Biological availability and efficacy of folates from bioengineered rice

The basic concept of folate rice is to develop a means for rural areas to combat folate deficiency. Folate deficiency is of concern given the relation between folate status and birth defects, ranging in severity from mild back ache to stillbirth, due to a malformation of the neural tube. While western populations benefit from diverse and nutrient rich diets, even these populations are at risk for folate deficiency. One method to alleviate the health burden associated with folate deficiency is to add synthetic folic acid to commonly consumed food items such as breakfast cereals or bread. However, remote, mostly rural communities, do not have free access to folic acid fortification. As such, a rice variety with a high natural folate content that could be grown locally may serve to achieve an adequate folate status. However, since folate rice is a genetically engineered rice variety, public and governmental acceptance depends on scientific evidence that the folates contained within these rice grains are released into the bloodstream when consumed. This is because some genetically engineered food items have been criticized as a result of their limited influence on nutrient status. It is therefore of paramount importance to evaluate the impact of folate rice consumption on folate status. Given the need for strict control of nutrient intake and regulatory constraints, a rodent feeding trial was devised to evaluate the efficacy of folate rice as a dietary folate source. Due to the complexity of folate metabolism and body distribution, a long term study was performed including regular evaluation of folate status, folate related clinical parameters and general health. These results were compared between groups receiving no folate, a small amount of folate present in ‘normal’ rice, a larger amount of folate in either folate rice or folic acid fortified rice or ample folate as an optimal scenario. To quantify the biological outcome of folate rice consumption, an analytical method was developed to measure the amount of folates in 2 separate blood fractions, i.e. plasma and red blood cells. Given the low concentrations in which folates are present, a sensitive method using liquid chromatography coupled to tandem mass spectrometry was used. Since the term folate signifies several different molecules with different chemical and biological properties, the developed method required the measurement of individual folate species. Also, to exclude the influence of folates which might be present in the rodent diets used during the rodent trial, a separate method was developed to determine the folate concentration in the rodent diets used. Due to the necessity of folates for DNA-synthesis in white blood cells, the influence of folate concentration and speciation on the functioning of the immune system was investigated as well

Determination of five folate monoglutamates in rodent diets

A method for the quantitative determination of folates in rodent diets is very important for correct interpretation of folate intake during feeding trials, given the possible discrepancy between the actual folate concentration in the diet and that mentioned on the product sheet. Liquid chromatography tandem-mass spectrometry is the method of choice to differentiate and quantify the individual folate species present. This discrepancy may be accounted for by, e.g., inaccurate folic acid supplementation and/or the presence of endogenous reduced and substituted folates. We developed a method, validated based on FDA guidelines, that allows the measurement of added and endogenous folates by quantitative determination of 5 folate monoglutamates with linear ranges from 8 mu g to 2 mg/kg feed. This information, combined with feed intake data, allows insight into the actual folate intake in animal feeding studies. The relevance of this method was illustrated by the analysis of several feed samples of varying composition, by the investigation of the effect of casein incorporation, and by evaluating the variability of the folate content between pellets and production batches

An automated system for the measurement of hydrogen peroxide in industrial applications

An automated sensor system for the continuous and in-line measurement of hydrogen peroxide in industrial applications is described. The hydrogen peroxide concentration can be measured over the entire pH range, over a wide concentration range of hydrogen peroxide (10-3 70 g/l), from 0 to 70°C, and with high precision and accuracy (errors less than 1% ). The system consists of a bypass in which the necessary electrodes are positioned and electronically controlled. The sensor is very selective for hydrogen peroxide, easy to instal, and it is stable for at least two months after calibration. The calibration can be done in the process solution during a running process

The small RNA ncS35 regulates growth in Burkholderia cenocepacia J2315

Burkholderia cenocepacia J2315 is a member of the B. cepacia complex. It has a large genome with three replicons and one plasmid; 7,261 genes code for annotated proteins, while 113 code for functional RNAs. Small regulatory RNAs of B. cenocepacia have not yet been functionally characterized. We investigated a small regulatory RNA, designated ncS35, that was discovered by differential RNA sequencing. Its expression under various conditions was quantified, and a deletion mutant, Delta ncS35, was constructed. Compared to planktonic growth in a rich medium, the expression of ncS35 was elevated when B. cenocepacia 12315 was grown in biofilms and in minimal medium. Cells of the deletion mutant showed increased aggregation, higher metabolic activity, a higher growth rate, and an increased susceptibility to tobramycin. A transcriptomic analysis revealed upregulation of the phenylacetic acid and tryptophan degradation pathways in Delta ncS35. Computational target prediction indicated that ncS35 likely interacts with the first gene of the tryptophan degradation pathway. Overall, we demonstrated that small RNA ncS35 is a noncoding RNA with an attenuating effect on the metabolic rate and growth. It is possible that slower growth protects B. cenocepacia J2315 against stressors acting on fast-dividing cells and enhances survival under unfavorable conditions. IMPORTANCE Small RNAs play an important role in the survival of bacteria in diverse environments. We explored the physiological role of ncS35, a small RNA expressed in B. cenocepacia J2315, an opportunistic pathogen in cystic fibrosis patients. In cystic fibrosis patients, infections can lead to "cepacia syndrome," a rapidly progressing and often fatal pneumonia. Infections with Burkholderia spp. are difficult to threat with antibiotics because of their high intrinsic resistance and ability to form biofilms. We show that ncS35 attenuates the growth and reduces the metabolic rate of B. cenocepacia and influences biofilm structure. This demonstrates that as-yet-uncharacterized small RNAs with regulatory function can influence physiological traits of B. cenocepacia that are relevant for infection

Clinical determination of folates : recent analytical strategies and challenges

Since the introduction of liquid chromatography tandem mass spectrometry in clinical laboratories, folate analysis has shifted from microbiological or protein-binding assays to chromatographic methods. Now, it is possible to sensitively and selectively determine several folate species in clinical samples where only a total folate content could be quantified using a microbiological or a binding assay. Although several chromatographic methods have been developed, validated, and published, interlaboratory variability limits the comparability of the results. In this review, we provide an overview of the latest strategies for sampling, sample treatment, and analysis and how these may influence the final analytical result. Among the variables covered are the effect of pH, temperature, and storage and the use of antioxidants and anticoagulants on analyte stability. In addition, we highlight the importance of correct assay calibration and the use of (labeled) certified reference materials in order to obtain correct and comparable results among different laboratories

Comparative studies on osmosis based encapsulation of sodium diclofenac in porcine and outdated human erythrocyte ghosts

The objective of our study was to develop controlled drug delivery system based on erythrocyte ghosts for amphiphilic compound sodium diclofenac considering the differences between erythrocytes derived from two readily available materials - porcine slaughterhouse and outdated transfusion human blood. Starting erythrocytes, empty erythrocyte ghosts and diclofenac loaded ghosts were compared in terms of the encapsulation efficiency, drug releasing profiles, size distribution, surface charge, conductivity, surface roughness and morphology. The encapsulation of sodium diclofenac was performed by an osmosis based process - gradual hemolysis. During this process sodium diclofenac exerted mild and delayed antihemolytic effect and increased potassium efflux in porcine but not in outdated human erythrocytes. FTIR spectra revealed lack of any membrane lipid disorder and chemical reaction with sodium diclofenac in encapsulated ghosts. Outdated human erythrocyte ghosts with detected nanoscale damages and reduced ability to shrink had encapsulation efficiency of only 8%. On the other hand, porcine erythrocyte ghosts had encapsulation efficiency of 37% and relatively slow drug release rate. More preserved structure and functional properties of porcine erythrocytes related to their superior encapsulation and release performances, define them as more appropriate for the usage in sodium diclofenac encapsulation process

The transcription factor ETS1 is an important regulator of human NK cell development and terminal differentiation

Natural killer (NK) cells are important in the immune defense against tumor cells and pathogens, and regulate other immune cells by cytokine secretion. Whereas murine NK cell biology has been extensively studied, knowledge about transcriptional circuitries controlling human NK cell development and maturation is limited. By generating ETS1-deficient human embryonic stem cells (hESC) and by expressing the dominant-negative ETS1 p27 isoform in cord blood (CB) hematopoietic progenitor cells (HPCs), we show that the transcription factor ETS1 is critically required for human NK cell differentiation. Genome-wide transcriptome analysis determined by RNA-sequencing combined with chromatin immunoprecipitation-sequencing (ChIP-seq) analysis reveals that human ETS1 directly induces expression of key transcription factors that control NK cell differentiation, i.e. E4BP4, TXNIP, TBET, GATA3, HOBIT and BLIMP1. In addition, ETS1 regulates expression of genes involved in apoptosis and NK cell activation. Our study provides important molecular insights into the role of ETS1 as an important regulator of human NK cell development and terminal differentiation

Lactobacilli have a niche in the human nose

Although an increasing number of beneficial microbiome members are characterized for the human gut and vagina, beneficial microbes are underexplored for the human upper respiratory tract (URT). In this study, we demonstrate that taxa from the beneficial Lactobacillus genus complex are more prevalent in the healthy URT than in patients with chronic rhinosinusitis (CRS). Several URT-specific isolates are cultured, characterized, and further explored for their genetic and functional properties related to adaptation to the URT. Catalase genes are found in the identified lactobacilli, which is a unique feature within this mostly facultative anaerobic genus. Moreover, one of our isolated strains, Lactobacillus casei AMBR2, contains fimbriae that enable strong adherence to URT epithelium, inhibit the growth and virulence of several URT pathogens, and successfully colonize nasal epithelium of healthy volunteers. This study thus demonstrates that specific lactobacilli are adapted to the URT and could have a beneficial keystone function in this habitat

IRF2 is required for development and functional maturation of human NK cells

Natural killer (NK) cells are cytotoxic and cytokine-producing lymphocytes that play an important role in the first line of defense against malignant or virus-infected cells. A better understanding of the transcriptional regulation of human NK cell differentiation is crucial to improve the efficacy of NK cell-mediated immunotherapy for cancer treatment. Here, we studied the role of the transcription factor interferon regulatory factor (IRF) 2 in human NK cell differentiation by stable knockdown or overexpression in cord blood hematopoietic stem cells and investigated its effect on development and function of the NK cell progeny. IRF2 overexpression had limited effects in these processes, indicating that endogenous IRF2 expression levels are sufficient. However, IRF2 knockdown greatly reduced the cell numbers of all early differentiation stages, resulting in decimated NK cell numbers. This was not caused by increased apoptosis, but by decreased proliferation. Expression of IRF2 is also required for functional maturation of NK cells, as the remaining NK cells after silencing of IRF2 had a less mature phenotype and showed decreased cytotoxic potential, as well as a greatly reduced cytokine secretion. Thus, IRF2 plays an important role during development and functional maturation of human NK cells