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PubMed2002

A reverse transcription comparative real-time PCR method for quantitative detection of angiogenic growth factors in head and neck cancer patients.

OBJECTIVES: Head and neck cancer is one of the ten most frequent cancers in the world. The angiogenic growth factors VEGF, PDGF and bFGF play a role in cancer aggressiveness. We developed a sensitive method to quantify the gene expression of these factors in the tissues of head and neck cancer patients. DESIGN AND METHODS: All assays were performed using real-time RT-PCR, which yields a value (Ct) denoting the threshold cycle of PCR amplification at which product is first detected by fluorescence. The Ct is dependent on the quantity of the target molecule in the sample. To control for variation in RNA quantity and quality, we used 18S ribosome RNA as an internal control to calculate a relative Ct for the target molecules of interest, VEGF, PDGF and bFGF. A serially diluted positive control sample was analyzed by linear regression to determine the sensitivity and linearity of the assay. Paired normal and cancerous tissue samples from 115 head and neck cancer patients were assayed to ascertain the relative levels of the growth factors. RESULTS: The CVs of within-run and between-run assays for VEGF, PDGF and bFGF were all less than 3%. The correlation coefficient of the RNA concentrations and Ct values were 0.9987, 0.9977, and 0.9996 respectively for VEGF, PDGF and bFGF. The assay was sensitive to as little as 10(-3) ng of RNA. All three growth factors were significantly increased in tumor tissue as compared to normal tissue. VEGF, PDGF and bFGF levels were elevated in 71.3%, 58.2% and 54.0% of cancerous tissue samples, with average levels of over-expression of 35.1, 24.6 and 13. sixfold, respectively. CONCLUSION: This method provides sensitive, quantitative, high-throughput analysis for direct comparison of gene expression levels between samples, while adjusting for factors that may influence quantity determination. It should be applicable to molecules other than angiogenic growth factors, as well.

Endothelial Growth Factors↗
PubMed1980

Phase-space description of the cell cycle: application to noncycling, senescent, and transformed cells.

The behavior of a cellular biochemical reaction system can be portrayed by its trajectory in phase space. Phase-plane trajectories are proposed which depict the concentration of a characteristic chemical species in normally reproducing diploid cells, and in their noncycling, transformed, and senescent counterparts. It is assumed that the biochemical reaction system which determines the concentration of the characteristic species is nonlinear and nonconservative; the trajectories are therefore analyzed with the help of established qualitative mathematical techniques which are applicable to such systems. Mitotic cycles are presented as stable limit cycles; noncycling and senescent states are represented as isolated stable singular points. The concentration trajectory traversed in the transformed cell cycle surrounds the trajectory traversed in the normal cell cycle, which itself surrounds the singular point corresponding to the noncycling or senescent state. Transitions between the normal cycle and the noncycling state are associated with changes in the concentration of the characteristic species which exceed cyclically varying rate-dependent threshold values. Transitions from the normal cycle to the transformed cycle or to the senescent state are described by the coalescence and disappearance of the normal stable limit cycle with one of two adjacent unstable limit cycles.

Cell Cycle↗
PubMed2000

Basal antral follicle number and mean ovarian diameter predict cycle cancellation and ovarian responsiveness in assisted reproductive technology cycles.

OBJECTIVE: To determine the predictive value and define threshold levels for basal antral follicle number and mean ovarian diameter in patients undergoing ART cycles. DESIGN: Retrospective. SETTING: Tertiary care center. PATIENTS: Two hundred seventy-eight patients who had ovarian measurements performed on cycle day 3 before beginning treatment with gonadotropins. INTERVENTION: Pretreatment ovarian ultrasound measurements. MAIN OUTCOME MEASURE: Number of oocytes retrieved, hormone levels, and cycle outcomes. RESULTS: A direct linear correlation was observed between mean ovarian diameter and basal follicle number. Both measures demonstrated a positive linear correlation with recovered oocytes, basal E(2), and peak E(2). Both demonstrated a negative linear correlation with ampules of gonadotropins administered, days of stimulation, patient age, cycle day 3 FSH, and FSH:LH ratio. An antral follicle count of </=10 or a mean ovarian diameter of <20 mm was associated with an increased risk of cycle cancellation. CONCLUSIONS: Ovarian diameter and basal antral follicle number identify patients who may respond poorly to ART stimulation. These ovarian measures correlate well with ART screening and stimulation parameters. This knowledge allows physicians to evaluate and counsel patients immediately before an ART stimulation and to optimize stimulation protocols.

Female↗
PubMed2003

A prospective assessment of the predictive value of basal antral follicles in in vitro fertilization cycles.

OBJECTIVE: To determine the predictive value and define threshold values for basal antral follicle count in patients undergoing IVF. DESIGN: Prospective cohort analysis. Tertiary care center. Two hundred eighty-nine patients. Transvaginal ultrasonography before starting gonadotropin administration. MAIN OUTCOME MEASURES: Number of oocytes retrieved, basal hormone levels, and cycle outcomes. RESULTS: Pregnant patients had significantly more antral follicles (13.8 +/- 7.5 vs. 12.4 +/- 10.0). Patients in whom cycles were canceled had significantly fewer antral follicles (7.6 +/- 4.8 vs. 13.7 +/- 8.8). Antral follicle count significantly correlated with most prestimulation and poststimulation IVF variables. Threshold analysis demonstrated a lower pregnancy rate (23.5% vs. 57.6%) and a higher cancellation rate (41% vs. 6.4%) associated with having four or fewer antral follicles. CONCLUSION(S): The basal antral follicle count identified patients who responded poorly to IVF stimulation. Having four or fewer antral follicles was associated with a high cancellation rate (41%) and, in patients without a cancelled cycle, a low pregnancy rate (23%). However, no antral follicle count absolutely predicted pregnancy or cycle cancellation.

Cohort Studies↗
PubMed2006

Evaluation of post-polymerase chain reaction melting temperature analysis for meat species identification in mixed DNA samples.

Real-time uniplex and duplex polymerase chain reaction (PCR) assays with a SYBR Green I post-PCR melting curve analysis were evaluated for the identification and quantification of bovine, porcine, horse, and wallaroo DNA in food products. Quantitative values were derived from threshold-cycle (C(t)) data obtained from serial dilutions of purified DNA. The limits of detection in uniplex reactions were 0.04 pg for porcine and wallaroo DNA and 0.4 pg for cattle and horse DNA. Species specificity of the PCR products was tested by the identification of peaks in DNA melting curves, measured as the decrease of SYBR Green I fluorescence at the dissociation temperature. The peaks could be distinguished above the background even at the lowest amount of template DNA detected by the C(t) method. The system was also tested in duplex reactions, by use of either single-species DNA or DNA admixtures containing different shares of two species. The minimum proportions of each DNA species allowing the resolution of T(m) peaks in the duplex reactions were 5% (cattle or wallaroo) in cattle/wallaroo mixtures, 5% porcine and 1% horse in porcine/horse mixtures, 60% porcine and 1% wallaroo in porcine/wallaroo mixtures, and 1% cattle and 5% horse in cattle/horse mixtures. A loss in the sensitivity of the method was observed for some DNA combinations in the duplex assay. In contrast, the results obtained from SYBR Green I uniplex and duplex reactions with single-species DNA were largely comparable to those obtained previously with species-specific TaqMan probes, showing the suitability of that simpler experimental approach for large-scale analytical applications.

Animals↗
PubMed2005

Validation of pharmacodynamic assays to evaluate the clinical efficacy of an antisense compound (AEG 35156) targeted to the X-linked inhibitor of apoptosis protein XIAP.

The inhibitor of apoptosis protein, XIAP, is frequently overexpressed in chemoresistant human tumours. An antisense oligonucleotide (AEG 35156/GEM 640) that targets XIAP has recently entered phase I trials in the UK. Method validation data are presented on three pharmacodynamic assays that will be utilised during this trial. Quantitative RT-PCR was based on a Taqman assay and was confirmed to be specific for XIAP. Assay linearity extended over four orders of magnitude. MDA-MB-231/U6-E1 cells and clone X-G4 stably expressing an RNAi vector against XIAP were chosen as high and low XIAP expression quality controls (QCs). Within-day and between-day coefficients of variation (CVs) in precision for cycle threshold (CT) and delta CT values (employing GAPDH and beta 2 microglobulin as housekeepers) were always less than 10%. A Western blotting technique was validated using a GST-XIAP fusion protein as a standard and HeLa cells and SF268 (human glioblastoma) cells as high and low XIAP expression QCs. Specificity of the final choice of antibody for XIAP was evaluated by analysing a panel of cell lines including clone X-G4. The assay was linear over a 29-fold range of protein concentration and between-day precision was 29% for the low QC and 23% for the high QC when normalised to GAPDH. XIAP protein was also shown to be stable at -80 degrees C for at least 60 days. M30-Apoptosense plasma Elisa detects a caspase-cleaved fragment of cytokeratin 18 (CK18), believed to be a surrogate marker for tumour cell apoptosis. Generation of an independent QC was achieved through the treatment of X-G4 cells with staurosporine and collection of media. Measurements on assay precision and kit-to-kit QC were always less than 10%. The M30 antigen (CK18-Asp396) was stable for 3 months at -80 degrees C, while at 37 degrees C it had a half-life of 80-100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited.

Antineoplastic Agents↗
PubMed2004

Effects of pulsed electromagnetic fields on cognitive processes - a pilot study on pulsed field interference with cognitive regeneration.

BACKGROUND: Due to the ubiquitous use of cellular phones much has been speculated on secondary effects of electromagnetic irradiation emitted by those. Additionally, several studies have reported vegetative alterations as well as effects on the neuronal and molecular levels in humans. Here, using a psycho-physiological test paradigm, we examined effects of exposure to pulsed electromagnetic fields on cognitive performance. MATERIALS AND METHODS: In 11 volunteers, we tested cognitive processing under field exposure (GSM standard) and under field-free conditions. To examine the hypothesized effect of pulsed fields, we applied an auditory discrimination task and determined the participant's current 'Order Threshold' value. Following a first test cycle, the volunteers had to relax for 50 min while being, or not, exposed to pulsed electromagnetic fields. Subsequently, the test was repeated. Data acquired before and after the resting phase were compared from both experimental conditions. RESULTS: We found that nine of the 11 test participants (81.8%) showed worse results in their auditory discrimination performance upon field exposure as compared with control conditions. Group data comparison revealed a statistical significance of P = 0.0105. CONCLUSION: We could show that the participants' cognitive performance was impaired after exposure to pulsed electromagnetic fields. With regard to this finding, we recommend that the use of cellular phones should be restricted generally and in particular in respect of physical hazard of high-risk groups, e.g. elderly, children and ill people.

Acoustic Stimulation↗
PubMed1990

The effects of superior laryngeal nerve stimulation on the respiratory rhythm: phase-resetting and aftereffects.

The effect of brief superior laryngeal nerve stimulation on the respiratory rhythm was investigated in midcollicular decerebrate, unanesthetized, artificially ventilated, paralysed, vagotomized and debuffered cats. Stimulus trains (50 ms, 200 Hz) delivered during inspiration (I) with intensities exceeding a threshold value, that was inversely related to the phase of the cycle, terminated I and shortened the following expiration (E) (irreversible I termination). Stimulus trains given during I with intensities just below this threshold value produced a transient suppression of I followed by resumption of activity, resulting in a slight prolongation of both I and the following E (reversible I termination). Stimulation during E produced a phase-dependent prolongation of E, but did not affect the next I. Phase-resetting curves were constructed by measuring the changes in respiratory cycle duration produced by stimuli given at phases throughout the cycle. A single stimulus produced aftereffects that lasted several cycles. The aftereffects were investigated by delivering stimuli at a fixed delay from cycle onset every n cycles (n is an integer). Certain combinations of delay, stimulus intensity, and n, resulted in (1) a variable combination of reversible and irreversible I terminations, rather than a consistent response, and (2) an increase in cycle duration. The stimulus aftereffects, that can last up to 9 cycles, may account for previously described unpredictability in the response of the respiratory oscillator to a given stimulus.

Animals↗
PubMed2006

Changes in climate and habitat suitability for the cattle tick Boophilus microplus in its southern Neotropical distribution range.

We addressed the possible effects of several climate scenarios on habitat suitability (HS) for the cattle tick Boophilus microplus and the probability of producing permanent populations from introduced females of that tick in central parts of Argentina, using both a correlative model (derived from climate predictors) and a mechanistic (life cycle) model. There was high correlation (R2 = 0.866) between HS-derived and life cycle outputs for HS values higher than 0.52, suggesting that HS is a good estimator of the life cycle of the tick above a critical threshold of HS values. Scenarios with increased temperatures increased suitable habitats for the tick in southern parts of the study region, extending below parallel 34 degrees S, but suitable habitats remained limited in the west. A concurrent increase in rainfall produced a further increase of HS in these areas. Results from the life cycle model suggest that in areas of suitable habitat, permanent cattle tick populations are most probable if engorged females are introduced during mid-summer.

Animals↗
PubMed1993

The relationship between follicle-stimulating hormone dose and level and its relevance for ovulation induction with adjuvant gonadotropin-releasing hormone-agonist treatment.

OBJECTIVE: To investigate the effect of a GnRH agonist (GnRH-a) on the FSH threshold level and the relationship between the FSH dose and the FSH level of patients suffering from polycystic ovarian syndrome (PCOS). DESIGN: The stimulation with low-dose FSH in PCOS (group 1) was compared with the subsequently performed stimulation with low-dose FSH combined with GnRH-a in another group of patients suffering from the same syndrome (group 2). SETTING: Specialist Reproductive Endocrine Unit. PATIENTS: Suffering from clomiphene citrate-resistant PCOS. MAIN OUTCOME MEASURES: The FSH threshold level for ongoing follicular growth and the relationship between dose and level of FSH. RESULTS: In 15 patients in group 1 and in 13 patients in group 2, respectively, 39 and 32 stimulation cycles were performed. Below and above threshold values of FSH of group 1 and 2 did not differ significantly. For the equation stable level of FSH (Y mIU/mL) = A X infusion rate of FSH (X IU/24 h) + basal level of FSH (B mIU/mL), the median A of group 1 was 0.027 and A of group 2 was 0.055 (significant difference). CONCLUSIONS: In PCOS, a change of the FSH threshold level for ongoing follicular growth induced by the GnRH-a could neither be proven nor ruled out. The use of a GnRH-a resulted in larger FSH level increases per IU/24 h of FSH administered and might therefore interfere with the effect of low-dose FSH treatment.

Adjuvants, Pharmaceutic↗
PubMed1969

Interference of moonlight with the photoperiodic measurement of time by plants, and their adaptive reaction.

Threshold values of photoperiodic time-measurements correspond approximately to moonlight intensities. Experiments with Glycine and Euglena reveal that this is also the threshold value for synchronization of the circadian cycle. Saturation of this reaction is reached with 10 lx in 12:12 hr light-dark cycles. Thus, moonlight might disturb time measurement.In Glycine, Arachis, and Trifolium the intensity of the light coming from the moon to the upper surface of the leaf is reduced by circadian leaf movement to values between 5 and 20 per cent (or even less than 5 per cent) of full-moon light intensity. Such a reduction eliminates the disturbing effects of moonlight. This finding indicates that leaf movements have an adaptive value of the kind that Darwin sought to identify. It also indicates that the behavior of the upper leaf epidermis as a "sense organ for light"(13) has an adaptive value.In the short-day plants Perilla ocymoides and Chenopodium amaranticolor, a specific photoperiodic phenomenon was found that counteracts the disturbing effect of moonlight. Here light intensities similar to those of moonlight, introduced during the night, promote flowering instead of inhibiting it.

Journal Article↗
PubMed2006

Psychophysically determined thresholds for thermal perception and pain perception in healthy women across the menstrual cycle.

OBJECTIVES: Several studies have indicated changes in sensation and/or pain sensitivity among women across the menstrual cycle, but the pattern of the changes varies considerably. One reason for the reported discrepancies could reside in lack of biochemical definition of menstrual cycle phase. The aim was to quantify temperature and temperature pain thresholds at biochemically defined stages of the menstrual cycle. METHODS: Nineteen healthy women were included in the study. Temperature and temperature pain thresholds were evaluated by quantitative sensory testing, performed at 3 occasions during the menstrual cycle (early follicular phase, late follicular phase, and mid-luteal phase). At each test session, serum concentrations of estradiol and progesterone were assessed. RESULTS: Thermal cold perception threshold at the mammilla was significantly lower in the late follicular and mid-luteal phases, compared with the early follicular phase (P<0.05, respectively). For the remaining test sites, no cycle related differences in thermal perception or thermal thresholds could be documented. CONCLUSIONS: The present study has indicated no major changes in thermal pain thresholds related to phase of the menstrual cycle for the tested locations, although thermal cold perception threshold at the mammilla was a significantly lower in the late follicular and mid-luteal phases, compared with the early follicular phase. The findings of the present study further underlines the need for strict criteria for menstrual cycle phase when studying pain sensitivity in relation to hormonal events in women.

Adult↗
PubMed1998

Improved quantitation of minimal residual disease in multiple myeloma using real-time polymerase chain reaction and plasmid-DNA complementarity determining region III standards.

The complementarity determining region III of the rearranged immunoglobulin heavy chain gene has been the target for tumor-specific PCR assays for the detection and follow-up of B-cell malignancies. Previously, these assays have relied on gel-based end point data collection methods (i.e., band densitometry) and, thus, have provided at best a semiquantitative assessment of tumor levels. We show the development of a novel, real-time TaqMan PCR assay to quantitate residual multiple myeloma cells in clinical samples after high-dose chemotherapy and autologous stem cell transplantation. We provide evidence that real-time PCR is reproducible, sensitive, and quantitative. In a 40-replicate PCR experiment targeting the beta-actin gene, the coefficient of variation for threshold cycle data was 1.6%, whereas it increased to 13.6% and 31%, respectively, for end point fluorescence and gel densitometry. Moreover, in an experiment directly comparing standard curves obtained from band densitometry and threshold cycle data, the standard curve constructed from threshold cycle data had a multiple R2 value of 1.00 and demonstrated a dynamic range >4 logs, compared with the 2-log linear range of gel densitometry. Finally, we show that when a complementarity determining region III-specific PCR primer is used in conjunction with a consensus primer for the immunoglobulin heavy chain joining gene, plasmid DNA can be used as a readily available and effective substitute for clonal plasma-cell genomic DNA when preparing standards. By applying real-time PCR to the analysis of clinical samples, we are able to quantitate levels of tumor involvement with unparalleled reproducibility and statistical confidence. Real-time PCR technology may well provide the accuracy and reliability necessary for minimal residual disease detection to have real prognostic significance.

Base Sequence↗
PubMed2003

The effects of stages of the estrous cycle on pain thresholds in female white rats.

The effects of stages in the estrous cycle on electrical pain thresholds were studied in white rats in conditions of chronic measurement. On recording day 3, females in the diestrus and estrus stages showed sharp increases in shudder and paw-shuffling thresholds. Females in proestrus and metestrus showed no change in thresholds as compared with those in the first days of recording. Starting from day 7, there were progressive decreases in electrical pain thresholds regardless of the stage of the estrous cycle. Data for all animals showed that regular measurement of pain thresholds induces a smooth two-fold increase in body resistance from day 1 to day 7. This was followed by a sharp drop on day 8, with parallel decreases in body resistance and electrical pain thresholds. Thus, chronic measurement of electrical pain thresholds and body resistance changed in a complex fashion, and threshold values depended on the stage of the estrous cycle.

Animals↗
PubMed2001

Evaluation of 5' nuclease assay for detection of Actinobacillus pleuropneumoniae.

Sequence detection by the 5' nuclease TaqMan assay uses online detection of internal fluorogenic probes in closed PCR tubes. Primers and probe were chosen from a part of the omlA gene common to all serotypes of Actinobacillus pleuropneumoniae, which gave an amplicon of 92 bp. The test was evaluated with 73 lung isolates and 120 tonsil isolates of A. pleuropneumoniae as well as with a collection of reference strains. By using a C(t) value (cycle number in which the fluorescence exceeds the threshold defined by the software) of 30 as the cutoff limit, the 5' nuclease assay represents a test with 100% sensitivity and 100% specificity. A high degree of reproducibility of the test was demonstrated. If samples with C(t) values of </=30 are considered positive, the detection limit of the assay was 1 CFU/reaction tube, corresponding to a 10-fold higher number of DNA templates. After cycle 30, nonspecific reactions appeared when testing dilutions of DNA templates or pure cultures of A. pleuropneumoniae, as well as when testing tonsil scrapings from specific-pathogen-free herds. The diagnostic sensitivity, as evaluated with 586 tonsil scrapings from animals infected with A. pleuropneumoniae, is the same level as that of a PCR test based on the omlA gene described previously. The 5' nuclease assay represents a fast method for species-specific detection and identification of A. pleuropneumoniae in pure and mixed cultures. The evaluation shows, however, that a C(t) value cutoff limit of </=30 must be chosen in order to obtain reliable results. The investigation emphasizes that a thorough evaluation of the criteria used to define a positive test result is necessary.

Actinobacillus Infections↗
PubMed2005

Night-time melatonin secretion and seasonally delayed puberty in gilts.

This study was conducted to determine whether the seasonal delay in puberty in autumn is driven by individual differences in night-time melatonin secretion in domestic gilts at the attainment of puberty. A group of spring-born gilts (n = 30) were expected to reach puberty in autumn by the age of 7 months. Eighteen of these gilts were selected in pairs on the basis of matched days of birth. By the expected time, half of the animals showed oestrous symptoms (group CYCLING, n = 9) with the rest remaining silent (group SILENT, n = 9). Afterwards, all gilts were fitted with indwelling jugular catheters for frequent blood sampling. Blood samples were collected from all animals three times during the day followed by three times in the night at 2-h intervals for 48 h. The samples were analysed by a commercial radioimmunoassay (RIA). The results show a consistent 25-fold rise (on average) in night-time melatonin concentration in every animal sampled with group averages ranging from 0.28 +/- 0.04 to 0.37 +/- 0.06 pg/ml at day and from 10.20 +/- 2.16 to 10.67 +/- 0.05 pg/ml at night. Night-time group mean values between CYCLING and SILENT gilts did not differ significantly (10.26 +/- 0.67 and 10.38 +/- 0.94 for the CYCLING; 10.67 +/- 0.05 and 10.20 +/- 2.16 for the SILENT). When 10 pg/ml was used as a threshold value, six individuals did not reach it during the night (low responders). Two of these gilts were CYCLING and four were SILENT. In conclusion, the results presented imply no involvement of the level of night-time melatonin concentration in the seasonal delay of puberty in gilts.

Animals↗
PubMed2001

Rhinomanometric and olfactometric variations throughout the menstrual cycle.

A prospective study was performed to evaluate the rhinomanometric values and the changes in the olfactory threshold that occur in women throughout the menstrual cycle. The subjects were 60 healthy volunteer premenopausal women 18 to 40 years of age. We performed rhinomanometric and olfactometric measurements during the follicular, periovular, and luteal phases of the menstrual cycle. Rhinomanometry showed a higher (not statistically significant) airflow during the periovular phase than during the follicular and luteal phases. Olfactometry showed a higher sensitivity during the follicular phase (p < .05) and the periovular phase (p < .001) than during the luteal phase. We conclude that airflow and transnasal pressure during spontaneous respiration, as well as the olfactory threshold to odors, seem to depend on the variations of the ovarian steroids that occur during the menstrual cycle.

Adolescent↗
PubMed1985

Bone remodeling in response to in vivo fatigue microdamage.

It has been suggested that osteonal remodeling is triggered by bone microdamage. The validity of this theory rests on the assumption that loading within the physiological range will produce substantial microdamage with relatively few load cycles. The object of the first experiment was to determine threshold values required to consistently produce fatigue microdamage in vivo. The left forelimb of five groups of dogs, characterized by different strain levels and different numbers of load cycles, were loaded in three point bending. The number of microscopic fields which contained some microdamage was calculated as a percentage of the total number of fields. This experiment indicated that loads producing strains as low as 1500 microstrain on the radius and 1400 microstrain on the ulna for 10,000 cycles will produce significant bone microdamage. A second experiment was performed to verify this threshold and to determine whether microcracks are associated with the initiation of bone remodeling. Procedures in this experiment were the same as those in the first, except that all dogs were loaded in such a way as to produce strains on the radius of 1500 microstrain for 10,000 cycles, and the dogs were sacrificed 1-4 days after loading. The loaded limb demonstrated significantly more microdamage than the control limb (p = 0.03). Moreover, we observed 44 times as many microcracks in association with resorption spaces as expected by chance alone. These data support the hypothesis that fatigue microdamage is a significant factor in the initiation of intracortical bone remodeling.

Animals↗