Construction of a highly bioluminescent Nitrosomonas as a probe for nitrification conditions

Cloned luciferase-encoding operons were transferred by conjugation to a natural isolate of the ammonia-oxidizing bacterial strain Nitrosomonas sp. RST41–3, thereby establishing conjugation as a tool for gene transfer into Nitrosomonas strains. Luminescence was dependent on the pH of the medium and the concentration of the substrate ammonium chloride. Moreover, the luminescence of the transconjugants was reduced immediately by micromolar concentrations of nitrapyrin and allylthiourea, which are specific inhibitors of nitrification. Our results indicate that luminescent Nitrosomonas strains may be useful as a probe to detect nitrification conditions in the natural environment as well as in sewage plants.     

Methods of collection for salivary cortisol measurement in dogs

Salivary cortisol has been increasingly used as a measure of stress response in studies of welfare, reaction to stress and human-animal interactions in dogs and other species. While it can be a very useful measure, there are a number of saliva collection issues made evident through studies in the human and animal fields which have not been investigated in the canine species. Collection materials and the volume of saliva that is collected; the use of salivary stimulants; and the effect of food contamination can all dramatically impact cortisol measurement, leading to spurious results. In order to further examine the limitations of the collection method and the effects of collection material and salivary stimulant on salivary cortisol levels, a series of clinical, in vitro and in vivo studies were performed. It was found that there is a large amount of inter- and intra-individual variation in salivary cortisol measurement. Beef flavoring of collection materials leads to unpredictable variability in salivary cortisol concentration. Using salivary stimulants such as citric acid also has the potential to affect cortisol concentration measurement in saliva. Hydrocellulose appears to be a useful collection material for salivary cortisol determination. Recommendations for collection materials and use of salivary stimulants are presented.     

Responses of salivary cortisol levels to stress-situations

A procedure is described for determining salivary cortisol levels by a competitive protein-binding assay using horse transcortin. The collection of saliva was performed by means of filter paper-strips. Filter paper samples are more than 5 days stable after air-drying. In this form, the samples could be stored without refrigerator or deep-freezer and, if necessary, sent by post to the laboratory without any special precaution. Stressful situation of either painful or anxious origin were associated with an adequate increase of salivary cortisol levels. The increases were 157 to 230% of the initial or normal values dependent on the kind of stress. The mean values in 4 cases of Cushing's syndrome were 380% and 1 hour after 25 I.U. ACTH 690% higher than those in normal persons. In normal persons, a well-defined circadian rhythm has been observed.     

A bioluminescent assay for measuring glucose uptake

Identifying activators and inhibitors of glucose uptake is critical for both diabetes management and anticancer therapy. To facilitate such studies, easy-to-use nonradioactive assays are desired. Here we describe a bioluminescent glucose uptake assay for measuring glucose transport in cells. The assay is based on the uptake of 2-deoxyglucose and the enzymatic detection of the 2-deoxyglucose-6-phosphate that accumulates. Uptake can be measured from a variety of cell types, it can be inhibited by known glucose transporter inhibitors, and the bioluminescent assay yields similar results when compared with the radioactive method. With HCT 116 cells, glucose uptake can be detected in as little as 5000 cells and remains linear up to 50,000 cells with signal-to-background values ranging from 5 to 45. The assay can be used to screen for glucose transporter inhibitors, or by multiplexing with viability readouts, changes in glucose uptake can be differentiated from overall effects on cell health. The assay also can provide a relevant end point for measuring insulin sensitivity. With adipocytes and myotubes, insulin-dependent increases in glucose uptake have been measured with 10- and 2-fold assay windows, respectively. Significant assay signals of 2-fold or more have also been measured with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and skeletal myoblasts.     

A bioluminescent sensor for high throughput toxicity classification

A high throughput toxicity monitoring and classification biosensor system has been successfully developed using four immobilized bioluminescent Escherichia coli strains, DPD2511, DPD2540, DPD2794 and TV1061, which have plasmids bearing a fusion of a specific promoter to the luxCDABE operon. The bioluminescence of DPD2511 increases in the presence of oxidative damage, DPD2540 by membrane damage, DPD2794 by DNA damage and TV1061 by protein damage. In the developed biosensor these strains are immobilized in a single 96 well plate using an LB-agar matrix, and are able to detect the toxicities of hydrogen peroxide, phenol and mitomycin C in water samples. As the concentration of each chemical was increased, the bioluminescence levels from the corresponding wells, containing either DPD2511, DPD2540, DPD2794 or TV1061, increased. This increase in bioluminescence followed a dose dependent response to the toxic chemicals within a specific concentration range. In particular, each test requires only 4 h to give clear bioluminescent response signature. Storage of the biosensor at 4 degrees C for 2 weeks caused no change in its dose-dependent response. The fast and easy detection of oxidative, membrane, protein and DNA damaging agents in aqueous environments is possible due to the high throughput capability of this biosensor.     

A sensitive bioluminescent immunoassay for dinitrophenol and trinitrotoluene

A bioluminescent immunoassay for measuring dinitrophenol and trinitrotoluene (TNT) has been developed. The DNP and TNT were covalently linked to firefly luciferase, resulting in a conjugate containing 1 mol of DNP or trinitrophenyl (TNP) per mole of luciferase. The conjugate retained 90% of its original catalytic activity. When the conjugate was incubated with immobillzed anti-TNT or anti-DNP and varying concentrations of free TNT or DNP-leucine, the amount of conjugate bound was inversely proportional to the concentration of the free compound. Using this procedure it is possible to detect 2.5 pmol of DNP-leucine and 1.0 pmol of TNT. If the TNP or DNP is linked to glucose-6-phosphate dehydrogenase instead of luciferase, much lower quantities of antigen can be detected. This is due to the fact that this enzyme has a large turnover number so that amplification is possible. The NADH produced is measured using immobilized bacterial NADH:FMN oxidoreductase and luciferase. With this procedure, 10 amol (10^?17 mol) of antigen can be measured. These procedures should be suitable for measuring any antigen.     

A bioluminescent assay for the sensitive detection of proteases

A bioluminescent general protease assay was developed using a combination of five luminogenic peptide substrates. The peptide-conjugated luciferin substrates were combined with luciferase to form a homogeneous, coupled-enzyme assay. This single-reagent format minimized backgrounds, gave stable signals, and reached peak sensitivity within 30 min. The bioluminescent assay was used to detect multiple proteases representing serine, cysteine, and metalloproteinase classes. The range of proteases detected was broader and the sensitivity greater, when compared with a standard fluorescent assay based on cleavage of the whole protein substrate casein. Fifteen of twenty proteases tested had signal-to-background ratios >10 with the bioluminescent method, compared with only seven proteases with the fluorescent approach. The bioluminescent assay also achieved lower detection limits (≤100 pg) than fluorescent methods. During protein purification processes, especially for therapeutic proteins, even trace levels of contamination can impact the protein's stability and activity. This sensitive, bioluminescent, protease assay should be useful for applications in which contaminating proteases are detrimental and protein purity is essential.     

A bioluminescent reporter for the halophilic archaeon Haloferax volcanii

Haloarchaea have evolved to thrive in hypersaline environments. Haloferax volcanii is of particular interest due to its genetic tractability; however, few in vivo reporters exist for halophiles. Haloarchaeal proteins evolved characteristics that promote proper folding and function at high salt concentrations, but many mesophilic reporter proteins lack these characteristics. Mesophilic proteins that acquire salt-stabilizing mutations, however, can lead to proper function in haloarchaea. Using laboratory-directed evolution, we developed and demonstrated an in vivo luciferase that functions in the hypersaline cytosol of H. volcanii.

     

A luxCDABE-based bioluminescent bioreporter for the detection of phenol

A bioluminescent reporter strain, Acinetobacter sp. DF4-8, was constructed for the detection of phenol by inserting a mopR-like promoter upstream of the Vibrio fischeri bioluminescent luxCDABE gene cassette in a modified mini-Tn5 construct. When introduced into the chromosome of Acinetobacter sp. DF4, the bioreporter produced a sensitive bioluminescent response to phenol at concentrations ranging from 2.5 to 100 ppm. This response was linear (R ²=0.986) in the range from 20 to 90 ppm. A significant bioluminescent response was also recorded when strain DF4-8 was incubated with slurries from aged, phenol-contaminated soil. Received 13 February 2002/ Accepted in revised form 11 July 2002     

Electronic monitoring of salivary cortisol sampling compliance in daily life

Naturalistic research methods have been developed to collect data in the daily environment, providing ecological valid measures. Recent reports suggest, however, that compliance with fixed time sampling protocols may be problematic and can bias results. This study investigated compliance with an intensive, random time sampling protocol for salivary cortisol and effects of non-compliance on cortisol results. Twenty female twin pairs and nineteen of their sisters were instructed to take saliva samples when signaled at ten unpredictable moments on each of five consecutive days. Subjects recorded collection times, unaware that compliance with the sampling protocol was being investigated by means of electronic monitoring devices. Samples taken < or = 15 min after the signal, according to self-report, were defined as adherent to the protocol. Samples taken < or = 10 min after the self-reported collection time, according to the monitor, were defined as accurate. Self-reported adherence to the sampling protocol was 96.4%. Verified compliance was somewhat lower, with 81% of all saliva samples accurately timed. Contrary to previous reports, inclusion of non-compliant samples in the analysis did not distort the cortisol diurnal profile. Intensive, random time sampling appears to have advantages over fixed time sampling for obtaining valid cortisol profiles when researchers do not have devices to monitor compliance. Results indirectly support the validity of momentary self-report data about daily experiences obtained with the same sampling methods.     

Effect of exercise at three exercise intensities on salivary cortisol

Changes in cortisol concentration in response to exercise at 3 different intensities were quantified. Ten apparently healthy, recreationally active males participated. On 4 separate occasions, subjects were assigned a random order of 1-hour cycle ergometer bouts of exercise at 44.5 +/- 5.5%, 62.3 +/- 3.8%, and 76.0 +/- 6.0% (mean +/- SD) of VO2peak and a resting control session. Saliva samples were collected before exercise at 10, 20, 40, and 59 minutes of exercise and at 20 minutes of recovery. Differences in cortisol concentration were assessed via multivariate analysis of variance (alpha = 0.05) Tukey post hoc analysis when indicated. During the highest-intensity exercise session, cortisol was significantly higher at 59 minutes of exercise (p = 0.004) and at 20 minutes of recovery (p = 0.016) than at those same time points during the resting control session. No significant differences in cortisol concentration were noted among resting, low-, and moderate-intensity exercise. Exercise <40 minutes in duration elicited no significant differences at any intensity. These data indicate that only exercise of high intensity and long duration results in significant elevations of salivary cortisol.     

Circadian rhythms of salivary cortisol content during long-term space flight

Adaptation mechanisms of adrenal function related to secretion of cortisol were studied under conditions of microgravity. Parameters of diurnal rhythms of salivary cortisol were studied by Russian cosmonauts on board orbital station Mir during long-term space flights (SF). The preflight circadian rhythms of salivary cortisol in cosmonauts were characterized by the morning maximum occurring at 9∶43 a.m., the fluctuation amplitude 6.05 nmol/1, and the daily average concentration 8.79 nmol/l. The characteristics of cortisol diurnal rhythm changed under conditions of long-term space flight. On average, the rhythm measure and amplitude decreased after two months of flight. The postflight maximum concentration of free cortisol tended to occur later in the day. Evidently, the motor activity during SF, i.e., prophylactic exercises along with other factors, significantly influenced the parameters of cortisol circadian rhythm that was revealed by the individual variability of findings during the flight. After the long-term SF, individual ratios of salivary and plasma cortisol levels increased against the background of increased plasma content of the hormone, i.e., the fraction of free, physiologically active hormone in the total pool of circulating molecules decreased.     

Diagnostic value of salivary cortisol in end stage renal disease

OBJECTIVES: Salivary cortisol has been proposed a surrogate marker for free serum cortisol measurements. The aim of this study was to ascertain the diagnostic value of basal and stimulated salivary cortisol for the detection of adrenal insufficiency (AI) in hypotensive end stage renal disease (ESRD) patients. Basal salivary cortisol and basal total serum cortisol were studied in order to determine the accuracy of both biomarkers in predicting AI. PATIENTS AND METHODS: Twenty-nine ESRD patients with sustained hypotension were investigated for possible AI. Salivary cortisol was assessed at baseline and 30min after 25microg ACTH i.m. (LDTs). The dosage of salivary aldosterone was performed in salivary cortisol hypo-responders. Basal blood samples were drawn for steroids, renin and ACTH measurements. RESULTS: A clear separation between patients with normal and impaired adrenal function was obtained through salivary cortisol levels at 30min after ACTH. AI was detected in six cases (21%) through impaired salivary cortisol responses; stimulated salivary aldosterone helped to differentiate primary (n=3) from secondary AI (n=3). ROC curves showed that cutoff values for basal SAF < or =4.4nM and serum cortisol < or =232.0nM suggest AI (sensitivities: 93% and 69%; specificities: 86.4% and 91%, respectively). CONCLUSIONS: We conclude that ACTH stimulated SAF is an accurate biomarker for the diagnosis of AI in hypotensive ESRD patients. Neither basal salivary cortisol nor serum cortisol showed 100% sensitivities for the detection of AI.     

The salivary testosterone and cortisol response to three loading schemes

This aim of this study was to examine the free hormone (in saliva) responses to squat workouts performed by recreationally weight-trained males, using either a power (8 sets of 6 reps, 45% 1 repetition maximum [1RM], 3-minute rest periods, ballistic movements), hypertrophy (10 sets of 10 reps, 75% 1RM, 2-minute rest periods, controlled movements), or maximal strength scheme (6 sets of 4 reps, 88% 1RM, 4-minute rest periods, explosive intent). To determine the relative importance of the different training variables, these schemes were equated by workout duration with the power and strength schemes also equated by load volume. Salivary testosterone (T) and cortisol (C) both increased following the hypertrophy scheme (P < 0.05), with little to no hormonal change across the power and maximal strength schemes (P > 0.05). In general, the postexercise T and C responses to the hypertrophy scheme exceeded the other two schemes (P < 0.05). The greater volume of load lifted in the hypertrophy protocol over the same workout duration may explain the endocrine differences observed. The similar T and C responses to the power and maximal strength schemes (of equal volume) support such a view and suggest that differences in load intensity, rest periods, and technique are secondary to volume. Because the acute hormonal responses to resistance exercise contribute to protein metabolism, then load volume may be the most important workout variable activating the endocrine system and stimulating muscle growth.     

Abolished circadian rhythm of salivary cortisol in elite artistic gymnasts

Objective

The aim of this study was to evaluate the effects of intensive physical exercise and acute psychological stress during high level athletic competition as reflected on the levels of salivary cortisol in elite artistic gymnasts (AGs).

Design

The study included 239 AGs (142 females-97 males) who participated in the European Championship of Gymnastics in 2006 and 81 adolescents (40 females-41 males), matched for age, as controls. All athletes participated voluntarily in all or parts of the study, providing samples or data for each of the variables measured. Height, weight, body fat, lean body mass (LBM), bone age and Tanner stage of puberty were assessed and data concerning the time of thelarche, adrenarche and menarche as well as, the onset and the intensity (hours per week) of training were obtained.

Methods

Saliva samples were collected, the morning before training and in the afternoon shortly after the competition. From controls, the saliva samples were collected in the morning. Cortisol concentrations were measured using a chemiluminescence method. Acute stress was assessed using a questionnaire designed for the study.

Results

No difference was found between morning and afternoon salivary cortisol levels in both male and female AGs (females: AM: 15.45 ± 7.45 nmol/l vs PM: 15.73 ± 9.38 nmol/l; males: AM: 10.21 ± 5.52 nmol/l vs PM: 9.93 ± 13.8 nmol/l, p > 0.05).Female AGs presented higher levels of morning salivary cortisol than female controls (p < 0.05). Both male and female AGs had higher degree of psychological stress in comparison with controls (p < 0.001, p < 0.013, respectively). Female AGs had higher morning and afternoon salivary cortisol levels (p < 0.01, p < 0.01, respectively) and higher degree of stress (p < 0.003) than males.

Conclusions

In elite AGs the diurnal rhythm of salivary cortisol has been abolished, probably due to the strenuous training and competition conditions. Female AGs presented higher levels of morning salivary cortisol and psychological stress compared to both male AGs and female controls. The long term consequences of these modifications of the HPA axis remain to be elucidated.     

A multienzyme bioluminescent time-resolved pyrophosphate assay

We have developed a high-sensitivity assay for measurement of inorganic pyrophosphate (PPi) in adenosine 5'-triphosphate (ATP)-contaminated samples. The assay is based on time-resolved measurements of the luminescence kinetics and implements multiple enzymes to convert PPi to ATP that is, in turn, utilized to produce light and to hydrolyze PPi for measurement of the steady state background luminescence. A theoretical model for describing luminescence kinetics and optimizing composition of the assay detection mixture is presented. We found that the model is in excellent agreement with the experimental results. We have developed and evaluated two algorithms for PPi measurement from luminescence kinetics acquired from ATP-contaminated samples. The first algorithm is considered to be the method of choice for analysis of long, i.e., 3-5 min, kinetics. The activity of enzymes is controlled during the experiment; the sensitivity of PPi detection is about 7 pg/ml or 15 pM of PPi in ATP-contaminated samples. The second algorithm is designed for analysis of short, i.e., less than 1-min, luminescence kinetics. It has about 20 pM PPi detection sensitivity and may be the better choice for assays in microplate format, where a short measurement time is required. The PPi assay is primarily developed for RNA expression analysis, but it also can be used in various applications that require high-sensitivity PPi detection in ATP-contaminated samples.     

Lack of adult-type salivary cortisol circadian rhythm in hospitalized preterm infants

BACKGROUND/AIMS: Knowledge of the presence or absence of cortisol (F) circadian rhythm in preterm infants is important for the interpretation of F measurements made in samples taken for both clinical and research purposes. Little is known about its emergence in very preterm infants. This study examines circadian rhythm in F secretion in hospitalized infants born before 30 weeks' gestation. METHODS: Design: Prospective longitudinal observational study. Subjects: 11 infants admitted consecutively and born before 30 completed weeks of gestation. Measurements: F was measured by highly specific radioimmunoassay on morning and evening saliva samples gathered at weekly intervals until discharged home. Circadian rhythm was defined as > or =40% reduction from morning to evening level. RESULTS: For all data, the median salivary F was 10.3 nmol/l (range <0.5-372.8). F levels were highest in the first 3 weeks of life. No infants displayed classical circadian rhythm for 4 weeks or more prior to being discharged from hospital. The other infants showed randomly distributed morning and evening F values with a trend in 4 infants towards periods of consistently higher evening than morning values. CONCLUSION: Adult-type F circadian rhythm is rarely evident in hospitalized preterm infants born before 30 weeks' gestation.