Characterization of PbPga1, an Antigenic GPI-Protein in the Pathogenic Fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by β-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection.     

Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.     

Pharmacological undertreatment of coronary risk factors in patients with psoriasis: observational study of the Danish nationwide registries

Background

Patients with psoriasis have increased prevalence of coronary risk factors and limited recent results have suggested that these risk factors are undertreated in patients with psoriasis. This may contribute to the increased risk of cardiovascular diseases observed in patients with psoriasis.

Objective

To examine the pharmacological treatment of coronary risk factors in patients with severe psoriasis treated with biologic agents in a real-world setting.

Methods and Findings

Medical history of patients with severe psoriasis treated with biologic agents in the time period 2007–09 was retrieved from a Danish nationwide registry (DERMBIO). Individual-level linkage of nationwide administrative registries of hospitalizations, concomitant medications, and socioeconomic status was performed to gain insights into the use of pharmacological treatment. A total of 693 patients (mean age 46.1±12.7 years, 65.7% male) with severe psoriasis treated with biologic agents were identified. Hypertension, hypercholesterolemia, and diabetes mellitus were identified in 16.6%, 9.2%, and 6.7% of cases, respectively. Patients with severe psoriasis were significantly less likely to receive cardiovascular pharmacotherapy compared to age, sex, and coronary risk factor matched controls. In psoriatic patients with hypertension 27.7% received no antihypertensive pharmacotherapy. Patients with dyslipidemia received cholesterol-lowering medications in 55.8% of cases and patients with diabetes mellitus received angiotensin converting enzyme inhibitors/angiotensin II receptor blockers and cholesterol-lowering medications in 42.1% and 23.7% of cases, respectively. Similar results were found for the subset of patients with >1 coronary risk factor and for high risk patients with established atherosclerotic disease.

Conclusion

This nationwide study of patients with severe psoriasis demonstrated substantial undertreatment of coronary risk factors. Increased focus on identifying cardiovascular risk factors and initiation of preventive cardiovascular pharmacotherapy in patients with psoriasis is warranted.     

Development of quantitative metabolomics for <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Accurate, reliable and reproducible measurement of intracellular metabolite levels has become important for metabolic studies of microbial cell factories. A first critical step for metabolomic studies is the establishment of an adequate quenching and washing protocol, which ensures effective arrest of all metabolic activity and removal of extracellular metabolites, without causing leakage of metabolites from the cells. Five different procedures based on cold methanol quenching and cell separation by filtration were tested for metabolomics of Pichia pastoris regarding methanol content and temperature of the quenching solution as key parameters. Quantitative evaluation of these protocols was carried out through mass balance analysis, based on metabolite measurements in all sample fractions, those are whole broth, quenched and washed cells, culture filtrate and quenching and washing solution. Finally, the optimal method was used to study the time profiles of free amino acid and central carbon metabolism intermediates in glucose-limited chemostat cultures. Acceptable recoveries (>90%) were obtained for all quenching procedures tested. However, quenching at −27°C in 60% v/v methanol performed slightly better in terms of leakage minimization. We could demonstrate that five residence times under glucose limitation are enough to reach stable intracellular metabolite pools. Moreover, when comparing P. pastoris and S. cerevisiae metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both yeasts, except for the lower glycolysis, where the levels of these metabolites in P. pastoris suggested an enzymatic capacity limitation in that part of the metabolism.     

Leakage-free rapid quenching technique for yeast metabolomics

Accurate determination of intracellular metabolite levels requires reliable, reproducible techniques for sampling and sample treatment. Quenching in 60% (v/v) methanol at −40°C is currently the standard method for sub-second arrest of metabolic activity in microbial metabolomics but there have been contradictory reports in the literature on whether leakage of metabolites from the cells occurs. We have re-evaluated this method in S. cerevisiae using a comprehensive, strictly quantitative approach. By determining the levels of a large range of metabolites in different sample fractions and establishing mass balances we could trace their fate during the quenching procedure and confirm that leakage of metabolites from yeast cells does occur during conventional cold methanol quenching, to such an extent that the levels of most metabolites have been previously underestimated by at least twofold. In addition, we found that the extent of leakage depends on the time of exposure, the temperature and the properties of the methanol solutions. Using the mass balance approach we could study the effect of different quenching conditions and demonstrate that leakage can be entirely prevented by quenching in pure methanol at ≤−40°C, which we propose as a new improved method. Making use of improved data on intracellular metabolite levels we also re-evaluated the need of sub-second quenching of metabolic activity and of removing the extracellular medium. Our findings have serious implications for quantitative metabolomics-based fields such as non-stationary ¹³C flux analysis, in vivo kinetic modeling and thermodynamic network analysis.

Non-linear viscoelastic behavior of abdominal aortic aneurysm thrombus

The objective of this work was to determine the linear and non-linear viscoelastic behavior of abdominal aortic aneurysm thrombus and to study the changes in mechanical properties throughout the thickness of the thrombus. Samples are gathered from thrombi of seven patients. Linear viscoelastic data from oscillatory shear experiments show that the change of properties throughout the thrombus is different for each thrombus. Furthermore the variations found within one thrombus are of the same order of magnitude as the variation between patients. To study the non-linear regime, stress relaxation experiments are performed. To describe the phenomena observed experimentally, a non-linear multimode model is presented. The parameters for this model are obtained by fitting this model successfully to the experiments. The model cannot only describe the average stress response for all thrombus samples but also the highest and lowest stress responses. To determine the influence on the wall stress of the behavior observed the model proposed needs to implemented in the finite element wall stress analysis.     

A prospective study of dairy consumption in relation to changes in metabolic risk factors: the Hoorn Study

Objective: Higher dairy consumption has been suggested to reduce the risk of obesity and metabolic disturbances. The aim of our study was to investigate the prospective association between dairy consumption and changes in weight and metabolic disturbances. Methods and Procedures: Baseline dairy intake (servings/day) was assessed by a semi‐quantitative food‐frequency questionnaire in 1,124 participants of the Hoorn Study. Linear and logistic regression analyses were performed to investigate the association between dairy intake and 6.4‐year change in weight, fat distribution, and metabolic risk factors (glucoses, lipids, blood pressure) and the incidence of metabolic syndrome (MS). Results: Baseline dairy consumption was not associated with changes in fasting and post‐load glucose concentrations, serum lipid levels (high‐density lipoprotein‐cholesterol, low‐density lipoprotein‐cholesterol, and triglycerides), or blood pressure, nor with the risk of developing the MS in 6.4 years (odds ratio with 95% confidence interval was 0.86 (0.52–1.42) comparing highest with lowest quartile of dairy consumption). In subjects with BMI < 25 kg/m², higher dairy consumption was significantly associated with an increase in BMI, weight, waist, and a decrease in high‐density lipoprotein. Discussion: Our results do not support the hypothesis that a higher dairy consumption protects against weight gain and development of metabolic disturbances in a Dutch elderly population.     

Ecological Risk Assessment of Tebuthiuron Following Application on Tropical Australian Wetlands

The present study assessed the ecological risks of the herbicide tebuthiuron to freshwater fauna and flora of northern Australia's tropical wetlands. Effects characterization utilized acute and chronic toxicity data of tebuthiuron to local freshwater species (three animals and two plants) as well as toxicity data derived from northern hemisphere species. Species sensitivity distributions (SSDs) for four effects scenarios—plant chronic toxicity (NOEC data), plant chronic toxicity (EC/IC₅₀ data), invertebrate and vertebrate chronic toxicity (NOEC data), and vertebrate acute toxicity (LC₅₀ data)—were used to characterize effects and calculate 10, 5, and 1% hazardous concentrations (HCs). Tebuthiuron concentrations affecting 5% of species (i.e., HC₅s) for the earlier scenarios were 0.013, 0.093, 9.0, and 97 mg L^?1, respectively. Exposure characterization involved the use of historical field monitoring data of tebuthiuron concentrations following application of tebuthiuron to a large infestation of the wetland weed Mimosa pigra (Mimosa). Tebuthiuron concentrations in surface water ranged from below detection to 2.05 mg L^?1 and were still measurable up to 10 months following application. A breakpoint regression model was fitted to the field monitoring data, providing a time-dependent estimate of exposure to tebuthiuron. Risk characterization involved the comparison of the SSDs and associated HCs for each of the effects scenarios, with the time-dependent model of tebuthiuron exposure. Modeled tebuthiuron concentrations over the first 12 days post-application were in excess of concentrations required to cause major (i.e., 50% reductions in population numbers) effects to over 85% of freshwater plant species (based on data for phytoplankton and floating macrophytes). Beyond this period and up to 300 d post-application, 10–20% of species were still predicted to be affected. To quantify the probability of prolonged effects, the plant SSDs were compared to a cumulative probability distribution of tebuthiuron measured from 70 d to 293 d post-application. The probability of at least 5% of freshwater plant species experiencing chronic effects due to tebuthiruon at ≥70 d post-application was 58% based on NOEC data and 8% based on EC/IC₅₀ data. Overlap of the 95% confidence limits of the exposure distribution and plant SSDs indicated substantial uncertainty in the risk estimates. Risks of effects to freshwater invertebrates and vertebrates were generally < 1%. It was concluded that tebuthiuron appears to represent a significant and prolonged risk to native freshwater plant species, particularly phytoplankton and floating macrophytes, whereas the risks to freshwater invertebrates and vertebrates appear low. However, from a management perspective, the risks of tebuthiuron (and other herbicides) must be weighed against the known, serious environmental and economic impacts of the target weed, Mimosa. Overall, the outcomes of the risk assessment support the various management options that have been implemented with regard to the use of tebuthiuron to control Mimosa.     

How does global change affect the strength of trophic interactions?

Recent research has generally shown that a small change in the number of species in a food web can have consequences both for community structure and ecosystem processes. However ‘change’ is not limited to just the number of species in a community, but might include an alteration to such properties as precipitation, nutrient cycling and temperature, all of which are correlated with productivity. Here we argue that predicted scenarios of global change will result in increased plant productivity. We model three scenarios of change using simple Lotka–Volterra dynamics, which explore how a global change in productivity might affect the strength of local species interactions and detail the consequences for community and ecosystem level stability. Our results indicate that (i) at local scales the average population size of consumers may decline because of poor quality food resources, (ii) that the strength of species interactions at equilibrium may become weaker because of reduced population size, and (iii) that species populations may become more variable and may take longer to recover from environmental or anthropogenic disturbances. At local scales interaction strengths encompass such properties as feeding rates and assimilation efficiencies, and encapsulate functionally important information with regard to ecosystem processes. Interaction strengths represent the pathways and transfer of energy through an ecosystem. We examine how such local patterns might be affected given various scenarios of ‘global change’ and discuss the consequences for community stability and ecosystem functioning.

Zusammenfassung

Die neuere Forschung hat im Allgemeinen gezeigt, dass eine kleine Veränderung in der Zahl der Arten in einem Nahrungsnetz sowohl Konsequenzen für die Gemeinschaftsstruktur als auch für die Ökosystemprozesse haben kann. „Wandel“ist jedoch nicht nur auf die Anzahl der Arten in einer Gemeinschaft beschränkt, sondern kann auch Veränderungen von Eigenschaften wie Niederschlag, Nährstoffkreisläufe und Temperatur beinhalten, die alle mit der Produktivität korreliert sind. Hier argumentieren wir, dass vorhergesagte Szenarios des globalen Wandels in einer erhöhten Pflanzenproduktivität resultieren werden. Wir modellieren drei Szenarien des Wandels unter der Verwendung einfacher Lotka–Volterra–Dynamiken, die erkunden, wie ein globaler Wandel in der Produktivität die Stärke von lokalen Arteninteraktionen beeinflusst, und wir beschreiben detailliert die Konsequenzen für die Gemeinschaft und die Stabilität des Ökosystemlevels. Unsere Ergebnisse deuten darauf hin, (i) dass auf einer lokalen Skala die durchschnittliche Populationsgröße der Konsumenten aufgrund der geringen Qualität der Nahrungsressourcen abnehmen könnte, (ii) dass die Stärke der Arteninteraktionen im Gleichgewicht aufgrund der reduzierten Populationsgröße schwächer werden könnte und (iii) dass die Populationen der Arten variabler werden und länger brauchen könnten, um sich von umweltbedingten oder anthropogenen Störungen zu erholen. Auf lokalen Skalen umfassen Interaktionsstärken Eigenschaften wie Fraßraten und Assimilationseffizienzen und enthalten wichtige funktionelle Information in Bezug auf Ökosystemprozesse. Interaktionsstärken repräsentieren die Energiewege und den Energietransfer in einem Ökosystem. Wir untersuchen, wie solche lokalen Muster unter der Voraussetzung verschiedener gegebener Szenarios des „globalen Wandels“beeinflusst werden könnten und diskutieren die Konsequenzen für die Gemeinschaftsstabilität und Ökosystemfunktion.