48.0%) (P = 0.013). The median (interquartile range) follow-up after SRARP was 16.5 (10.0-30.0) months and 37.0 (18.5-64.0) months after SRT. SRT appeared to confer greater biochemical recurrence-free survival at 1, 2 and 3 years compared with SRARP in high-risk patients (year 3: 86.3% versus 66.0%), but biochemical recurrence-free survival was similar for intermediate-risk patients (year 3: 90.0% versus 75.6%). There was no statistical difference in pad-free continence at 12 and 24 months between SRARP (77.2 and 84.7%) and SRT (75.0 and 74.0%) (P = 0.724, 0.114). Erectile function was more likely to be preserved in men who underwent SRT. After SRT, cumulative bowel and urinary Radiation Therapy Oncology Group toxicity grade I were 25.0 and 45.0%, grade II were 11.0 and 11.0% and grade III or IV complications were 4.0 and 5.0%, respectively. CONCLUSION: We report the first comparative analyses of salvage prostatectomy and radiotherapy following ablative therapy. Men with high-risk disease appear to have superior oncological outcomes after SRT; however, treatment allocation does not appear to influence oncological outcomes for men with intermediate-risk disease. Treatment allocation was associated with a different spectrum of toxicity profile. Our data may inform shared decision-making when considering salvage treatment following focal or whole-gland ablative therapy.

Phagoptosis - Cell Death By Phagocytosis - Plays Central Roles in Physiology, Host Defense and Pathology.

Cell death by phagocytosis - termed 'phagoptosis' for short - is a form of cell death caused by the cell being phagocytosed i.e. recognised, engulfed and digested by another cell. Phagocytes eat cells that: i) expose 'eat-me' signals, ii) lose 'don't-eat-me' signals, and/or iii) bind opsonins. Live cells may express such signals as a result of cell stress, damage, activation or senescence, which can result in phagoptosis. Phagoptosis may be the most abundant form of cell death physiologically as it mediates erythrocyte turnover. It also regulates: reproduction by phagocytosis of sperm, development by removal stem cells and excess cells, and immunity by removal of activated neutrophils and T cells. Phagoptosis mediates the recognition of non-self and host defence against pathogens and cancer cells. However, in inflammatory conditions, excessive phagoptosis may kill our cells, leading to conditions such as hemophagy and neuronal loss.

Not all facial swellings are angioedemas

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Chlorhexidine: a retrospective observational study of a potentially life-threatening molecule

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Foraging by a wood-decomposing fungus is ecologically adaptive.

We show that fungi that forage for wood do not conform to the paradigm of symmetric radial growth and grow asymmetrically by default. Asymmetry is further accentuated by contact with a resource that also partially polarizes growth in the direction of the resource. Despite marked changes at the perimeter, overall growth allocation on an area basis is, however, unchanged implying sophisticated regulation at the colony level. Using mathematical models, we show that this behaviour is best explained as a local response of the immediate segment contacting the resource. The model reveals that foraging behaviour is adaptive but only for resources that are clustered in space and is selectively neutral for randomly scattered resources. This clustered spatial distribution matches that found in the natural environment. Modelling also shows that the foraging strategy used by these fungi involves substantial risks as well as benefits.

An increase in serum tryptase even below 11.4 ng/mL may indicate a mast cell-mediated hypersensitivity reaction: a prospective study in Hymenoptera venom allergic patients.

BACKGROUND: During a systemic hypersensitivity reaction (SR), an increase in serum tryptase compared to the baseline value is an indicator of mast cell activation, most often due to an IgE-mediated mechanism. OBJECTIVE: To study the relevance of an increase in serum tryptase below the upper normal value of 11.4 ng/mL. METHODS: Serum tryptase levels were measured in 35 patients with Hymenoptera venom hypersensitivity before and during venom exposure. Of these, 20 developed SR to stings or following venom injections during immunotherapy (reactors), while 15 tolerated reexposure to stings or venom injections during immunotherapy without SR (non-reactors). Serum tryptase was estimated at 2, 5 and 24 h after exposure and was compared to a baseline value obtained before or at least 72 h after exposure. RESULTS: Considering circadian variation of serum tryptase, a relative increase to ≥135% of the baseline value (relative delta bound) was defined to indicate mast cell activation. Such an increase was observed in 17 of 20 reactors (85%), but none of 15 non-reactors. A serum tryptase of ≥11.4 ng/mL following venom exposure was observed in eight of the 20 reactors (40%) and 2 (13.3%) of the 15 non-reactors. Both these non-reactors also had an elevated baseline serum tryptase. CONCLUSIONS AND CLINICAL RELEVANCE: Serum tryptase values obtained during a suspected hypersensitivity reaction must always be compared to a baseline value. A relative tryptase increase to ≥135% of the baseline value during a suspected hypersensitivity reaction indicates mast cell activation even below 11.4 ng/mL.

Prevalence of emetic Bacillus cereus in different ice creams in Bavaria.

In this study, 809 samples of ice cream from different sources were investigated by using cultural methods for the presence of presumptive Bacillus cereus. Isolates from culture-positive samples were examined with a real-time PCR assay targeting a region of the cereulide synthetase gene (ces) that is highly specific for emetic B. cereus strains. The samples were collected from ice cream parlors and restaurants that produced their own ice cream and from international commercial ice cream companies in different regions of Bavaria during the summer of 2008. Presumptive B. cereus was found in 508 (62.7%) ice cream samples investigated, and 24 (4.7%) of the isolates had the genetic background for cereulide toxin production. The level of emetic B. cereus in the positive samples ranged from 0.1 to 20 CFU/g of ice cream.

Phosphorylation of Puma modulates its apoptotic function by regulating protein stability.

Puma is a potent BH3-only protein that antagonises anti-apoptotic Bcl-2 proteins, promotes Bax/Bak activation and has an essential role in multiple apoptotic models. Puma expression is normally kept very low, but can be induced by several transcription factors including p53, p73, E2F1 and FOXO3a, whereby it can induce an apoptotic response. As Puma can to bind and inactivate all anti-apoptotic members of the Bcl-2 family, its activity must be tightly controlled. We report here, for the first time, evidence that Puma is subject to post-translational control through phosphorylation. We show that Puma is phosphorylated at multiple sites, with the major site of phosphorylation being serine 10. Replacing serine 10 with alanine causes reduced Puma turnover and enhanced cell death. Interestingly, Puma turnover occurs through the proteasome, and substitution of serine 10 causes elevated Puma levels independently of macroautophagy, Bcl-2 family member binding, caspase activity and apoptotic death. We conclude, therefore, that phosphorylation of Puma at serine 10 promotes Puma turnover, represses Puma's cell death potential and promotes cell survival. Owing to the highly pro-apoptotic nature of Puma, these studies highlight an important additional regulatory step in the determination of cellular life or death.

Evidence for nickel/proton antiport activity at the tonoplast of the hyperaccumulator plant Alyssum lesbiacum.

The mechanism of nickel uptake into vacuoles isolated from leaf tissue of Alyssum lesbiacum was investigated to help understand the ability of this species to hyperaccumulate Ni. An imaging system was designed to monitor Ni uptake by single vacuoles using the metal-sensitive fluorescent dye, Newport Green. Nickel uptake into isolated vacuoles from leaf tissue of A. lesbiacum was enhanced by the presence of Mg/ATP, presumably via energisation of the vacuolar H(+)-ATPase (V-ATPase). This ATP-stimulated Ni uptake was abolished by bafilomycin (a diagnostic inhibitor of the V-ATPase) and by dissipation of the transmembrane pH difference with an uncoupler. These observations are consistent with Ni(2+)/nH(+) antiport activity at the tonoplast driven by a proton electrochemical gradient established by the V-ATPase, which would provide a mechanism for secondary active transport of Ni(2+) into the vacuole. This study provides insights into the molecular basis of Ni tolerance in Alyssum, and may aid in the identification of genes involved in Ni hyperaccumulation.

Confocal imaging of glutathione redox potential in living plant cells.

Reduction-oxidation-sensitive green fluorescent protein (roGFP1 and roGFP2) were expressed in different sub-cellular compartments of Arabidopsis and tobacco leaves to empirically determine their performance as ratiometric redox sensors for confocal imaging in planta. A lower redox-dependent change in fluorescence in combination with reduced excitation efficiency at 488 nm resulted in a significantly lower dynamic range of roGFP1 than for roGFP2. Nevertheless, when targeted to the cytosol and mitochondria of Arabidopsis leaves both roGFPs consistently indicated redox potentials of about -320 mV in the cytosol and -360 mV in the mitochondria after pH correction for the more alkaline matrix pH. Ratio measurements were consistent throughout the epidermal cell layer, but results might be attenuated deeper within the leaf tissue. Specific interaction of both roGFPs with glutaredoxin in vitro strongly suggests that in situ both variants preferentially act as sensors for the glutathione redox potential. roGFP2 targeted to plastids and peroxisomes in epidermal cells of tobacco leaves was slightly less reduced than in other plasmatic compartments, but still indicated a highly reduced glutathione pool. The only oxidizing compartment was the lumen of the endoplasmic reticulum, in which roGFP2 was almost completely oxidized. In all compartments tested, roGFP2 reversibly responded to perfusion with H(2)O(2) and DTT, further emphasizing that roGFP2 is a reliable probe for dynamic redox imaging in planta. Reliability of roGFP1 measurements might be obscured though in extended time courses as it was observed that intense irradiation of roGFP1 at 405 nm can lead to progressive photoisomerization and thus a redox-independent change of fluorescence excitation ratios.

Imaging complex nutrient dynamics in mycelial networks.

Transport networks are vital components of multi-cellular organisms, distributing nutrients and removing waste products. Animal cardiovascular and respiratory systems, and plant vasculature, are branching trees whose architecture is thought to determine universal scaling laws in these organisms. In contrast, the transport systems of many multi-cellular fungi do not fit into this conceptual framework, as they have evolved to explore a patchy environment in search of new resources, rather than ramify through a three-dimensional organism. These fungi grow as a foraging mycelium, formed by the branching and fusion of threadlike hyphae, that gives rise to a complex network. To function efficiently, the mycelial network must both transport nutrients between spatially separated source and sink regions and also maintain its integrity in the face of continuous attack by mycophagous insects or random damage. Here we review the development of novel imaging approaches and software tools that we have used to characterise nutrient transport and network formation in foraging mycelia over a range of spatial scales. On a millimetre scale, we have used a combination of time-lapse confocal imaging and fluorescence recovery after photobleaching to quantify the rate of diffusive transport through the unique vacuole system in individual hyphae. These data then form the basis of a simulation model to predict the impact of such diffusion-based movement on a scale of several millimetres. On a centimetre scale, we have used novel photon-counting scintillation imaging techniques to visualize radiolabel movement in small microcosms. This approach has revealed novel N-transport phenomena, including rapid, preferential N-resource allocation to C-rich sinks, induction of simultaneous bi-directional transport, abrupt switching between different pre-existing transport routes, and a strong pulsatile component to transport in some species. Analysis of the pulsatile transport component using Fourier techniques shows that as the colony forms, it self-organizes into well demarcated domains that are identifiable by differences in the phase relationship of the pulses. On the centimetre to metre scale, we have begun to use techniques borrowed from graph theory to characterize the development and dynamics of the network, and used these abstracted network models to predict the transport characteristics, resilience, and cost of the network.

Quantifying dynamic resource allocation illuminates foraging strategy in Phanerochaete velutina.

Saprotrophic woodland fungi forage for mineral nutrients and woody resources by extension of a mycelial network across the forest floor. Different species explore at different rates and establish networks with qualitatively differing architecture. However, detailed understanding of fungal foraging behaviour has been hampered by the absence of tools to quantify resource allocation and growth accurately and non-invasively. To solve this problem, we have used photon-counting scintillation imaging (PCSI) to map and quantify nutrient allocation and localised growth simultaneously in heterogeneous resource environments. We show that colonies spontaneously shift to an asymmetric growth pattern, even in the absence of added resources, often with a distinct transition between the two growth phases. However, the extent of polarisation was much more pronounced and focussed in the presence of an additional cellulose resource. In this case, there was highly localised growth, often at the expense of growth elsewhere in the colony, and marked accumulation of (14)C-AIB in the sector of the colony with the added resource. The magnitude of the response was greatest when resource was added around the time of the endogenous developmental transition. The focussed response required a metabolisable resource, as only limited changes were seen with glass fibre discs used to mimic the osmotic and thigmotropic stimuli upon resource addition. Overall the behaviour is consistent with an adaptive foraging strategy, both to exploit new resources and also to redirect subsequent foraging effort to this region, presumably with an expectation that the probability of finding additional resources is increased.

Emergence of self-organised oscillatory domains in fungal mycelia.

Fungi play a central role in the nutrient cycles of boreal and temperate forests. In these biomes, the saprotrophic wood-decay fungi are the only organisms that can completely decompose woody plant litter. In particular, cord-forming basidiomycete fungi form extensive mycelial networks that scavenge scarce mineral nutrients and translocate them over long distances to exploit new food resources. Despite the importance of resource allocation, there is limited information on nutrient dynamics in these networks, particularly for nitrogen, as there is no suitable radioisotope available. We have mapped N-translocation using photon-counting scintillation imaging of the non-metabolised amino acid analogue, (14)C-aminoisobutyrate. We describe a number of novel phenomena, including rapid, preferential N-resource allocation to C-rich sinks, induction of simultaneous bi-directional N-transport, abrupt switching between different pre-existing transport routes, and emergence of locally synchronised, oscillatory phase domains. It is possible that such self-organised oscillatory behaviour is a mechanism to achieve global co-ordination in the mycelium.

Fourier-based spatial mapping of oscillatory phenomena in fungi.

Microorganisms display a range of oscillatory phenomena that operate over different temporal scales. Fourier analysis provides a compact description of such oscillations in terms of their frequency, magnitude and phase. However, in the majority of studies there is no explicit consideration of the spatial organisation of the oscillation. Here we describe procedures and a software package to map oscillatory phenomena in microorganisms in both the time and frequency domains. Key parameters of interest, such as frequency, phase or magnitude of the oscillations, are presented as pseudo-colour coded maps. This maintains the spatial information in the image and greatly facilitates understanding of potentially complex propagating waves or development of oscillatory domains with distinct behaviour. We illustrate the utility of this system with reference to spatial analysis of the pulsatile component to amino acid transport in mycelial systems of Phanerochaete velutina and Coniophora puteana, and actin-myosin based contractions in Physarum polycephalum.