The interplay between transport and metabolism in fungal itaconic acid production.

Besides enzymatic conversions, many eukaryotic metabolic pathways also involve transport proteins that shuttle molecules between subcellular compartments, or into the extracellular space. Fungal itaconate production involves two such transport steps, involving an itaconate transport protein (Itp), and a mitochondrial tricarboxylate transporter (Mtt). The filamentous ascomycete Aspergillus terreus and the unicellular basidiomycete Ustilago maydis both produce itaconate, but do so via very different molecular pathways, and under very different cultivation conditions. In contrast, the transport proteins of these two strains are assumed to have a similar function. This study aims to investigate the roles of both the extracellular and mitochondrial transporters from these two organisms by expressing them in the corresponding U. maydis knockouts and monitoring the extracellular product concentrations. Both transporters from A. terreus complemented their corresponding U. maydis knockouts in mediating itaconate production. Surprisingly, complementation with At_MfsA from A. terreus led to a partial switch from itaconate to (S)-2-hydroxyparaconate secretion. Apparently, the export protein from A. terreus has a higher affinity for (S)-2-hydroxyparaconate than for itaconate, even though this species is classically regarded as an itaconate producer. Complementation with At_MttA increased itaconate production by 2.3-fold compared to complementation with Um_Mtt1, indicating that the mitochondrial carrier from A. terreus supports a higher metabolic flux of itaconic acid precursors than its U. maydis counterpart. The biochemical implications of these differences are discussed in the context of the biotechnological application in U. maydis and A. terreus for the production of itaconate and (S)-2-hydroxyparaconate.

Current climate for digital game-based learning of science in further and higher education.

Digital game-based learning (DGBL) is being used increasingly as an alternative learning tool to teach science in further and higher education. A variety of digital game formats currently exist for science learning, alongside diverse methods for their implementation and evaluation. This paper aims to provide a broad summary of the field by discussing the current platforms for DGBL and examples of games played on them. These include gamified simulations and traditional digital games delivered through personal computer and online software; mobile games delivered through downloaded applications for devices such as tablets and mobile phones; and educational modifications of commercial games, known amongst gamers as 'mods'. To conclude the summary, the paper discusses the current challenges and barriers associated with DGBL in further and higher science education, and potential strategies researchers may consider to overcome them.

Online in vivo monitoring of cytosolic NAD redox dynamics in Ustilago maydis.

Maintenance of metabolic redox homeostasis is essential to all life and is a key factor in many biotechnological processes. Changes in the redox state of NAD affect metabolic fluxes, mediate regulation and signal transduction, and thus determine growth and productivity. Here we establish an in vivo monitoring system for the dynamics of the cytosolic NADH/NAD+ ratio in the basidiomycete Ustilago maydis using the ratiometric fluorescent sensor protein Peredox-mCherry. Metabolic redox dynamics were determined in the cytosol of living cells with high time resolution under biotechnologically relevant conditions, i.e. with high cell density and high aeration. Analytical boundary conditions for reliable analysis were determined, and perturbations in C-, N- or O- availability had marked impact on the cytosolic NADH/NAD+ ratio. NAD redox dynamics could be manipulated in lines inducibly expressing a water-forming NADH oxidase as a synthetic reductant sink. The establishment of Peredox-mCherry in U. maydis and the analysis of NAD redox dynamics provides a versatile methodology for the in vivo investigation of cellular metabolism, and contributes fundamental knowledge for rational design and optimization of biocatalysts.

Promoters from the itaconate cluster of Ustilago maydis are induced by nitrogen depletion.

BACKGROUND: Ustilago maydis is known for its natural potential to produce a broad range of valuable chemicals, such as itaconate, from both industrial carbon waste streams and renewable biomass. Production of itaconate, and many other secondary metabolites, is induced by nitrogen limitation in U. maydis. The clustered genes responsible for itaconate production have recently been identified, enabling the development of new expression tools that are compatible with biotechnological processes. RESULTS: Here we report on the investigation of two of the native promoters, P tad1 and P mtt1 , from the itaconate cluster of U. maydis MB215. For both promoters the specific activation upon nitrogen limitation, which is known to be the trigger for itaconate production in Ustilago, could be demonstrated by gfp expression. The promoters cover a broad range of expression levels, especially when combined with the possibility to create single- and multicopy construct integration events. In addition, these reporter constructs enable a functional characterization of gene induction patterns associated with itaconate production. CONCLUSIONS: The promoters are well suited to induce gene expression in response to nitrogen limitation, coupled to the itaconate production phase, which contributes towards the further improvement of organic acid production with Ustilago.

Interaction of Influenza A Viruses with Oviduct Explants of Different Avian Species.

Infection of poultry with low pathogenic avian influenza viruses (LPAIV) is often associated with mild respiratory symptoms but may also lead to loss in egg production in laying birds. In vivo susceptibility of the reproductive tract for LPAIV infection was reported for turkeys and chickens, but virus-interaction with epithelial cells of the oviduct and possible stimulation of the local antiviral immune responses have not been characterized. In this study, we wanted to investigate the suitability of magnum organ cultures (MOC) as an in vitro model to study virus-host interactions. We compared the susceptibility of duck (Du), chicken (Ch), and turkey (Tu) MOC for three different influenza A viruses (IAV). Overall, the course of infection and the antiviral immune response varied between strains as well as host cell origin, but MOC gave reproducible results for all investigated parameters within each species. While pandemic (p) H1N1 and H9N2 efficiently replicated in MOC-Ch and MOC-Tu, MOC-Du were significantly less susceptible to infection as indicated by a reduced replication level for both viruses (p < 0.05). Overall, virus replication levels did not correlate with interferonα (IFNα) mRNA-expression levels in neither species. H9N2-infection led to a significant upregulation of interferonλ (IFNλ) mRNA expression in MOC of all species compared to the non-infected controls (p < 0.05), while a correlation with replication levels was only seen for MOC-Tu. pH1N1-infection induced only significant upregulation of IFNλ mRNA expression in MOC-Tu at 48 hours post infection (p < 0.05), but the expression pattern did not correlate with replication levels. Our results show that MOC are a suitable model to study IAV-interaction with the mucosal surface of the avian reproductive tract. The data suggest that the reproductive tract may play a role in the pathobiology of IAV in poultry.

Mycoplasma gallisepticum modifies the pathogenesis of influenza A virus in the avian tracheal epithelium.

Multiple respiratory infections have a significant impact on health and economy. Pathogenesis of co-infecting viruses and bacteria and their interaction with mucosal surfaces are poorly characterized. In this study we established a co-infection model based on pre-incubation of tracheal organ cultures (TOC) with Mycoplasma (M.) gallisepticum and a subsequent infection with avian influenza virus (AIV). Mycoplasma gallisepticum modified the pathogenesis of AIV as demonstrated in TOC of two different avian species (chickens and turkeys). Co-infection promoted bacterial growth in tracheal epithelium. Depending on the interaction time of M. gallisepticum with the host cells, AIV replication was either promoted or suppressed. M. gallisepticum inhibited the antiviral gene expression and affected AIV attachment to the host cell by desialylation of α-2,3 linked sialic acids. Ultrastructural analysis of co-infected TOC suggests that both pathogens may attach to and possibly infect the same epithelial cell. The obtained results contribute to better understanding of the interaction dynamics between M. gallisepticum and AIV. They highlight the importance of the time interval between infections as well as the biological properties of the involved pathogens as influencing factors in the outcome of respiratory infections.

Avian metapneumovirus infection of chicken and turkey tracheal organ cultures: comparison of virus-host interactions.

Avian metapneumovirus (aMPV) is a pathogen with worldwide distribution, which can cause high economic losses in infected poultry. aMPV mainly causes infection of the upper respiratory tract in both chickens and turkeys, although turkeys seem to be more susceptible. Little is known about virus-host interactions at epithelial surfaces after aMPV infection. Tracheal organ cultures (TOC) are a suitable model to investigate virus-host interaction in the respiratory epithelium. Therefore, we investigated virus replication rates and lesion development in chicken and turkey TOC after infection with a virulent aMPV subtype A strain. Aspects of the innate immune response, such as interferon-α and inducible nitric oxide synthase mRNA expression, as well as virus-induced apoptosis were determined. The aMPV-replication rate was higher in turkey (TTOC) compared to chicken TOC (CTOC) (P < 0.05), providing circumstantial evidence that indeed turkeys may be more susceptible. The interferon-α response was down-regulated from 2 to 144 hours post infection in both species compared to virus-free controls (P < 0.05); this was more significant for CTOC than TTOC. Inducible nitric oxide synthase expression was significantly up-regulated in aMPV-A-infected TTOC and CTOC compared to virus-free controls (P < 0.05). However, the results suggest that NO may play a different role in aMPV pathogenesis between turkeys and chickens as indicated by differences in apoptosis rate and lesion development between species. Overall, our study reveals differences in innate immune response regulation and therefore may explain differences in aMPV - A replication rates between infected TTOC and CTOC, which subsequently lead to more severe clinical signs and a higher rate of secondary infections in turkeys.

Development of stable isotope dilution assays for the quantitation of Amadori compounds in foods.

During thermal processing of foods, reducing carbohydrates and amino acids may form 1-amino-1-desoxyketoses named Amadori rearrangement products after the Italian chemist Mario Amadori. Although these compounds are transient intermediates of the Maillard reaction, they are often used as suitable markers to measure the extent of a thermal food processing, such as for spray-dried milk or dried fruits. Several methods are already available in the literature for their quantitation, but measurements are often done with external calibration without addressing losses during the workup procedure. To cope with this challenge, stable isotope dilution assays in combination with LC-MS/MS were developed for the glucose-derived Amadori products of the seven amino acids valine, leucine, isoleucine, phenylalanine, tyrosine, methionine, and histidine using the respective synthesized [(13)C6]-labeled isotopologues as internal standards. The quantitation of the analytes added to a model matrix showed a very good sensitivity with the lowest limits of detection for the Amadori compound of phenylalanine of 0.1 µg/kg starch and 0.2 µg/kg oil, respectively. Also, the standard deviation measured in, for example, wheat beer was only ±2% for this analyte. Application of the method to several foods showed the highest concentrations of the Amadori product of valine in unroasted cocoa (342 mg/kg) as well as in dried bell pepper (3460 mg/kg). In agreement with literature data, drying of foods led to the formation of Amadori products, whereas they were degraded during roasting of, for example, coffee or cocoa. The study presents for the first time results on concentrations of the Amadori compounds of tyrosine and histidine in foods.

A nipple shield delivery system for oral drug delivery to breastfeeding infants: microbicide delivery to inactivate HIV.

A new drug delivery method for infants is presented which incorporates an active pharmaceutical ingredient (API)-loaded insert into a nipple shield delivery system (NSDS). The API is released directly into milk during breastfeeding. This study investigates the feasibility of using the NSDS to deliver the microbicide sodium dodecyl sulfate (SDS), with the goal of preventing mother-to-child transmission (MTCT) of HIV during breastfeeding in low-resource settings, when there is no safer alternative for the infant but to breastfeed. SDS has been previously shown to effectively inactivate HIV in human milk. An apparatus was developed to simulate milk flow through and drug release from a NSDS. Using this apparatus milk was pulsed through a prototype device containing a non-woven fiber insert impregnated with SDS and the microbicide was rapidly released. The total SDS release from inserts ranged from 70 to 100% of the average 0.07 g load within 50 ml (the volume of a typical breastfeed). Human milk spiked with H9/HIV(IIIB) cells was also passed through the same set-up. Greater than 99% reduction of cell-associated HIV infectivity was achieved in the first 10 ml of milk. This proof of concept study demonstrates efficient drug delivery to breastfeeding infants is achievable using the NSDS.

Do locked doors in psychiatric hospitals preventpatients from absconding?

Background and Objectives: In the acute treatment of acute psychiatric patients coercive measures are often required and therapeutic relationships can be affected by such measures. In this study we assessed whether opening the entrance door of an acutepsychiatric ward influences absconding behaviour. Methods: An acute psychiatric ward was primarily closed (91.4%) for six months andprimarily open (75.6%) for six months over the time period of one year. In this one year period, 337 patients were treated (206 male, age: 40 ± 16 years): 60.2% of the patients hadschizophrenia, 13.6% had affective disorders, 11.6% suffered from addiction and 14.5%displayed other diagnoses. Results: In terms of age (t = 0.026, df = 335, p = 0.979), gender (chi2 = 1.6, df = 1, p = 0.13), diagnoses(chi2 = 7.337, df = 1, p = 0.062) and duration of stay (t = -0.90, df = 335, p = 0.928),we found no significant differences between the patients admitted in the closed and those admittedin the open ward period. Absconding (df = 1, chi2 = 5.107, p = 0.029), aggressive incidents(chi2 = 4.46, df = 1, p = 0.050) and coercive medications (chi2 = 4.646, df = 1, p =0.037) were observed significantly more often in the closed door period. Moreover, the duration up to readmission was reduced in the closed time period (t = 2.314, df = 54, p = 0.025).Conclusions: We hypothesize that open doors reduce patient´s discomfort, improveward atmosphere and aggressive acts and do not appear to increase the risk of absconding (AU)

Biochemical analysis of human milk treated with sodium dodecyl sulfate, an alkyl sulfate microbicide that inactivates human immunodeficiency virus type 1.

Reduction of transmission of human immunodeficiency virus type 1 (HIV-1) through human milk is needed. Alkyl sulfates such as sodium dodecyl sulfate (SDS) are microbicidal against HIV-1 at low concentrations, have little to no toxicity, and are inexpensive. The authors have reported that treatment of HIV-1-infected human milk with < or = 1% (10 mg/mL) SDS for 10 minutes inactivates cell-free and cell-associated virus. The SDS can be removed with a commercially available resin after treatment without recovery of viral infectivity. In this article, the authors report results of selective biochemical analyses (ie, protein, immunoglobulins, lipids, cells, and electrolytes) of human milk subjected to SDS treatment and removal. The SDS treatment or removal had no significant effects on the milk components studied. Therefore, the use of alkyl sulfate microbicides to treat milk from HIV-1-positive women may be a simple, practical, and nutritionally sound way to prevent or reduce transmission of HIV-1 while still feeding with mother's own milk.

Alternative modified infant-feeding practices to prevent postnatal transmission of human immunodeficiency virus type 1 through breast milk: past, present, and future.

Preventing mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) through breastfeeding is important to reduce the number of infected children. Research on making breastfeeding safer is a high priority. The authors reviewed the attempts to develop alternative methods, other than antiretroviral (ARV) therapy of mothers and/or babies, to decontaminate breast milk of infectious HIV-1 (free and associated with lymphocytes). They also review how these methods affect milk constituents, as well as their current and prospective status. A PubMed search for English publications on methods to prevent MTCT through breast milk was completed. Methods that have been tested, other than systemicuse or ARV or immunoprophylaxis, to reduce or prevent MTCT of HIV-1 through breast milk were broadly classified into 5 groups: (1) modified feeding practices, (2) heat treatment of milk, (3) lipolysis, (4) antimicrobial treatment of the breastfeeding mother, and (5) microbicidal treatment of infected milk. Their advantages and disadvantages are discussed, as well as future directions in the prevention of MTCT through breastfeeding.