Targets of RNA-directed DNA methylation

RNA-directed DNA methylation contributes substantially to epigenetic regulation of the plant genome. Methylation is guided to homologous DNA target sequences by 24 nt 'heterochromatic' small RNAs produced by nucleolar-localized components of the RNAi machinery and a plant-specific RNA polymerase, Pol IV. Plants contain unusually large and diverse populations of small RNAs, many of which originate from transposons and repeats. These sequences are frequent targets of methylation, and they are able to bring plant genes in their vicinity under small RNA-mediated control. RNA-directed DNA methylation can be removed by enzymatic demethylation, providing plants with a versatile system that facilitates epigenetic plasticity. In addition to subduing transposons, RNA-directed DNA methylation has roles in plant development and, perhaps, stress responses.     

Respiratory Syncytial Virus Uses CX3CR1 as a Receptor on Primary Human Airway Epithelial Cultures

Respiratory syncytial virus (RSV) is the most frequent cause of lower respiratory disease in infants, but no vaccine or effective therapy is available. The initiation of RSV infection of immortalized cells is largely dependent on cell surface heparan sulfate (HS), a receptor for the RSV attachment (G) glycoprotein in immortalized cells. However, RSV infects the ciliated cells in primary well differentiated human airway epithelial (HAE) cultures via the apical surface, but HS is not detectable on this surface. Here we show that soluble HS inhibits infection of immortalized cells, but not HAE cultures, confirming that HS is not the receptor on HAE cultures. Conversely, a “non-neutralizing” monoclonal antibody against the G protein that does not block RSV infection of immortalized cells, does inhibit infection of HAE cultures. This antibody was previously shown to block the interaction between the G protein and the chemokine receptor CX3CR1 and we have mapped the binding site for this antibody to the CX3C motif and its surrounding region in the G protein. We show that CX3CR1 is present on the apical surface of ciliated cells in HAE cultures and especially on the cilia. RSV infection of HAE cultures is reduced by an antibody against CX3CR1 and by mutations in the G protein CX3C motif. Additionally, mice lacking CX3CR1 are less susceptible to RSV infection. These findings demonstrate that RSV uses CX3CR1 as a cellular receptor on HAE cultures and highlight the importance of using a physiologically relevant model to study virus entry and antibody neutralization.     

Legionella pneumophila in commercial bottled mineral water

Sixty-eight commercial bottled mineral waters (64 brands, 68 different 'best-before dates') were tested for the presence of bacteria and fungi. Six samples were Legionella antigen positive and six were Legionella pneumophila PCR positive. Two samples were both Legionella antigen and L. pneumophila PCR positive. Legionella cultures were negative. Although the PCR might have detected only dead Legionella cells, the PCR has been described to detect specifically viable but not culturable (VBNC) L. pneumophila cells as well. Whether VBNC bacteria may be present in bottled mineral waters and the risk for infection this may pose for severely immunocompromised patients should be investigated.     

Catalase Overexpression Reduces Lactic Acid-Induced Oxidative Stress in Saccharomyces cerevisiae

Industrial production of lactic acid with the current pyruvate decarboxylase-negative Saccharomyces cerevisiae strains requires aeration to allow for respiratory generation of ATP to facilitate growth and, even under nongrowing conditions, cellular maintenance. In the current study, we observed an inhibition of aerobic growth in the presence of lactic acid. Unexpectedly, the cyb2Δ reference strain, used to avoid aerobic consumption of lactic acid, had a specific growth rate of 0.25 h⁻¹ in anaerobic batch cultures containing lactic acid but only 0.16 h⁻¹ in identical aerobic cultures. Measurements of aerobic cultures of S. cerevisiae showed that the addition of lactic acid to the growth medium resulted in elevated levels of reactive oxygen species (ROS). To reduce the accumulation of lactic acid-induced ROS, cytosolic catalase (CTT1) was overexpressed by replacing the native promoter with the strong constitutive TPI1 promoter. Increased activity of catalase was confirmed and later correlated with decreased levels of ROS and increased specific growth rates in the presence of high lactic acid concentrations. The increased fitness of this genetically modified strain demonstrates the successful attenuation of additional stress that is derived from aerobic metabolism and may provide the basis for enhanced (micro)aerobic production of organic acids in S. cerevisiae.Lactic acid is an organic acid with a wide range of applications. In the food industry, lactic acid has traditionally been used as an antimicrobial as well as a flavor enhancer. Besides having applications in textile, cosmetic, and pharmaceutical industries (5), lactic acid has been applied for the manufacture of lactic acid polymers (11, 40). These polymers have properties that are similar to those of petroleum-derived plastics. Skyrocketing oil prices caused by dwindling fossil fuel reserves coupled with pressures to tackle environmental issues are creating increased demand for bioderived, and often biodegradable, polymers, such as poly-lactic acid.Current industrial lactic acid fermentations are based on different species of lactic acid bacteria. These bacteria have complex nutrient requirements due to their limited ability to synthesize B vitamins and amino acids (8) and are intolerant to acidic conditions with a pH between 5.5 and 6.5 required for growth (40). Acidification of the growth medium during lactic acid fermentation is typically counteracted by the addition of neutralizing agents (e.g., CaCO₃), resulting in the formation of large quantities of insoluble salts, such as gypsum, during downstream processing.Saccharomyces cerevisiae has received attention as a possible alternative biocatalyst. This organism is relatively tolerant to low pH and has simple nutrient requirements. The production of lactic acid with metabolically engineered S. cerevisiae was achieved by introducing a NAD⁺-dependent lactate dehydrogenase, leading to the simultaneous formation of both ethanol and lactate (1a, 12, 31, 32, 36). Further improvements were made by constructing a pyruvate decarboxylase-negative (Pdc⁻) S. cerevisiae strain (1a, 31, 44) that converted glucose to lactic acid as the sole fermentation product.Although the redox balance and ATP generation in lactic acid fermentation are analogous to those in alcoholic fermentation, engineered homolactic S. cerevisiae strains could not sustain anaerobic growth (44). In addition, the lactate formation rate under anaerobic conditions in the presence of excess glucose was significantly lower than the specific ethanol production rate of the wild-type strain. Moreover, exposure of the anaerobic cell suspension to oxygen immediately led to a 2.5-fold increase in the lactate formation rate. The stimulatory effect of oxygen on lactic acid fermentation may reflect an energetic constraint in lactate fermentation, probably as a consequence of energy-dependent product export (42, 44). In agreement with this hypothesis, intracellular ATP concentrations and the related energy charge decrease rapidly during anaerobic homolactic fermentation by S. cerevisiae (1). Consequently, industrial production of lactic acid with S. cerevisiae may require (micro)aerobic conditions to allow for the generation of sufficient ATP to enable cell growth and, even under nongrowing conditions, maintenance.The formation of reactive oxygen species (ROS) during cellular respiration is an unavoidable side effect of aerobic life relying on oxygen as the final electron acceptor. At least 2% of oxygen consumed in mitochondrial respiration undergoes only one electron reduction, mainly by the semiquinone form of coenzyme Q, generating superoxide radicals (O₂⁻) (26). In addition, the prooxidant effects of organic acids have been demonstrated using sod mutants (30). An in vitro study by Ali et al. (3) also linked ROS formation to weak organic acids and showed enhanced hydroxy radical (OH) generation in the presence of lactic acid.Among different ROS, the hydroxy radical that originates from H₂O₂ in the metal-mediated Fenton/Haber-Weiss reactions is especially reactive. It indiscriminately oxidizes intracellular proteins, nucleic acids, and lipids in the cell membranes (4, 38). Lactate interacts with the ferric ion (Fe³⁺) to form a stable complex of Fe³⁺-lactate at a molar ratio of 1:2. This complex then reacts with H₂O₂ to enhance the OH generation via the Fenton reaction (2, 3). Although a similar in vivo mechanism has not yet been proven, previous research indicates that lactic acid and other weak organic acids enhance oxidative stress of aerobic yeast cultures.Like other eukaryotic organisms, S. cerevisiae possesses enzymatic defense mechanisms, including several crucial antioxidant enzymes, such as catalase and superoxide dismutase (SOD). SOD removes O₂⁻ by converting it to H₂O₂, which, in turn, can be disproportionated to water by catalase or glutathione peroxidase. Cytosolic catalase, Ctt1p, is thought to play a general role, as CTT1 expression is regulated by various stresses, including oxidative stress, osmotic stress, and starvation (15, 23, 33). More recently, catalase has also been implicated in response to acetic acid tolerance and acetic acid-induced programmed cell death (17, 47).The goals of the present study were to assess the in vivo relevance of lactate-mediated oxidative stress in S. cerevisiae and to investigate whether its effects could be ameliorated by enhanced expression of catalase.     

Novel Evolutionary Engineering Approach for Accelerated Utilization of Glucose,Xylose, and Arabinose Mixtures by Engineered Saccharomyces cerevisiae Strains

Lignocellulosic feedstocks are thought to have great economic and environmental significance for future biotechnological production processes. For cost-effective and efficient industrial processes, complete and fast conversion of all sugars derived from these feedstocks is required. Hence, simultaneous or fast sequential fermentation of sugars would greatly contribute to the efficiency of production processes. One of the main challenges emerging from the use of lignocellulosics for the production of ethanol by the yeast Saccharomyces cerevisiae is efficient fermentation of d-xylose and l-arabinose, as these sugars cannot be used by natural S. cerevisiae strains. In this study, we describe the first engineered S. cerevisiae strain (strain IMS0003) capable of fermenting mixtures of glucose, xylose, and arabinose with a high ethanol yield (0.43 g g⁻¹ of total sugar) without formation of the side products xylitol and arabinitol. The kinetics of anaerobic fermentation of glucose-xylose-arabinose mixtures were greatly improved by using a novel evolutionary engineering strategy. This strategy included a regimen consisting of repeated batch cultivation with repeated cycles of consecutive growth in three media with different compositions (glucose, xylose, and arabinose; xylose and arabinose; and only arabinose) and allowed rapid selection of an evolved strain (IMS0010) exhibiting improved specific rates of consumption of xylose and arabinose. This evolution strategy resulted in a 40% reduction in the time required to completely ferment a mixture containing 30 g liter⁻¹ glucose, 15 g liter⁻¹ xylose, and 15 g liter⁻¹ arabinose.In recent years, the need for biotechnological manufacturing based on lignocellulosic feedstocks has become evident (6, 10). In contrast to the readily fermentable, mainly starch- or sucrose-containing feedstocks used in current biotechnological production processes, lignocellulosic biomass requires intensive pretreatment and hydrolysis, which yield complex mixtures of sugars (3, 7, 14, 27). For cost-effective and efficient industrial processes, complete and fast conversion of all sugars present in lignocellulosic hydrolysates is a prerequisite. The major hurdles encountered in implementing these production processes are the conversion of substrates that cannot be utilized by the organism of choice and, even more importantly, the subsequent improvement of sugar conversion rates and product yields.The use of evolutionary engineering has proven to be very valuable for obtaining phenotypes of (industrial) microorganisms with improved properties, such as an expanded substrate range, increased stress tolerance, and efficient substrate utilization (16, 17). Also, for the yeast Saccharomyces cerevisiae, the preferred organism for large-scale ethanol production for the past few decades, evolutionary engineering has been extensively used to select for industrially relevant phenotypes. For ethanol production from lignocellulose by S. cerevisiae, one of the main challenges is efficient conversion of the pentoses d-xylose and l-arabinose to ethanol. To deal with this challenge, S. cerevisiae strains have been metabolically engineered since the early 1990s for the conversion of xylose into ethanol by the introduction of heterologous xylose utilization pathways (for recent reviews, see references 9 and 20). Arabinose utilization, however, has been addressed only quite recently. The most successful approach for obtaining arabinose consumption in S. cerevisiae has been the introduction of a bacterial arabinose utilization pathway (5, 26). In addition to metabolic engineering, extensive evolutionary engineering (by prolonged cultivation of recombinant S. cerevisiae strains in either anaerobic chemostat or repeated anaerobic batch cultures) was required to obtain S. cerevisiae strains that ferment either xylose (13, 19) or arabinose (5, 26) fast or to improve fermentation performance with mixtures containing glucose and xylose (12). In contrast, (evolutionary) engineering has still not resulted in fast and efficient fermentation of both xylose and arabinose to ethanol by a single recombinant S. cerevisiae strain. At best, simultaneous utilization of xylose and arabinose yielded large amounts of the undesirable side products xylitol and arabinitol (11). Hence, a major remaining challenge is the conversion of both xylose and arabinose with high ethanol production rates and yields.In a previous study, an S. cerevisiae strain was metabolically engineered to obtain both xylose and arabinose utilization. For this, the Piromyces XylA, S. cerevisiae XKS1, and Lactobacillus plantarum araA, araB, and araD genes, as well as the endogenous genes of the pentose phosphate pathway (RPE1, RKI1, TKL1, and TAL1), were overexpressed. Selection by sequential batch cultivation under conditions with arabinose as the sole carbon source resulted in strain IMS0002, which is capable of fermenting arabinose to ethanol under anaerobic conditions (26). Unfortunately, the ability to ferment xylose to ethanol was largely lost during long-term selection for improved l-arabinose fermentation. During anaerobic batch cultivation of strain IMS0002 in a glucose-xylose-arabinose mixture, xylose was not consumed completely and was converted to almost equimolar amounts of xylitol. This loss of xylose metabolism illustrates the limitations of selection in media supplemented with a single carbon and energy source.The goal of the present study was to evaluate and optimize selection strategies for evolutionary optimization of the utilization of substrate mixtures. Fermentation of glucose, xylose, and arabinose mixtures by engineered S. cerevisiae strains was used as the model.     

Germ band retraction as a landmark in glucose metabolism during <Emphasis Type="Italic">Aedes aegypti</Emphasis> embryogenesis

Background  

The mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown.     

Measurement of 25-OH-vitamin D in human serum using liquid chromatography tandem-mass spectrometry with comparison to radioimmunoassay and automated immunoassay

The plasma 25-OH vitamin D concentration is a reliable biomarker for vitamin D status but assay's variability makes adequate monitoring of vitamin D status difficult. We employed isotope-dilution liquid chromatography (LC) tandem-mass spectrometry (MS/MS) for the measurement of both 25-OH vitamin D₃ and 25-OH vitamin D₂ in human serum. Hexadeuterium labelled 25-OH vitamin D₃ internal standard (IS) was added to calibrators (prepared in phosphate-buffered saline with 60 g/L albumin), controls or patient sera and 25-OH vitamin D metabolites were released from vitamin D binding protein by adding sodium hydroxide prior to protein precipitation by acetonitrile/methanol (9:1, v/v). Subsequent off-line solid-phase extraction was followed by chromatographic separation on a C-18 column using a water/methanol/ammonium acetate gradient. Detection was by Atmospheric Pressure Electrospray Ionisation (AP-EI) followed by selected reaction monitoring. We compared the LC-MS/MS assay to the DiaSorin radioimmunoassay (RIA) and a recently re-standardised version of an automated electrochemiluminescent immunoassay (ECLIA) from Roche Diagnostics. We also analysed external quality control samples from the International Vitamin D External Quality Assessment Scheme (DEQAS) for comparison with other participating laboratories using LC-MS. The method was linear from 5 to at least 550 nmol/L with intra- and interday CV's ≤6% for both 25-OH vitamin D₃ and 25-OH vitamin D₂. Recoveries ranged between 94.9 and 106.9% for 25-OH vitamin D₃ and 82.7 and 100.3% for 25-OH vitamin D₂. Our results for the DEQAS serum pools averaged ?7.2% from the overall LC-MS method mean. The DiaSorin RIA agreed well with the LC-MS/MS method (r² = 0.90; average bias 1.61 nmol/L), the Roche ECLIA considerably disagreed (r² = 0.58; bias 10.13 nmol/L). This LC-MS/MS method is reliable and robust for the measurement of both 25-OH vitamin D₃ and 25-OH vitamin D₂ in human serum.     

大鼠心肌缺血再灌注损伤模型的实验研究

目的 制备一种新型的心肌急性缺血再灌注损伤模型,以探讨一种更符合临床实际需求的实验方法.方法 将20只雌性SD(Sprague-Dawley)大鼠随机分成2组(对照组、实验组),采用结扎主动脉根部引起心肌缺血5min再灌注30 min建立心肌急性缺血再灌注模型;通过应用透射电镜观察心肌细胞超微结构的改变,同时检测心肌组织匀浆丙二醛(Maleic Dialdehyde,MDA)含量、超氧化物歧化酶(Superoxide Dismutase,SOD)活力.结果 透射电镜下超微结构显示实验组较对照组明显加重了心肌组织结构和线粒体的损害;实验组心肌组织MDA明显高于对照组(P<0.01),而SOD明显低于对照组(P<0.01).结论 本实验成功建立了方法简便、易于操作、取材范围广泛的心肌缺血再灌注损伤模型,为心肌缺血再灌注损伤研究提供了一种更为可行的模型.