Effect of Cultivation Time on Production of Antifungal Metabolite(s) by Streptomyces hygroscopicus in Laboratory‐Scale Bioreactor

Biological control agents offer one of the best alternatives to reduce the use of pesticides . Fungi from the genera Alternaria, Colletotrichum and Fusarium are listed among the most important storage pathogens of apple fruits. During storage, transport and marketing, pathogenic fungi can cause significant losses of apple fruits. This investigation studied the potential of Streptomyces hygroscopicus as a biocontrol agent against pathogenic fungi obtained from apple fruit samples expressing rot symptoms. Production of antifungal metabolites by S. hygroscopicus was carried out in 3‐l bench‐scale bioreactor (Biostat^® Aplus, Sartorius AG, Germany) during 7 days. Fermentation was carried out at 27°C with aeration rate of 0.5 vvm and agitation rate of 200 rpm. The aim was to analyse bioprocess parameters of batch biofungicide production in medium containing glucose as a carbon source and to examine at which stage of bioprocess production of antifungal metabolite(s) against six phytopathogenic fungi occurs. In vitro antifungal activity of the produced metabolites against six fungi of the genera Colletotrichum, Fusarium and Alternaria grown on potato dextrose agar were determined every 24 h using wells technique. Antifungal activity of cell‐free culture filtrate and filtrate treated with high temperature were tested. The filtrate treated with high temperature did not show any antifungal activity suggesting that active components are thermo unstable. Stationary phase of growth occurred between the third and fourth day of cultivation when production of secondary metabolites begins. Obtained results showed that maximal antifungal activity is achieved on fifth and sixth day of S. hygroscopicus cultivation under defined conditions (inhibition zone diameter higher than 30 mm for all test fungi).     

Molecular species of oxidized phospholipids in brain differentiate between learning- and memory impaired and unimpaired aged rats

Amino Acids - Loss of cognitive function is a typical consequence of aging in humans and rodents. The extent of decline in spatial memory performance of rats, assessed by a hole-board test, reaches...     

Visualisation of Leishmania donovani fluorescent hybrids during early stage development in the sand fly vector

Background

The Leishmania protozoan parasites cause devastating human diseases. Leishmania have been considered to replicate clonally, without genetic exchange. However, an accumulation of evidence indicates that there are inter-specific and intra-specific hybrids among natural populations. The first and so far only experimental proof of genetic exchange was obtained in 2009 when double drug resistant Leishmania major hybrids were produced by co-infecting sand flies with two strains carrying different drug resistance markers. However, the location and timing of hybridisation events in sand flies has not been described.

Methodology/Principal Findings

Here we have co-infected Phlebotomus perniciosus and Lutzomyia longipalpis with transgenic promastigotes of Leishmania donovani strains carrying hygromycin or neomycin resistance genes and red or green fluorescent markers. Fed females were dissected at different times post bloodmeal (PBM) and examined by fluorescent microscopy or fluorescent activated cell sorting (FACS) followed by confocal microscopy. In mixed infections strains LEM3804 and Gebre-1 reached the cardia and stomodeal valves more rapidly than strains LEM4265 and LV9. Hybrids unequivocally expressing both red and green fluorescence were seen in single flies of both vectors tested, co-infected with LEM4265 and Gebre-1. The hybrids were present as short (procyclic) promastigotes 2 days PBM in the semi-digested blood in the endoperitrophic space. Recovery of a clearly co-expressing hybrid was also achieved by FACS. However, hybrids could not sustain growth in vitro.

Conclusions/Significance

For the first time, we observed L. donovani hybrids in the sand fly vector, 2 days PBM and described the morphological stages involved. Fluorescence microscopy in combination with FACS allows visualisation and recovery of the progeny of experimental crosses but on this occasion the hybrids were not viable in vitro. Nevertheless, genetic exchange in L. donovani has profound epidemiological significance, because it facilitates the emergence and spread of new phenotypic traits.     

Experimental transmission of Leishmania infantum by two major vectors: a comparison between a viscerotropic and a dermotropic strain

We quantified Leishmania infantum parasites transmitted by natural vectors for the first time. Both L. infantum strains studied, dermotropic CUK3 and viscerotropic IMT373, developed well in Phlebotomus perniciosus and Lutzomyia longipalpis. They produced heavy late-stage infection and colonized the stomodeal valve, which is a prerequisite for successful transmission. Infected sand fly females, and especially those that transmit parasites, feed significantly longer on the host (1.5-1.8 times) than non-transmitting females. Quantitative PCR revealed that P. perniciosus harboured more CUK3 strain parasites, while in L. longipalpis the intensity of infection was higher for the IMT373 strain. However, in both sand fly species the parasite load transmitted was higher for the strain with dermal tropism (CUK3). All but one sand fly female infected by the IMT373 strain transmitted less than 600 promastigotes; in contrast, 29% of L. longipalpis and 14% of P. perniciosus infected with the CUK3 strain transmitted more than 1000 parasites. The parasite number transmitted by individual sand flies ranged from 4 up to 4.19×10(4) promastigotes; thus, the maximal natural dose found was still about 250 times lower than the experimental challenge dose used in previous studies. This finding emphasizes the importance of determining the natural infective dose for the development of an accurate experimental model useful for the evaluation of new drugs and vaccines.     

Characterization of advanced glycation end products for biochemical studies: side chain modifications and fluorescence characteristics

Advanced glycation end products (AGEs) are known to be involved in the pathogenesis of several diseases and therefore effects of AGEs on cells are the objective of numerous investigations. Since AGEs used in biochemical studies are usually not chemically characterized, comparison of data is difficult if not impossible. To find a suitable characterization protocol, human serum albumin was reacted with different concentrations of glucose, methyl glyoxal, and glyoxylic acid. The obtained AGEs were characterized with respect to the extent of side chain modifications (lysine and arginine), the carboxymethyl lysine and carbonyl content, and the fibrillar state. Additionally, their fluorescence and absorbance characteristics were extensively studied. Although we found significant differences in the degree of modification and in AGE-specific fluorescence when using different modifiers, the results provide important information and allow comparing AGEs derived from different modifier concentrations. The results also suggest strong conformational changes within the modified proteins. In the present paper we propose a set of parameters that is sufficient to partially characterize AGEs used for biochemical studies.     

Circulating isoflavonoid levels in CD-1 mice: effect of oral versus subcutaneous delivery and frequency of administration

The CD-1 mouse is a commonly used animal model to understand the biological effects of early-life exposure to soy isoflavones in infants. Most studies using CD-1 mice have administered isoflavones by daily subcutaneous injection, while infants receive oral feeds every few hours. The study objectives were to compare the total serum levels of genistein (GEN), daidzein (DAI) and the DAI metabolites equol and O-desmethyl-angolensin (O-DMA), after subcutaneous injection and oral dosing and to determine if frequency of oral administration results in different circulating levels of isoflavones using the CD-1 mouse model. From postnatal days 1 to 5, pups randomly received corn oil or soy isoflavones (total daily dose, 0.010 mg DAI+0.025 mg GEN) by subcutaneous injection once a day, orally once a day or orally every 4 hours. On postnatal day 5, 1 h posttreatment, mice were killed and serum was collected. Mice treated with soy isoflavones had higher (P<.05) serum GEN (female: 1895–3391 ng/ml and male: 483–578 ng/ml) and DAI (female: 850–1580 ng/ml and male: 248–322 ng/ml) concentrations versus control (5–20 ng/ml) mice, regardless of route or frequency of administration, and were similar among dosing strategies. Total serum concentrations of GEN and DAI were higher (P<.05) among females (GEN: 2714 ± 393 ng/ml and DAI: 1205 ± 164 ng/ml) than males (GEN: 521 ± 439 ng/ml and DAI: 288 ± 184 ng/ml) across treatment groups. Serum equol and O-DMA concentrations were negligible (<3 ng/ml) across groups. In conclusion, different routes of delivery and frequency of administration resulted in similar total serum levels of GEN, DAI¸ equol or O-DMA.     

The C-terminal domain of Fcj1 is required for formation of crista junctions and interacts with the TOB/SAM complex in mitochondria

Crista junctions (CJs) are tubular invaginations of the inner membrane of mitochondria that connect the inner boundary with the cristae membrane. These architectural elements are critical for mitochondrial function. The yeast inner membrane protein Fcj1, called mitofilin in mammals, was reported to be preferentially located at CJs and crucial for their formation. Here we investigate the functional roles of individual domains of Fcj1. The most conserved part of Fcj1, the C-terminal domain, is essential for Fcj1 function. In its absence, formation of CJ is strongly impaired and irregular, and stacked cristae are present. This domain interacts with full-length Fcj1, suggesting a role in oligomer formation. It also interacts with Tob55 of the translocase of outer membrane β-barrel proteins (TOB)/sorting and assembly machinery (SAM) complex, which is required for the insertion of β-barrel proteins into the outer membrane. The association of the TOB/SAM complex with contact sites depends on the presence of Fcj1. The biogenesis of β-barrel proteins is not significantly affected in the absence of Fcj1. However, down-regulation of the TOB/SAM complex leads to altered cristae morphology and a moderate reduction in the number of CJs. We propose that the C-terminal domain of Fcj1 is critical for the interaction of Fcj1 with the TOB/SAM complex and thereby for stabilizing CJs in close proximity to the outer membrane. These results assign novel functions to both the C-terminal domain of Fcj1 and the TOB/SAM complex.     

SWAN: Subset-quantile Within Array Normalization for Illumina Infinium HumanMethylation450 BeadChips

DNA methylation is the most widely studied epigenetic mark and is known to be essential to normal development and frequently disrupted in disease. The Illumina HumanMethylation450 BeadChip assays the methylation status of CpGs at 485,577 sites across the genome. Here we present Subset-quantile Within Array Normalization (SWAN), a new method that substantially improves the results from this platform by reducing technical variation within and between arrays. SWAN is available in the minfi Bioconductor package.