Executive function subdomains are associated with post-stroke functional outcome and permanent institutionalization.

BACKGROUND AND PURPOSE: Impairment of executive functions (EFs) is a common cognitive symptom post-stroke and affects independence in daily activities. Previous studies have often relied on brief cognitive tests not fully considering the wide spectrum of EF subdomains. A detailed assessment of EFs was used to examine which of the subdomains and tests have the strongest predictive value on post-stroke functional outcome and institutionalization in long-term follow-up. METHODS: A subsample of 62 patients from the Helsinki Stroke Aging Memory Study was evaluated with a battery of seven neuropsychological EF tests 3 months post-stroke and compared to 39 healthy control subjects. Functional impairment was evaluated with the modified Rankin Scale (mRS) and Instrumental Activities of Daily Living (IADL) scale at 3 months, and with the mRS at 15 months post-stroke. Institutionalization was reviewed from the national registers of permanent hospital admissions in up to 21-year follow-up. RESULTS: The stroke group performed more poorly than the control group in multiple EF tests. Tests of inhibition, set shifting, initiation, strategy formation and processing speed were associated with the mRS and IADL scale in stroke patients. EF subdomain scores of inhibition, set shifting and processing speed were associated with functional outcome. In addition, inhibition was associated with the risk for earlier institutionalization. CONCLUSIONS: Executive function was strongly associated with post-stroke functional impairment. In follow-up, poor inhibition was related to earlier permanent institutionalization. The results suggest the prognostic value of EF subdomains after stroke.

Cold temperature decreases bacterial species richness in nitrogen-removing bioreactors treating inorganic mine waters.

Explosives used in mining, such as ammonium nitrate fuel oil (ANFO), can cause eutrophication of the surrounding environment by leakage of ammonium and nitrate from undetonated material that is not properly treated. Cold temperatures in mines affect nitrogen removal from water when such nutrients are treated with bioreactors in situ. In this study we identified bacteria in the bioreactors and studied the effect of temperature on the bacterial community. The bioreactors consisted of sequential nitrification and denitrification units running at either 5 or 10°C. One nitrification bioreactor running at 5°C was fed with salt spiked water. From the nitrification bioreactors, sequences from both ammonia- and nitrite-oxidizing bacteria were identified, but the species were distinct at different temperatures. The main nitrifiers in the lower temperature were closely related to the genera Nitrosospira and Candidatus Nitrotoga. 16S rRNA gene sequences closely related to halotolerant Nitrosomonas eutropha were found only from the salt spiked nitrification bioreactor. At 10°C the genera Nitrosomonas and Nitrospira were the abundant nitrifiers. The results showed that bacterial species richness estimates were low, <150 operational taxonomic units (OTUs), in all bioreactor clone libraries, when sequences were assigned to operational taxonomic units at an evolutionary distance of 0.03. The only exception was the nitrification bioreactor running at 10°C where species richness was higher, >300 OTUs. Species richness was lower in bioreactors running at 5°C compared to those operating at 10°C.

Interaction between CD14-159C>T polymorphism and Helicobacter pylori is associated with serum total immunoglobulin E.

BACKGROUND: Total serum IgE is regulated by both environmental and genetic factors. Association and linkage studies have suggested a role of CD14-159C>T polymorphism in the regulation of serum total IgE, but the results have been contradictory. It seems that gene-environment interactions are involved in this regulation. OBJECTIVE: The aim of this study was to examine the possible gene-environment interactions among Toxoplasma gondii, Helicobacter pylori, CD14-159C>T and Toll-like receptor (TLR) 4+896A>G polymorphism on serum total IgE. For this study, we expanded the scope of our earlier comparison of allergic sensitization and microbial load between Finland and Russian Karelia by studying the CD14-159C>T and TLR4+896A>G polymorphism in a cohort of Russian Karelian children. METHODS: For this study, CD14-159C>T and TLR4+896A>G polymorphisms were analysed in 264 healthy Russian Karelian children. Serum total IgE levels and H. pylori and T. gondii antibodies were also measured. RESULTS: We constructed a multiway anova model to analyse the gene-environment interactions among T. gondii seropositivity, H. pylori seropositivity, CD14-159C>T and TLR4+896A>G polymorphisms on serum total IgE. The model showed that there was an interaction between the CD14-159 allele T carrier status and H. pylori antibodies on serum total IgE (P=0.004). No other interactions were found. CONCLUSION: Our results further emphasize the role of gene-environment interaction in the regulation of serum total IgE.

Temporal reprogramming of calcium signalling via crosstalk of gonadotrophin receptors that associate as functionally asymmetric heteromers.

Signal crosstalk between distinct G protein-coupled receptors (GPCRs) is one mechanism that underlies pleiotropic signalling. Such crosstalk is also pertinent for GPCRs activated by gonadotrophic hormones; follicle-stimulating hormone (FSH) and luteinising hormone (LH), with specific relevance to female reproduction. Here, we demonstrate that gonadotrophin receptor crosstalk alters LH-induced Gαq/11-calcium profiles. LH-induced calcium signals in both heterologous and primary human granulosa cells were prolonged by FSHR coexpression via influx of extracellular calcium in a receptor specific manner. LHR/FSHR crosstalk involves Gαq/11 activation as a Gαq/11 inhibitor abolished calcium responses. Interestingly, the enhanced LH-mediated calcium signalling induced by FSHR co-expression was dependent on intracellular calcium store release and involved Gßγ. Biophysical analysis of receptor and Gαq interactions indicated that ligand-dependent association between LHR and Gαq was rearranged in the presence of FSHR, enabling FSHR to closely associate with Gαq following LHR activation. This suggests that crosstalk may occur via close associations as heteromers. Super-resolution imaging revealed that LHR and FSHR formed constitutive heteromers at the plasma membrane. Intriguingly, the ratio of LHR:FSHR in heterotetramers was specifically altered following LH treatment. We propose that functionally significant FSHR/LHR crosstalk reprograms LH-mediated calcium signalling at the interface of receptor-G protein via formation of asymmetric complexes.

Luminescent yeast cells entrapped in hydrogels for estrogenic endocrine disrupting chemical biodetection.

In the construction of luminescent yeast cell based fibre-optic biosensors, we demonstrate a novel approach for estrogenic endocrine disrupting chemical (EDC) biodetection by entrapping genetically modified Saccharomyces cerevisiae cells, containing the estrogen receptor alpha-mediated expression of the luc reporter gene, in hydrogel matrices based on calcium alginate or PVA. In order to insure a significant signal, an optimal immobilization ratio of 1:2 alginate 3% (w/v): 5 x 10(6) [cells/ml], respectively, was used with the highest 17-beta-estradiol (beta-E2) induction factor after 2.5 h of incubation with 10[nM] beta-E2. It was shown that biocompatible alginate beads, 4.27-4.55 x 10(5) [CFU/bead], which were characterized by a detection limit of 0.08[microg l(-1)] and an EC50 of 0.64[microg l(-1)] for beta-E2, retained their viability for luminescence measurements after 1 month of storage at -80 degrees C slow freeze condition, and thus repeated cell cultivations were not required. The assay reproducibility for each tested EDC, represented by the coefficients of variation (CV), ranged from 4.35 to 18.47%. An alternative immobilization method, based on a room temperature partial drying of polyvinyl alcohol (PVA) solution (LentiKat Liquid) and cell suspension mix, was investigated with only a slightly lower detection limit for beta-E2 than that reported with alginate beads. Alginate yeast based hydrogels may also be applicable to the analysis of environmental water samples since the trend of detected estrogenic activities with alginate beads roughly correlated with LC-MS-MS analytical results.

Kinetic measurement of the membranolytic activity of serum complement using bioluminescent bacteria.

A method for studying the kinetics of serum complement activity is presented. The assay utilises Escherichia coli and Bacillus subtilis cells which have been made bioluminescent by expressing an insect luciferase gene from Pyrophorus plagiophthalamus. The diffusion of the luciferase enzyme substrate through the cell membranes is very slow, and therefore a change in membrane permeability is seen as a change of the in vivo luminescence of the cells. Treatment of the bacteria with human serum resulted in a bell-shaped curve of in vivo luminescence since this procedure facilitates passage of the substrate to the cytoplasm. The time point of maximum light emission was dependent on serum dilution. In vivo luminescence also proved to be a good indicator of the viability of bacterial cells. Using C1q deficient serum, or following treatment of normal serum with EGTA and Mg2+ it was possible to separate the membranolytic activities of alternative and classical pathways.

Luminescent bacterial sensor for cadmium and lead.

A sensor plasmid was constructed by inserting the regulation unit from the cadA determinant of plasmid pI258 to control the expression of firefly luciferase. The resulting sensor plasmid pTOO24 is capable of replicating in Gram-positive and Gram-negative bacteria. The expression of the reporter gene as a function of added extracellular heavy metals was studied in Staphylococcus aureus strain RN4220 and Bacillus subtilis strain BR151. Strain RN4220(pTOO24) mainly responded to cadmium, lead and antimony, the lowest detectable concentrations being 10 nM, 33 nM and 1 nM respectively. Strain BR151(pTOO24) responded to cadmium, antimony, zinc and tin at concentrations starting from 3.3 nM, 33 nM, 1 microM, and 100 microM, respectively. The luminescence ratios between induced and uninduced cells, the induction coefficients, of strains RN4220(pTOO24) and BR151(pTOO24) were 23-50 and about 5, respectively. These results were obtained with only 2-3 h incubation times. Freeze-drying of the sensor strains had only moderate effects on the performance with respect to sensitivity or induction coefficients.

Construction and Use of Broad Host Range Mercury and Arsenite Sensor Plasmids in the Soil Bacterium Pseudomonas fluorescens OS8.

We have generated new sensors for the specific detection and studies of bioavailability of metals by engineering Pseudomonas fluorescens with reporter gene systems. One broad host range mercury (pTPT11) and two arsenite (pTPT21 and pTPT31) sensor plasmids that express metal presence by luminescence phenotype were constructed and transferred into Escherichia coli DH5a and Pseudomonas fluorescens OS8. The maximal induction was reached after 2 h of incubation in metal solutions at room temperature (22 degrees C). In optimized conditions the half maximal velocity of reaction was achieved at acidic pH using a d-luciferin substrate concentration that was nearly sixfold lower for P. fluorescens OS8 than for E. coli DH5a. When using a luciferin concentration (150 mM) that was optimal for E. coli the luminescence declined rapidly in the case of Pseudomonas, for which the substrate level 25 mM gave a stable reading between about 20 min and 3 h. The ability of the strain OS8 to quantitatively detect specific heavy metals in spiked soil and soil extracts is as good, or even better in being a real-time reporter system, than that of a traditional chemical analysis. The Pseudomonas strain used is an isolate from pine rhizosphere in oil and heavy metal contaminated soil. It is also a good humus soil colonizer and is therefore a good candidate for measuring soil heavy metal bioavailability.

A group-specific microbiological test for the detection of tetracycline residues in raw milk.

The potentiality of using a luminescent Escherichia coli strain for the specific detection of tetracycline residues in raw bovine milk was investigated. The sensor cells contain a reporter plasmid carrying the bacterial luciferase operon of Photorhabdus luminescens under the control of the tetracycline responsive control region from transposon Tn10. Incubation of the cells with the sample containing tetracyclines increases the light emission of the sensor cells. The most sensitive tetracycline detection was achieved in 120 min and by using CDTA as a chelating agent in the assay. Heat-treatment of milk before the assay decreased the variations in background luminescence signals and in tetracycline-induced luminescence between different milk samples. The detection limits for tetracycline, oxytetracycline, chlortetracycline, doxycycline, methacycline, demeclocycline, and minocycline were between 2 and 35 ng/mL. Nontetracycline antibiotics did not significantly interfere with the detection of tetracyclines.

Qualitative detection of tetracycline residues in milk with a luminescence-based microbial method: the effect of milk composition and assay performance in relation to an immunoassay and a microbial inhibition assay.

Performance of Tet-Lux, a newly developed microbiological test for the detection of tetracycline residues in raw milk, based on tetracycline-controlled luminescence activation of the test bacteria, was evaluated in bovine milks with variable amounts of somatic cells, bacteria, fat, protein, and natural inhibitory compounds. The sensitivity of Tet-Lux was also compared to a commercially available tetracycline immunoassay (Snap, Idexx Laboratories Inc.) and to a microbial inhibition test (Delvotest SP, Gist-Brogades). There were slight differences in the luminescence signals between different milk samples, but no single factor could be pointed out to be responsible for them. There appeared to be a modest inverse relationship between luminescence and increasing fat and protein content. The amount of somatic cells, bacteria, and the natural inhibitors lysozyme and lactoferrin did not affect the luminescence response. The test fulfilled the sensitivity requirement specified by the European Union (maximum residue limit 100 ng/ml for tetracyclines). The Tet-Lux test was clearly more sensitive to all tetracyclines tested (oxytetracycline, tetracycline, chlortetracycline, doxycycline, demeclocycline, methacycline, minocycline) than Delvotest SP, and for five tetracyclines out of seven more sensitive than Snap. The test provides a fast, simple, and robust microbial method for the qualitative detection of tetracycline residues in milk.

The interaction between concrete pavement and corrosion-induced copper runoff from buildings.

Changes in chemical speciation of copper and the capacity of concrete pavement to retain copper in runoff water from external buildings have been investigated at urban field conditions, and in parallel laboratory experiments simulating outdoor scenarios. The research study showed the concrete surface to form a copper rich surface layer ( approximately 50 microm thick) upon exposure, and a high capacity to significantly reduce the bioavailable fraction of released copper (20-95%). The retention capacity of copper varied between 5 and 20% during single runoff events in the laboratory, and between 10 and 40% of the total copper release during single natural runoff events. The capacity to retain and reduce the bioavailable fraction of non-retained copper increased with increasing wetness of the concrete surfaces, increasing pH of the runoff water and decreasing flow rates. Bioassay testing with bacterial and yeast bioreporters showed the bioavailable fraction of non-retained copper to be significantly lower than the total copper concentration in the runoff water, between 22 and 40% for bacteria and between 8 and 31% for yeast. The application of generated data to simulate a fictive outdoor scenario, suggests a significant reduction of bioavailable and total copper to background values during environmental entry as a result of dilution, and the interaction with solid surfaces, organic matter and complexing agents already in the drainage system.

Allergic sensitization and microbial load--a comparison between Finland and Russian Karelia.

Epidemiological data have indicated that some infections are associated with a low risk of allergic diseases, thus supporting the idea (hygiene hypothesis) that the microbial load is an important environmental factor conferring protection against the development of allergies. We set out to test the hygiene hypothesis in a unique epidemiological setting in two socio-economically and culturally markedly different, although genetically related, populations living in geographically adjacent areas. The study cohorts included 266 schoolchildren from the Karelian Republic in Russia and 266 schoolchildren from Finland. The levels of total IgE and allergen-specific IgE for birch, cat and egg albumen were measured. Microbial antibodies were analysed against enteroviruses (coxsackievirus B4), hepatitis A virus, Helicobacter pylori and Toxoplasma gondii. Although total IgE level was higher in Russian Karelian children compared to their Finnish peers, the prevalence of allergen-specific IgE was lower among Russian Karelian children. The prevalence of microbial antibodies was, in turn, significantly more frequent in the Karelian children, reflecting the conspicuous difference in socio-economic background factors. Microbial infections were associated with lower risk of allergic sensitization in Russian Karelian children, enterovirus showing the strongest protective effect in a multivariate model. The present findings support the idea that exposure to certain infections, particularly in childhood, may protect from the development of atopy. Enterovirus infections represent a new candidate to the list of markers of such a protective environment. However, possible causal relationship needs to be confirmed in further studies.

Genetic and environmental factors in the immunopathogenesis of atopy: interaction of Helicobacter pylori infection and IL4 genetics.

BACKGROUND: Both genetic and environmental factors, e.g. early childhood infections, have a role in the pathogenesis of atopic diseases. OBJECTIVE: To examine simultaneously the strength and possible interactions of two known such factors, IL4 genetics and Helicobacter pylori infection, on the risk of atopy and asthma. METHODS: Gene polymorphism analyses and skin prick tests (SPT) were determined in 245 adult asthmatics and 405 nonasthmatic controls of population-based case-control study. SPTs were used as an indicator of atopy. H. pylori infection was verified by detecting anti-H. pylori IgG antibodies in sera. RESULTS: A significant negative association was seen between the presence H. pylori antibodies and SPT positivity (> or =1 positive reactions) in both asthmatics and controls (p = 0.002 and p = 0.025, respectively) but the effect of IL-4 polymorphism (SNP -590C/T) was nonsignificant in both groups (p = 0.071 and p = 0.072, respectively). However, IL4 genetics had an effect on susceptibility to H. pylori: asthmatics carrying the IL4 -590 allele T had a diminished risk to be H. pylori infected (OR 0.485 95%CI 0.287-0.819). This effect was not seen in controls. Logistic regression analysis indicated that H. pylori and IL4 effects on atopy risk are not interdependent. CONCLUSIONS: This study showed that the effect of H. pylori infection on atopy risk is stronger than that of IL4 genetics. There is no interaction between these factors on the pathogenesis of atopy suggesting that these factors have distinct immunopathogenetic mechanisms. However, the genetic effect may modify the role of infective agents by effecting on susceptibility to disease.

Detection of organomercurials with sensor bacteria.

Mercury and its organic compounds, especially methylmercury, are hazardous compounds that concentrate in biota via biomagnification and cause severe neurological disorders in animals. In this paper, a recombinant whole-cell bacterial sensor for the detection of the organic compounds of mercury was constructed. The sensor carries firefly luciferase gene as a reporter under the control of the mercury-inducible regulatory part of broad spectrum mer operon from pDU1358. In addition, a gene-encoding organomercurial lyase (an enzyme necessary for cleavage of the mercury-carbon bond) was coexpressed in the sensor strain. The sensitivity of the sensor was evaluated on some environmentally important organomercurial compounds. The lowest detectable concentrations were 0.2 nM (50 ng/L), 1 nM (0.34 microg/L), and 10 microM (2.3 mg/L) for methylmercury chloride, phenylmercury acetate, and dimethylmercury, respectively. The sensor responded also to inorganic mercury and, therefore, using the sensor described here together with sensor bacteria responding only to inorganic mercury, it should be possible to characterize the mercury contamination, for example, in environmental samples.

Nitric oxide donor-mediated killing of bioluminescent Escherichia coli.

The antimicrobial activities of two nitric oxide-releasing compounds against Escherichia coli were investigated by using recombinant E. coli cloned with a luciferase gene from Pyrophorus plagiophthalamus. Since luciferase uses intracellular ATP to generate visible light which can be measured from living cells in real time, we wanted to compare the extent to which cell viability parallels light emission. Results from luminescence measurements and CFU counts were in good agreement, and the decrease in light emission was shown to provide a rapid and more sensitive indication of cytotoxicity.

Use of controlled luciferase expression to monitor chemicals affecting protein synthesis.

In this article, we present a new bioluminescent test system for the screening of chemical compounds with an inhibitory effect on protein synthesis. The test is based on the measurement of real-time in vivo light production by Escherichia coli strains expressing different luciferase genes. The eukaryotic lucGR gene from Pyrophorus plagiophthalamus was found to be the best of three types of luciferase genes tested. Chemicals with known inhibitory effects on protein synthesis were used as test chemicals together with some general toxicants. The incubation of a test chemical with cells was performed either prior to or after the induction of protein synthesis, and the difference in the results of the two methods distinguishes the possible influence on protein synthesis from direct metabolic inhibition. Using lyophilized bacteria, the test is performed in less than an hour without any bacterial cultivation, which makes the test suitable for rapid and sensitive screening of chemicals or environmental samples. Compared with the standardized 50% inhibitory concentration calculation method of the bioluminescent cytotoxicity test, the more direct approach of calculation developed in this study proved to be more convenient than and as reliable as the standard method.

One-step measurement of firefly luciferase activity in yeast.

Firefly luciferase is often used as a sensitive genetic reporter in various cell types. The pitfall in yeast, however, has been the need to break down the rigid cells in order to measure the enzyme activity. In this study we have removed the peroxisomal targeting codons from the Photinus pyralis luciferase gene (luc) and shown that in the yeast Saccharomyces cerevisiae this modified luciferase gives high levels of light emission that is easy to measure from intact living cells. Furthermore, cells with the modified luciferase grew essentially faster than those with the wild-type luciferase, indicating that peroxisomal targeting of a foreign enzyme puts some constraints to cellular viability. As a model system we used two different reporter constructs. In the first, expression of the luciferase gene is under control of CUP1-promoter, a well known yeast promoter that is inducible by copper ions. In the second, luciferase activity is dependent on activation of the human oestrogen receptor and its interaction with oestrogen-responsive elements incorporated in a yeast promoter. The luciferase activity measurement could be done on a 96-well plate by simple addition of the substrate, D-luciferin, at a moderately acidic pH of 5.0. The ease of use of the non-peroxisomal luciferase makes it an interesting alternative for reporter genes that are conventionally used in yeast, such as lacZ.

Recombinant luminescent bacteria for measuring bioavailable arsenite and antimonite.

Luminescent bacterial strains for the measurement of bioavailable arsenite and antimony were constructed. The expression of firefly luciferase was controlled by the regulatory unit of the ars operon of Staphylococcus aureus plasmid pI258 in recombinant plasmid pTOO21, with S. aureus RN4220, Bacillus subtilis BR151, and Escherichia coli MC1061 as host strains. Strain RN4220(pTOO21) was found to be the most sensitive for metal detection responding to arsenite, antimonite, and cadmium, the lowest detectable concentrations being 100, 33, and 330 nM, respectively. Strains BR151(pTOO21) and MC1061(pTOO21) responded to arsenite, arsenate, antimonite, and cadmium, the lowest detectable concentrations being 3.3 and 330 microM and 330 and 330 nM with BR151(pTOO21), respectively, and 3.3, 33, 3.3, and 33 microM with MC1061(pTOO21), respectively. In the absence of the mentioned ions, the expression of luciferase was repressed and only a small amount of background light was emitted. Other ions did not notably interfere with the measurement in any of the strains tested. Freeze-drying of the cells did not decrease the sensitivity of the detection of arsenite; however, the induction coefficients were somewhat lower.