Validation of putative biomarkers of furan exposure through quantitative analysis of furan metabolites in urine of F344 rats exposed to stable isotope labeled furan.

Humans are chronically exposed to furan, a potent liver toxicant and carcinogen that occurs in a variety of heat-processed foods. Assessment of human exposure based on the furan content in foods is, however, subject to some uncertainty due to the high volatility of furan. Biomarker monitoring is thus considered an alternative or complementary approach to furan exposure assessment. Previous work suggested that urinary furan metabolites derived from the reaction of cis-2-butene-1,4-dial (BDA), the reactive intermediate of furan, with glutathione (GSH) or amino acids may serve as potential biomarkers of furan exposure. However, some metabolites were also reported to occur in urine of untreated animals, indicating either background contamination via animal feed or endogenous sources, which may limit their suitability as biomarkers of exposure. The overall aim of the present study was to accurately establish the correlation between external dose and concentration of furan metabolites in urine over time and to discriminate against endogenous formation and furan intake via feed. To this end, the furan metabolites GSH-BDA (N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine), NAcLys-BDA (R-2-(acetylamino)-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid), NAcCys-BDA-NAcLys (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine) and NAcCys-BDA-NAcLys sulfoxide (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine sulfoxide) were simultaneously analyzed by stable isotope dilution ESI-LC-MS/MS as unlabeled and [13C4]-furan dependent metabolites following oral administration of a single oral dose of isotopically labelled [13C4]-furan (0.1, 1, 10, 100 and 1000 µg/kg bw) to male and female F344/DuCrl rats. Although a linear correlation between urinary excretion of [13C4]-furan-dependent metabolites was observed, analysis of unlabeled NAcLys-BDA, NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide revealed substantial, fairly constant urinary background levels throughout the course of the study. Analysis of furan in animal feed excluded feed as a source for these background levels. GSH-BDA was identified as the only furan metabolite without background occurrence, suggesting that it may present a specific biomarker to monitor external furan exposure. Studies in humans are now needed to establish if analysis of urinary GSH-BDA may provide reliable exposure estimates.

Development of a hardened THz energy meter for use on the kilojoule-scale, short-pulse OMEGA EP laser.

A highly adaptable and robust terahertz (THz) energy meter is designed and implemented to detect energetic THz pulses from high-intensity (>1018 W/cm2) laser-plasma interactions on the OMEGA EP. THz radiation from the laser driven target is detected by a shielded pyrometer. A second identical pyrometer is used for background subtraction. The detector can be configured to detect THz pulses in the 1 mm to 30 µm (0.3- to 10-THz) range and pulse energies from joules to microjoules via changes in filtration, aperture size, and position. Additional polarization selective filtration can also be used to determine the THz pulse polarization. The design incorporates significant radiation and electromagnetic pulse shielding to survive and operate within the OMEGA EP radiation environment. We describe the design, operational principle, calibration, and testing of the THz energy meter. The pyrometers were calibrated using a benchtop laser and show linear sensitivity to up to 1000 nJ of absorbed energy. The initial results from four OMEGA EP THz experiments detected up to ∼15µJ at the detector, which can correspond to hundreds of mJ depending on THz emission and reflection models.

Demonstration of plasma mirror capability for the OMEGA Extended Performance laser system.

A plasma mirror platform was developed for the OMEGA-EP facility to redirect beams, thus enabling more flexible experimental configurations as well as a platform that can be used in the future to improve laser contrast. The plasma mirror reflected a short pulse focusing beam at 22.5° angle of incidence onto a 12.5 µm thick Cu foil, generating Bremsstrahlung and kα x rays, and accelerating ions and relativistic electrons. By measuring these secondary sources, the plasma mirror key performance metrics of integrated reflectivity and optical quality are inferred. It is shown that for a 5 ± 2 ps, 310 J laser pulse, the plasma mirror integrated reflectivity was 62 ± 13% at an operating fluence of 1670 J cm-2, and that the resultant short pulse driven particle acceleration and x-ray generation indicate that the on target intensity was 3.1 × 1018 W cm-2, which is indicative of a good post-plasma mirror interaction beam optical quality. By deriving the plasma mirror performance metrics from the secondary source scalings, it was simultaneously demonstrated that the plasma mirror is ready for adoption in short pulse particle acceleration and high energy photon generation experiments using the OMEGA-EP system.

Within-herd prevalence threshold for the detection of Mycobacterium avium ssp. paratuberculosis antibody-positive dairy herds using pooled milk samples: A field study.

Herd-level diagnosis of paratuberculosis using a pool-milk ELISA (pool size: n ≤ 50) is a novel, economical, and convenient method to identify blood serological Mycobacterium avium ssp. paratuberculosis (MAP) antibody-positive herds. To date, the diagnostic performance of the pool-milk ELISA has been described only under laboratory conditions where herd prevalence was simulated by the preparation of milk pools consisting of milk samples of cows with a known MAP status determined by fecal culture. In our observational study, test performance under field conditions was studied using pooled milk and individual blood samples. A total of 486 herds within the MAP prevalence reduction program of Lower Saxony, from which pooled milk and individual blood ELISA results were available, were assigned to this study. Data were analyzed for the period between January 1 and December 31, 2018, the first year after herd testing became obligatory in this federal state of Germany. To evaluate whether pooled milk samples reliably distinguish between herds with a MAP-apparent blood serological within-herd prevalence (MAP-Ab-WHPapp) ≥5% and herds with a MAP-Ab-WHPapp <5%, the distribution of the MAP-Ab-WHPapp was compared between pool-positive and pool-negative herds. The MAP-Ab-WHPapp was 3.4% (median; 95% confidence interval = 0-11.4%) in pool-positive herds and 1.2% (median; 95% confidence interval = 0-6.4%) in pool-negative herds. Only 10.8% (n = 12) of the pool sample-negative herds had a MAP-Ab-WHPapp ≥5% and were therefore false negatives, given the aims of the MAP prevalence reduction program. Hence, the pool-milk sampling strategy seems well suited to distinguish between herds with a MAP-Ab-WHPapp ≥ 5% and herds with a MAP-Ab-WHPapp <5% since only 10% of serum MAP-ELISA positive herds were missed. Employing a logistic regression model, we estimated that the minimum blood serological MAP-Ab-WHPapp to detect a pool-positive herd with a probability of 95% was 8%, which fits well with the aim of the MAP prevalence reduction program to focus on herds with a MAP-Ab-WHPapp of ≥5%. Despite the limitations of the control approach, which include milk pool sample collection and a low sensitivity of the ELISA used in milk pools and serum samples, the aims of the MAP prevalence reduction program can be achieved. The results of these field data support that pool-milk sample ELISA is a useful, economical, and low labor-intensive tool to identify herds seropositive for MAP in a MAP prevalence reduction program.

Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates / Otimização de um método baseado em PCR em tempo real para rastreamento de bactérias em concentrados de plaquetas

Abstract Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.

Resumo A contaminação bacteriana dos componentes sanguíneos é um grande desafio na medicina transfusional, principalmente nos concentrados de plaquetas (PCs) devido às condições de armazenamento que favorecem a proliferação bacteriana. Neste estudo, desenvolvemos um protocolo de PCR em tempo real rápido, sensível e específico para a triagem bacteriana de PCs. Um método baseado em PCR em tempo real, controlado internamente, foi otimizado e validado com um Master Mix Universal PCR 16S (IBMP / Fiocruz), que detecta uma região conservada do gene 16S rRNA bacteriano. O background de DNA não específico foi completamente eliminado tratando a PCR Master Mix com monoazida de etídio (EMA). O limite de detecção inferior observado foi de 10 cópias equivalentes do genoma com um valor de Ct 34 ± 1,07, a curva de calibração foi gerada com diluições seriada de 10 vezes do DNA de E. coli. O tempo de processamento, incluindo a purificação microbiana do DNA, foi de aproximadamente 4 horas. O método desenvolvido mostrou alta sensibilidade sem amplificação inespecífica e menor tempo de detecção do que os métodos microbiológicos tradicionais, demonstrando ser um meio eficiente de triagem de PCs pré-transfusionais.

Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates.

Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.

Farm-specific failure costs of production disorders in European organic dairy herds.

On-farm decision support in animal health management requires a tailor-made failure costs (FCs) assessment of production disorders for the individual farm. In our study we defined a generic framework to estimate the FC of production disorders in dairy cows. We converted the framework to a practical tool in which the farm-specific FC of mastitis, ketosis, lameness and metritis were estimated for 162 organic dairy farms in four European countries. Along with the structure of the framework, the FC estimation required three distinct types of model input: performance input (related to herd performance parameters), consequential input (related to the consequences of the disorders) and economic input (related to price levels). Input was derived from official herd recordings (e.g. test-day records and animal health recordings) and farmers' responses (e.g. questionnaire replies). The average FC of mastitis, ketosis, lameness and metritis amounted to € 96, € 21, € 43 and € 10 per cow per year, respectively. The variation in FC outcomes was high among farmers and countries. Overall ranking of the disorders based on absolute values was the same for all countries, with mastitis being the costliest disorder followed in order by lameness, ketosis, and metritis. Farm specific estimates can be used to rank production related disorders in terms of their associated failure costs and thus provide valuable insights for herd health management. The practical calculation tool developed in this study should be considered by farmers or herd health advisors to support their animal health practices or advice.

The dependence of interplay effects on the field scan direction in PBS proton therapy.

The literature is controversial about the scan direction dependency of interplay effects in pencil beam scanning (PBS) treatment of moving targets. A directional effect is supported by many simulation studies, whereas the experimental data are mostly limited to simple geometries, not reflecting realistically clinical treatment plans. We have compared increasingly complex treatment fields, from a homogeneous single energy layer to a more modulated lung plan, under identical experimental settings, seeking evidence for differences in motion mitigation due to the selection of primary scanning direction. In total, 120 experimental samples were taken, combining two primary scan directions and three rescanning regimes with different motion scenarios. 4D dose distributions were measured in water with a moving ionisation chamber array and compared to those of a stationary delivery using 2D gamma analysis. Each plan has been verified twice for the same rescanning regime and motion scenario, changing the meandering direction in between to scan perpendicularly to, or along, the target motion. Additionally, machine log files of the lung plan, together with 4DCT data, were used to calculate the dose distribution that such deliveries would have produced in the patient. The primary meandering direction has a clear influence on measured dose distributions when considering a single energy layer. Introducing spot weight modulation and multiple energy layers however, makes the dynamic of interplay more complex and difficult to predict. Overall, gamma (3%/3 mm) differences between scanning along or orthogonal to the target motion follow a normal distribution [Formula: see text] when considering multiple motion scenarios and rescanning regimes. Nevertheless, data spread [Formula: see text] is significant enough such that, for individual experiments and set-ups, a dependency may be observed even if this is not a general result. Patient reconstructed doses follow the same trend, the two primary scan directions producing statistically insignificant differences in dose distributions in terms of conformity or homogeneity. Except for extremely simplified cases of mono-energetic and homogeneous treatment fields, the interplay effect has been found to be only marginally influenced by the choice of the primary scanning direction.

Effects of a participatory approach, with systematic impact matrix analysis in herd health planning in organic dairy cattle herds.

The animal health and welfare status in European organic dairy production does not in all aspects meet the organic principles and consumers' expectations and needs to be improved. To achieve this, tailored herd health planning, targeted to the specific situation of individual farms could be of use. The aim of this study was to apply herd health planning in a structured participatory approach, with impact matrix analysis, not previously used in this context, in European organic dairy farms and to assess changes in animal health and welfare. Herd health planning farm visits were conducted on 122 organic dairy farms in France, Germany and Sweden. The farmer, the herd veterinarian and/or an advisor took part in the farm discussions. The researcher served as facilitator. Baseline data on the animal health status of the individual farm, collected from national milk recording schemes, were presented as an input for the discussion. Thereafter a systematic impact matrix analysis was performed. This was to capture the complexity of individual farms with the aim to identify the farm-specific factors that could have a strong impact on animal health. The participants (i.e. farmer, veterinarian and advisor) jointly identified areas in need of improvement, taking the health status and the interconnected farm system components into account, and appropriate actions were jointly identified. The researcher took minutes during the discussions, and these were shared with the participants. No intervention was made by the researcher, and further actions were left with the participants. The number of actions per farm ranged from 0 to 22. The change in mortality, metabolic diseases, reproductive performance and udder health was assessed at two time points, and potential determinators of the change were evaluated with linear regression models. A significant association was seen between change in udder health, as measured by the somatic cell count, and country. At the first follow-up, a significant association was also found between change in the proportion of prolonged calving interval and the farmers' desire to improve reproductive health as well as with an increase in herd size, but this was not seen at the second follow-up. The degree of implementation of the actions was good (median 67%, lower quartile 40%, upper quartile 83%). To conclude, the degree of implementation was quite high, improvement of animal health could not be linked to the herd health planning approach. However, the approach was highly appreciated by the participants and deserves further study.

Portable Pocket colposcopy performs comparably to standard-of-care clinical colposcopy using acetic acid and Lugol's iodine as contrast mediators: an investigational study in Peru.

OBJECTIVE: Our goal was to develop a tele-colposcopy platform for primary-care clinics to improve screening sensitivity and access. Specifically, we developed a low-cost, portable Pocket colposcope and evaluated its performance in a tertiary healthcare centre in Peru. DESIGN AND SETTING: Images of the cervix were captured with a standard-of-care and Pocket colposcope at la Liga Contra el Cáncer in Lima, Peru. POPULATION: Two hundred Peruvian women with abnormal cytology and/or human papillomavirus positivity were enrolled. METHODS: Images were collected using acetic acid and Lugol's iodine as contrast agents. Biopsies were taken as per standard-of-care procedures. MAIN OUTCOME MEASURES: After passing quality review, images from 129 women were sent to four physicians who provided a diagnosis for each image. RESULTS: Physician interpretation of images from the two colposcopes agreed 83.1% of the time. The average sensitivity and specificity of physician interpretation compared with pathology was similar for the Pocket (sensitivity = 71.2%, specificity = 57.5%) and standard-of-care (sensitivity = 79.8%, specificity = 56.6%) colposcopes. When compared with a previous study where only acetic acid was applied to the cervix, results indicated that adding Lugol's iodine as a secondary contrast agent improved the percent agreement between colposcopes for all pathological categories by up to 8.9% and the sensitivity and specificity of physician interpretation compared with pathology by over 6.0 and 9.0%, respectively. CONCLUSIONS: The Pocket colposcope performance was similar to that of a standard-of-care colposcope when used to identify precancerous and cancerous lesions using acetic acid and Lugol's iodine during colposcopy examinations in Peru. TWEETABLE ABSTRACT: The Pocket colposcope performance was similar to that of a standard-of-care colposcope when identifying cervical lesions.

Detection of IgG3 antibodies specific to the human immunodeficiency virus type 1 (HIV-1) p24 protein as marker for recently acquired infection.

Reducing the risk of human immunodeficiency virus type 1 (HIV-1) transmission is still a public health priority. The development of effective control strategies relies on the quantification of the effects of prophylactic and therapeutic measures in disease incidence. Although several assays can be used to estimate HIV incidence, these estimates are limited by the poor performance of these assays in distinguishing recent from long-standing infections. To address such limitation, we have developed an assay to titrate p24-specific IgG3 antibodies as a marker of recent infection. The assay is based on a recombinant p24 protein capable to detect total IgG antibodies in sera using a liquid micro array and enzyme-linked immunosorbent assay. Subsequently, the assay was optimised to detect and titrate anti-p24 IgG3 responses in a panel of sequential specimens from seroconverters over 24 months. The kinetics of p24-specific IgG3 titres revealed a transient peak in the 4 to 5-month period after seroconversion. It was followed by a sharp decline, allowing infections with less than 6 months to be distinguished from older ones. The developed assay exhibited a mean duration of recent infection of 144 days and a false-recent rate of ca. 14%. Our findings show that HIV-1 p24-specific IgG3 titres can be used as a tool to evaluate HIV incidence in serosurveys and to monitor the efficacy of vaccines and other transmission control strategies.

Graph-based impact analysis as a framework for incorporating practitioner knowledge in dairy herd health management.

Production diseases in dairy cows are multifactorial, which means they emerge from complex interactions between many different farm variables. Variables with a large impact on production diseases can be identified for groups of farms using statistical models, but these methods cannot be used to identify highly influential variables in individual farms. This, however, is necessary for herd health planning, because farm conditions and associated health problems vary largely between farms. The aim of this study was to rank variables according to their anticipated effect on production diseases on the farm level by applying a graph-based impact analysis on 192 European organic dairy farms. Direct impacts between 13 pre-defined variables were estimated for each farm during a round-table discussion attended by practitioners, that is farmer, veterinarian and herd advisor. Indirect impacts were elaborated through graph analysis taking into account impact strengths. Across farms, factors supposedly exerting the most influence on production diseases were 'feeding', 'hygiene' and 'treatment' (direct impacts), as well as 'knowledge and skills' and 'herd health monitoring' (indirect impacts). Factors strongly influenced by production diseases were 'milk performance', 'financial resources' and 'labour capacity' (directly and indirectly). Ranking of variables on the farm level revealed considerable differences between farms in terms of their most influential and most influenced farm factors. Consequently, very different strategies may be required to reduce production diseases in these farms. The method is based on perceptions and estimations and thus prone to errors. From our point of view, however, this weakness is clearly outweighed by the ability to assess and to analyse farm-specific relationships and thus to complement general knowledge with contextual knowledge. Therefore, we conclude that graph-based impact analysis represents a promising decision support tool for herd health planning. The next steps include testing the method using more specific and problem-oriented variables as well as evaluating its effectiveness.

Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates

Abstract Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.

Resumo A contaminação bacteriana dos componentes sanguíneos é um grande desafio na medicina transfusional, principalmente nos concentrados de plaquetas (PCs) devido às condições de armazenamento que favorecem a proliferação bacteriana. Neste estudo, desenvolvemos um protocolo de PCR em tempo real rápido, sensível e específico para a triagem bacteriana de PCs. Um método baseado em PCR em tempo real, controlado internamente, foi otimizado e validado com um Master Mix Universal PCR 16S (IBMP / Fiocruz), que detecta uma região conservada do gene 16S rRNA bacteriano. O background de DNA não específico foi completamente eliminado tratando a PCR Master Mix com monoazida de etídio (EMA). O limite de detecção inferior observado foi de 10 cópias equivalentes do genoma com um valor de Ct 34 ± 1,07, a curva de calibração foi gerada com diluições seriada de 10 vezes do DNA de E. coli. O tempo de processamento, incluindo a purificação microbiana do DNA, foi de aproximadamente 4 horas. O método desenvolvido mostrou alta sensibilidade sem amplificação inespecífica e menor tempo de detecção do que os métodos microbiológicos tradicionais, demonstrando ser um meio eficiente de triagem de PCs pré-transfusionais.

Assessing, and understanding, European organic dairy farmers' intentions to improve herd health.

Many believe the health status of organic dairy herds in Europe should be improved to meet consumers' and legislators' expectations to improve animal welfare. This paper reports on a study in four countries that examined dairy farmers' intentions towards improving the health status of their organic herds through the use of the Theory of Planned Behaviour. It was found that farmers across the countries were positive about taking additional preventative measures to improve the health status of their herds. They believed this would not only improve herd physical performance, such as milk yield and fertility, but also achieve greater cost effectiveness and improved job satisfaction for them. Most study farmers would implement a tailored package of improvement measures designed by the study team with higher uptake most likely being by younger farmers, those who make greater use of veterinarians and professional advisory services, and those supplying specialist milk-marketing chains. Furthermore, farmers will be most likely to take-up additional health promotion if compatible with their everyday activities and if they have strong business performance goals aimed at maximising the physical performance of the herd.

Electrospinning induced surface activation (EISA) of highly porous PMMA microfiber mats for HIV diagnosis.

This work presents an Electrospray Induced Surface Activation (EISA) method generalization for electrospinning. It allows an easy way to produce surface functionalized microfiber mats for infectious disease diagnostic purposes. We present the details of both the production and characterization of surface functionalized highly porous poly methyl methacrylate (PMMA) microfiber mats produced using dry (DS) and wet substrate (WS) configurations. The characterization was performed using high-resolution scanning electron microscopy (HRSEM), Size Exclusion Chromatography (SEC), X-ray photoelectron spectroscopy (XPS) and biological essays attaching both the recombinant auto-fluorescent green fluorescent protein (GFP) and the anti-human Ig protein containing a fluorescent reporter R-phycoerythrin (AbPE). The final biological application assay was performed by positively detecting HIV contaminated human samples.

Gateless patterning of epitaxial graphene by local intercalation.

We present a technique to pattern the charge density of a large-area epitaxial graphene sheet locally without using metallic gates. Instead, local intercalation of the graphene-substrate interface can selectively be established in the vicinity of graphene edges or predefined voids. It provides changes of the work function of several hundred meV, corresponding to a conversion from n-type to p-type charge carriers. This assignment is supported by photoelectron spectroscopy, scanning tunneling microscopy, scanning electron microscopy and Hall effect measurements. The technique introduces materials contrast to a graphene sheet in a variety of geometries and thus allows for novel experiments and novel functionalities.

Proteomic analysis reveals differentially expressed proteins in macrophages infected with Leishmania amazonensis or Leishmania major.

CBA macrophages effectively control Leishmania major infection, yet are permissive to Leishmania amazonensis. Employing a transcriptomic approach, we previously showed the up-regulation of the genes involved in the classical pathway of macrophage activation in resistant mice. However, microarray analyses do not evaluate changes in gene expression that occur after translation. To circumvent this analytical limitation, we employed a proteomics approach to increase our understanding of the modulations that occur during infection and identify novel targets for the control of Leishmania infection. To identify proteins whose expression changes in CBA macrophages infected with L. major or L. amazonensis, protein extracts were obtained and digested and the peptides were characterized using multi-dimensional liquid chromatography coupled with tandem mass spectrometry analyses. A total of 162 proteins were selected as potentially modulated. Using biological network analyses, these proteins were classified as primarily involved in cellular metabolism and grouped into cellular development biological networks. This study is the first to use a proteomics approach to describe the protein modulations involved in cellular metabolism during the initial events of Leishmania-macrophage interaction. Based on these findings, we hypothesize that these differentially expressed proteins likely play a pivotal role in determining the course of infection.

Tailoring the graphene/silicon carbide interface for monolithic wafer-scale electronics.

Graphene is an outstanding electronic material, predicted to have a role in post-silicon electronics. However, owing to the absence of an electronic bandgap, graphene switching devices with high on/off ratio are still lacking. Here in the search for a comprehensive concept for wafer-scale graphene electronics, we present a monolithic transistor that uses the entire material system epitaxial graphene on silicon carbide (0001). This system consists of the graphene layer with its vanishing energy gap, the underlying semiconductor and their common interface. The graphene/semiconductor interfaces are tailor-made for ohmic as well as for Schottky contacts side-by-side on the same chip. We demonstrate normally on and normally off operation of a single transistor with on/off ratios exceeding 10(4) and no damping at megahertz frequencies. In its simplest realization, the fabrication process requires only one lithography step to build transistors, diodes, resistors and eventually integrated circuits without the need of metallic interconnects.

Involvement of host cell heparan sulfate proteoglycan in Trypanosoma cruzi amastigote attachment and invasion.

Cell surface glycosaminoglycans (GAGs) play an important role in the attachment and invasion process of a variety of intracellular pathogens. We have previously demonstrated that heparan sulfate proteoglycans (HSPG) mediate the invasion of trypomastigote forms of Trypanosoma cruzi in cardiomyocytes. Herein, we analysed whether GAGs are also implicated in amastigote invasion. Competition assays with soluble GAGs revealed that treatment of T. cruzi amastigotes with heparin and heparan sulfate leads to a reduction in the infection ratio, achieving 82% and 65% inhibition of invasion, respectively. Other sulfated GAGs, such as chondroitin sulfate, dermatan sulfate and keratan sulfate, had no effect on the invasion process. In addition, a significant decrease in infection occurred after interaction of amastigotes with GAG-deficient Chinese Hamster Ovary (CHO) cells, decreasing from 20% and 28% in wild-type CHO cells to 5% and 9% in the mutant cells after 2 h and 4 h of infection, respectively. These findings suggest that amastigote invasion also involves host cell surface heparan sulfate proteoglycans. The knowledge of the mechanism triggered by heparan sulfate-binding T. cruzi proteins may provide new potential candidates for Chagas disease therapy.