Analysis of miR-146a and miR-142-3p as Potential Markers of Freshly Isolated or In Vitro-Expanded Human Treg cells.

Regulatory CD4+ T cells (Tregs) are pivotal for prevention of autoimmunity. The use of Tregs is therefore of increasing interest in in vitro drug screening assays as well as for a cytotherapy per se against autoimmune disorders. For both purposes, in vitro expansion of peripheral blood Tregs is necessary and there is an increasing need to identify novel markers that can discriminate natural thymic-derived Tregs (tTregs) from other T cell subsets, and ideally, such markers should be stably expressed during in vitro expansion procedures. We screened for novel miRNAs differentially expressed in tTregs and identified miR-146a and 142-3p as possible candidates. We analysed freshly isolated naïve and activated tTregs and non-Treg subsets after or prior to in vitro expansion. We observed a tTreg-specific profile of these miRNAs together with FOXP3 and Helios in freshly isolated tTregs, but observed a decline in the same markers in activated tTregs as opposed to naïve tTregs. In vitro-expanded Tregs could be identified based on FOXP3 expression, but with loss of a discriminate profile for miRNA candidates and a decline in FOXP3 when activated tTregs were expanded. Our data demonstrate miR-146a and 142-3p as potential miRNA markers for discrimination between non-Treg cells and tTregs, but these miRNAs are not stable markers for in vitro-expanded Treg cells. In addition, the loss of FOXP3 in expansion of activated tTregs has implication for in vitro use of this cell subset in immunopharmacological assays and cytotherapy as FOXP3 is pivotal for suppressive function.

Sweet Corn Sentinel Monitoring for Lepidopteran Field-Evolved Resistance to Bt Toxins.

As part of an insect resistance management plan to preserve Bt transgenic technology, annual monitoring of target pests is mandated to detect susceptibility changes to Bt toxins. Currently Helicoverpa zea (Boddie) monitoring involves investigating unexpected injury in Bt crop fields and collecting larvae from non-Bt host plants for laboratory diet bioassays to determine mortality responses to diagnostic concentrations of Bt toxins. To date, this monitoring approach has not detected any significant change from the known range of baseline susceptibility to Bt toxins, yet practical field-evolved resistance in H. zea populations and numerous occurrences of unexpected injury occur in Bt crops. In this study, we implemented a network of 73 sentinel sweet corn trials, spanning 16 U.S. states and 4 Canadian provinces, for monitoring changes in H. zea susceptibility to Cry and Vip3A toxins by measuring differences in ear damage and larval infestations between isogenic pairs of non-Bt and Bt hybrids over three years. This approach can monitor susceptibility changes and regional differences in other ear-feeding lepidopteran pests. Temporal changes in the field efficacy of each toxin were evidenced by comparing our current results with earlier published studies, including baseline data for each Bt trait when first commercialized. Changes in amount of ear damage showed significant increases in H. zea resistance to Cry toxins and possibly lower susceptibility to Vip3a. Our findings demonstrate that the sentinel plot approach as an in-field screen can effectively monitor phenotypic resistance and document field-evolved resistance in target pest populations, improving resistance monitoring for Bt crops.

Phenotypic and functional markers for 1alpha,25-dihydroxyvitamin D(3)-modified regulatory dendritic cells.

The clinical use of dendritic cells (DCs) to induce antigen-specific immune tolerance has been hampered by the lack of a widely acknowledged method for generating human regulatory DCs but even more so by the non-existence of reliable markers. Thus, we set out to find reliable markers that can be measured with simple methods to identify regulatory DCs that are applicable for future clinical studies. Human DCs were generated from peripheral blood monocytes in the presence of 1alpha,25-dihydroxyvitamin D(3) (VD3), which gave rise to a phenotype that resembles immature DCs, with the exception of high CD14 and reduced CD1a on the cell surface. These VD3-treated DCs exert a long-lasting inefficient T cell stimulation and induce T cell hyporesponsiveness with regulatory potential. Importantly, such VD3-treated DCs were readily distinguishable from untreated DCs by low levels of interleukin-23 secretion and low expression of miR-155 upon exposure to maturation stimuli. Furthermore, VD3-treated DCs showed over-expression of miR-378. All these features can be used as robust markers for quality control of VD3-treated regulatory DCs in future clinical studies.

Effect of tablets containing probiotic candidate strains on gingival inflammation and composition of the salivary microbiome: a randomised controlled trial.

The aim of the study was to investigate clinical and microbial effects of probiotic candidate strains in patients with moderate gingivitis. The null hypothesis was that the clinical measurements with treatment would not differ from placebo. 47 adult patients were enrolled in a randomised placebo-controlled trial with a 4-week intervention of tablets containing a mix of Lactobacillus rhamnosus PB01, DSM 14869 and Lactobacillus curvatus EB10, DSM 32307 or placebo. Clinical examinations and samplings were done at baseline and after 2, 4 and 6 weeks. The clinical endpoints were general bleeding on probing (BOP), general plaque index (PI) and flow of gingival crevicular fluid (GCF). In addition, the concentration of selected cytokines (interleukin (IL)-1ß, IL-6, IL-8, IL-10, tumour necrosis factor alpha (TNF-α)) in GCF was determined with multiplex immunoassays. The profiles of the salivary microbiome were analysed with Next Generation Sequencing (NGS) and qPCR. In contrast to the placebo group, there was a significant reduction in BOP and amount of GCF (P<0.05) after 4 weeks in the probiotic test group when compared with baseline. The general PI was less affected although there was a tendency of decreased plaque levels in the probiotic group (P=0.05-0.09). The cytokines were unaffected by the intervention as well as the salivary microbiome. The Shannon index showed no significant differences between the groups or alterations over time. The occurrence of both probiotic strains increased in saliva of the test subjects during the intervention but returned to baseline levels within 2 weeks. Although a marked improvement in gingival health was recorded in the probiotic group, the null hypothesis could not be rejected.

Sleep-disordered breathing and myocardial ischemia in patients with coronary artery disease.

STUDY OBJECTIVES: To examine the occurrence of nocturnal myocardial ischemia and its relationship to sleep-disordered breathing (apneas and oxygen desaturations) in randomly selected men and women undergoing coronary angiography because of angina pectoris. DESIGN: An observational study using an overnight sleep study and Holter recording to examine disordered breathing (oxyhemoglobin desaturations > or = 4% and apnea-hypopneas), heart rates, and ST-segment depressions (> or = 1 mm, > or = 1 min). SETTING: University Hospital, Umeå, a teaching hospital in northern Sweden. PATIENTS: One hundred thirty-two men and 94 women referred for consideration of coronary intervention were randomly included, by lot. RESULTS: ST-segment depressions occurred in 59% (134 of 226) of the patients, and nocturnal ST-segment depressions occurred in 31% (69 of 226). A ST-segment depression occurred within 2 min after an apnea-hypopnea or desaturation in 12% (27 of 226) of patients. This temporal association was seen in 19% of nocturnal ST-segment depressions (71 of 366), more frequently in men (p < 0.01) and in more severely disordered breathing (p < 0.001). Most of these ST-segment depressions were preceded by a series of breathing events: three or more apnea-hypopneas or desaturations or both in 70% (50 of 71). CONCLUSION: Episodes of nocturnal myocardial ischemia are common in patients with angina pectoris. However, a temporal relationship between sleep-disordered breathing and myocardial ischemia is present only in a minority of the patients, but occurs more frequently in men and in more severely disordered breathing.

Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions.

We have tested the influence on the polymerase chain reaction (PCR) of a large number of compounds found in food, in media used for selective propagation of food-borne pathogens or in DNA-extraction methods. PCR was found to be sensitive to large volumes of complex food samples containing high amounts of fat and protein, however, an extraction procedure based on treatment with hot NaOH/SDS reduced the effect significantly. Some culture media (Fraser, MLEB, MRB and Rappaport) interfered with the analysis and for most of the media it was possible to assign the inhibitory effect to one or more individual components. Several compounds (detergents, lysozyme, NaOH, alcohols, EDGA, EGTA) used in DNA extraction procedures were found to have some inhibitory effect. The inhibitory effects need to be taken into consideration when designing new tests.

A rapid polymerase chain reaction (PCR)-based assay for the identification of Listeria monocytogenes in food samples.

A rapid and simple assay has been developed which allows specific detection of Listeria monocytogenes within 3.5 h in cultures prepared from suspect food samples and propagated 48 h in selective medium. The assay is based on PCR technology, and uses a specific primer set derived from sequences located down-stream of the hlyA gene. The specificity of the primer set was confirmed by testing 115 L. monocytogenes, 14 L. innocua, 5 L. seeligeri and 4 L. ivanovii isolates. The assay was compared to standard microbiological tests and gave identical results for 83 food samples, including 32 positives. These field trials indicate that the assay developed provides an alternative detection system for L. monocytogenes in foods, which can be used by the food industry.

A field survey of chemicals and biological products used in shrimp farming.

This study documented the use of chemicals and biological products in marine and brackish water shrimp farming in Thailand, the world's top producer of farmed shrimp. Interviews were conducted with 76 shrimp farmers in three major shrimp producing regions, the eastern Gulf coast, the southern Gulf coast and the Andaman coast area. Farmers in the study used on average 13 different chemicals and biological products. The most commonly used products were soil and water treatment products, pesticides and disinfectants. Farmers in the southern Gulf coast area used a larger number of products than farmers in the other two areas. In the study, the use of more than 290 different chemicals and biological products was documented. Many of the pesticides, disinfectants and antibiotics used by the farmers could have negative effects on the cultured shrimps, cause a risk for food safety, occupational health, and/or have negative effects on adjacent ecosystems. Manufacturers and retailers of the products often neglected to provide farmers with necessary information regarding active ingredient and relevant instructions for safe and efficient use.

Dopamine protects neurons against glutamate-induced excitotoxicity.

Glutamate excitotoxicity is responsible for neuronal death in acute neurological disorders including stroke, trauma and neurodegenerative disease. Loss of calcium homeostasis is a key mediator of glutamate-induced cell death. The neurotransmitter dopamine (DA) is known to modulate calcium signalling, and here we show that it can do so in response to physiological concentrations of glutamate. Furthermore, DA is able to protect neurons from glutamate-induced cell death at pathological concentrations of glutamate. We demonstrate that DA has a novel role in preventing delayed calcium deregulation in cortical, hippocampal and midbrain neurons. The effect of DA in abolishing glutamate excitotoxicity can be induced by DA receptor agonists, and is abolished by DA receptor antagonists. Our data indicate that the modulation of glutamate excitotoxicity by DA is receptor-mediated. We postulate that DA has a major physiological function as a safety catch to restrict the glutamate-induced calcium signal, and thereby prevent glutamate-induced cell death in the brain.

HtrA2 deficiency causes mitochondrial uncoupling through the F1F0-ATP synthase and consequent ATP depletion.

Loss of the mitochondrial protease HtrA2 (Omi) in mice leads to mitochondrial dysfunction, neurodegeneration and premature death, but the mechanism underlying this pathology remains unclear. Using primary cultures from wild-type and HtrA2-knockout mice, we find that HtrA2 deficiency significantly reduces mitochondrial membrane potential in a range of cell types. This depolarisation was found to result from mitochondrial uncoupling, as mitochondrial respiration was increased in HtrA2-deficient cells and respiratory control ratio was dramatically reduced. HtrA2-knockout cells exhibit increased proton translocation through the ATP synthase, in combination with decreased ATP production and truncation of the F1 α-subunit, suggesting the ATP synthase as the source of the proton leak. Uncoupling in the HtrA2-deficient mice is accompanied by altered breathing pattern and, on a cellular level, ATP depletion and vulnerability to chemical ischaemia. We propose that this vulnerability may ultimately cause the neurodegeneration observed in these mice.

Rapid identification of pathogenic yeast isolates by real-time PCR and two-dimensional melting-point analysis.

There is a need in the clinical microbiological laboratory for rapid and reliable methods for the universal identification of fungal pathogens. Two different regions of the rDNA gene complex, the highly polymorphic ITS1 and ITS2, were amplified using primers targeting conserved regions of the 18S, 5.8S and 28S genes. After melting-point analysis of the amplified products, the T(m) of the two PCR-products were plotted into a spot diagram where all the 14 tested, clinically relevant yeasts separated with good resolution. Real-time amplification of two separate genes, melting-point analysis and two-dimensional plotting of T(m) data can be used as a broad-range method for the identification of clinical isolates of pathogenic yeast such as Candida and Cryptococcus spp.

The sequence of a 32,420 bp segment located on the right arm of chromosome II from Saccharomyces cerevisiae.

The sequence of a 32,420 bp segment of Saccharomyces cerevisiae chromosome II has been deduced. The sequence data revealed 19 potential new genes covering 83.5% of the sequence. Four genes had already been cloned and sequenced: part of RIF1, DPB3, MRP-L27 and SNF5. Besides these four genes, 15 open reading frames (ORFs) of at least 100 amino acids encoding potential new genes were identified. Two of these ORFs are overlapping and a third is located within another ORF. The putative gene product of ORF YBR2039 was homologous to the group of uncoupling proteins involved in the mitochondrial energy transfer system. We propose a remapping of the MRP-L27 gene encoding the mitoribosomal protein YmL27 as it previously has been mapped on chromosome X. The ORF YBR2020 has a strong homology with a 31.9% identity in a 473 amino acid region to the yeast gene SEC61, suggesting that YBR2020 is a new gene encoding a protein involved in translocation of proteins in the yeast cell. Six of the potential genes do not exhibit any significant homology to previously sequenced genes as predicted in the Fast A analysis.

Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR.

An in situ PCR protocol by which we can monitor the presence or absence of lac mRNA in individual cells of a Salmonella typhimurium F' lac+ strain has been developed. In this protocol, fixed cells are permeabilized with lysozyme and subjected to a seminested reverse transcriptase PCR using reporter molecule-labeled primers, and subsequently, intracellular reporter molecules are detected microscopically at the individual-cell level by use of a horseradish peroxidase-conjugated antifluorescein antibody assay. In order to determine the sensitivity of the in situ PCR assay, the ability to detect lac mRNA in suboptimally isopropyl-beta-D-thiogalactopyranoside-induced cells was investigated. By use of a single-cell beta-galactosidase assay, it was confirmed that homogeneous suboptimally induced cultures of S. typhimurium F' lacY cells could be established, and the number of functional lac mRNAs in individual cells was estimated from standard population level beta-galactosidase assays. Cells estimated to contain a single lac mRNA were detected as containing lac mRNA by the in situ PCR method. Conclusively, we demonstrate the potential of in situ PCR for detection of even poorly expressed mRNA in individual bacterial cells.

Non-genetic population heterogeneity studied by in situ polymerase chain reaction.

Expression of a lac operon in Salmonella typhimurium single cells was monitored using lac mRNA targeting in situ reverse transcription-polymerase chain reaction (RT-PCR). It is demonstrated that suboptimal induction of the lac operon in a culture of S. typhimuriuml/F'lac+ cells generates a subpopulation in which transcription of the lac operon occurs and another subpopulation in which transcription of the lac operon is repressed, whereas suboptimal induction of the lac operon in a culture of S. typhimuriuml/F'lacY cells generates a population with uniform levels of lac mRNA. The outcome of the single-cell lac mRNA detection assay was compared with the outcome of a single-cell beta-galactosidase assay. In cultures grown under different suboptimal lac induction conditions, the fraction of cells in which transcription of the lac operon occurred was concurrent with the fraction of cells showing beta-galactosidase activity. Besides supporting the hypothesis that the lactose permease has a role in generating non-genetic heterogeneity in suboptimally induced cultures of Lac+ cells, these results demonstrate the usefulness of in situ RT-PCR for the study of non-genetic population heterogeneities.

A highly sensitive and fast nonradioactive method for detection of polymerase chain reaction products.

A novel system for the detection of polymerase chain reaction (PCR) products has been developed. The system is based on a PCR in which one of the primers is biotinylated and digoxigenin-11-dUTP is incorporated during elongation. Biotinylated PCR products are captured on streptavidin-coated solid supports, and alkaline phosphatase conjugated to anti-digoxigenin antibody is subsequently bound to the incorporated digoxigenin. The detection may be obtained with colorimetric, fluorescent, or luminescent substrates for alkaline phosphatase. The detection system can be performed in microtiter plates allowing easy handling of multiple samples. The total assay time following the PCR is between 1 and 2 h dependent on the type of substrate and the type of solid support applied in the system. Within this period of time the system is capable of detecting 1 template in 29 cycles of PCR.

Physiological states of individual Salmonella typhimurium cells monitored by in situ reverse transcription-PCR.

The possibility of using levels of specific mRNAs in individual bacteria as indicators of single-cell physiology was investigated. Estimates of the numbers of groEL and tsf mRNAs per cell in Salmonella typhimurium cells in different physiological states were obtained by Northern analysis. The average number of groEL mRNAs per cell was estimated to be 22 in fast-growing cultures and 197 in heat-shocked cultures. The average number of tsf mRNAs per cell was estimated to be 37 in fast-growing cultures, 4 in slow-growing cultures, and 0 in nongrowing cultures. The potential of mRNA-targeted in situ reverse transcription (RT)-PCR to monitor quantitatively different levels of groEL and tsf mRNA in individual cells and thus monitor both specific gene induction and general growth activity was assessed. Neither groEL nor tsf mRNA was present in stationary-phase cells, but it was shown that stationary-phase cells contain other RNA species at high levels, which may provide a possibility for monitoring directly stationary-phase individual cells by the use of in situ RT-PCR. The outcome of the in situ RT-PCR analyses indicated that a population of fast-growing cells is heterogeneous with respect to groEL mRNA single-cell contents, suggesting a cell-cycle-controlled expression of groEL in S. typhimurium, whereas a fast-growing culture is homogeneous with respect to tsf mRNA single-cell contents, suggesting that the level of tsf mRNA is relatively constant during the cell cycle.

Sleep-disordered breathing and coronary artery disease: long-term prognosis.

The evidence linking sleep-disordered breathing to increased mortality and cardiovascular morbidity has been conflicting and inconclusive. We hypothesized that a potential adverse effect of disordered breathing would be more obvious in patients with established vascular disease. In a prospective cohort study 408 patients aged 70 yr or younger with verified coronary disease were followed for a median period of 5.1 yr. An apnea-hypopnea index (AHI) of > or = 10 and an oxygen desaturation index (ODI) of > or = 5 were used as the diagnostic criteria for sleep-disordered breathing. The primary end point was a composite of death, cerebrovascular events, and myocardial infarction. There was a 70% relative increase and a 10.7% absolute increase in the primary composite end point in patients with disordered breathing defined as an ODI of > or = 5 (risk ratio 1.70, 95% confidence interval [CI] 1.15-2.52, p = 0.008). Similarly, patients with an AHI of > or = 10 had a 62% relative increase and a 10.1% absolute increase in the composite endpoint (risk ratio 1.62, 95% CI 1.09-2.41, p = 0.017). An ODI of > or = 5 and an AHI of > or = 10 were both independently associated with cerebrovascular events (hazard ratio 2.62, 95% CI 1.26-5.46, p = 0.01, and hazard ratio 2.98, 95% CI 1.43-6.20, p = 0.004, respectively). We conclude that sleep-disordered breathing in patients with coronary artery disease is associated with a worse long-term prognosis and has an independent association with cerebrovascular events.

Cardiac rhythm in patients with sleep-disordered breathing and coronary artery disease.

We examined the effect of sleep-disordered breathing on heart rates and arrhythmias in men and women with disabling angina pectoris and verified coronary artery disease by an overnight sleep study and Holter recording. The number of oxyhaemoglobin desaturations > or =4% (ODI) and number of apnoea-hypopnoeas per hour of sleep (AHI) were recorded. ODI > or =5 and AHI > or =10 were used as measures of disordered breathing and patients below these limits formed the control groups. One-hundred and forty-one men and 98 women < or =70 years of age were randomly included. Thirty-eight percent of the men and 36% of the women had an ODI > or =5. No serious ventricular arrhythmias occurred. Women with disordered breathing (ODI > or =5) had higher heart rates (mean 63.3 vs 59.1, p < 0.05) and a higher occurrence of ventricular premature contractions (VPCs) during sleep (75th percentiles 2.5 vs 0.5 per hour, p < 0.01). In men, however, no significant association between disordered breathing and heart rates or arrhythmias was found. We conclude that serious arrhythmias are infrequent in unselected patients with coronary artery disease and mild to moderate sleep-disordered breathing. Disordered breathing in women is associated with higher heart rates and a higher occurrence of VPCs during sleep.

Improved tolerance to salinity and low temperature in transgenic tobacco producing glycine betaine.

Glycine betaine is an osmoprotectant found in many organisms, including bacteria and higher plants. The bacterium Escherichia coli produces glycine betaine by a two-step pathway where choline dehydrogenase (CDH), encoded by betA, oxidizes choline to betaine aldehyde which is further oxidized to glycine betaine by the same enzyme. The second step, conversion of betaine aldehyde into glycine betaine, can also be performed by the second enzyme in the pathway, betaine aldehyde dehydrogenase (BADH), encoded by betB. Transformation of tobacco (Nicotiana tabacum), a species not accumulating glycine betaine, with the E. coli genes for glycine betaine biosynthesis, resulted in transgenic plants accumulating glycine betaine. Plants producing CDH were found to accumulate glycine betaine as did F1 progeny from crosses between CDH- and BADH-producing lines. Plants producing both CDH and BADH generally accumulated higher amounts of glycine betaine than plants producing CDH alone, as determined by 1H NMR analysis. Transgenic tobacco lines accumulating glycine betaine exhibited increased tolerance to salt stress as measured by biomass production of greenhouse-grown intact plants. Furthermore, experiments conducted with leaf discs from glycine betaine-accumulating plants indicated enhanced recovery from photoinhibition caused by high light and salt stress as well as improved tolerance to photoinhibition under low temperature conditions. In conclusion, introduction of glycine betaine production into tobacco is associated with increased stress tolerance probably partly due to improved protection of the photosynthetic apparatus.