FCSlib: an open-source tool for fluorescence fluctuation spectroscopy analysis for mobility, number and molecular brightness in R.

SUMMARY: FCSlib is an open-source R tool for fluorescence fluctuation spectroscopy data analysis. It encompasses techniques such as Fluorescence Correlation Spectroscopy, Number and Brightness, Pair Correlation Function and Pair Correlation of Molecular Brightness. AVAILABILITY AND IMPLEMENTATION: Source code available at https://cran.r-project.org/web/packages/FCSlib/ for Linux, Windows and macOS platforms. Sample data as well as a user's guide are available at https://github.com/FCSlib/FCSlib. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Terahertz magnetoplasmon resonances in coupled cavities formed in a gated two-dimensional electron gas.

We report on both experiments and theory of low-terahertz frequency range (up to 400 GHz) magnetoplasmons in a gated two-dimensional electron gas at low (<4K) temperatures. The evolution of magnetoplasmon resonances was observed as a function of magnetic field at frequencies up to ∼400 GHz. Full-wave 3D simulations of the system predicted the spatial distribution of plasmon modes in the 2D channel, along with their frequency response, allowing us to distinguish those resonances caused by bulk and edge magnetoplasmons in the experiments. Our methodology is anticipated to be applicable to the low temperature (<4K) on-chip terahertz measurements of a wide range of other low-dimensional mesoscopic systems.

Increasing the sensitivity of terahertz split ring resonator metamaterials for dielectric sensing by localized substrate etching.

We demonstrate a significant enhancement in the sensitivity of split ring resonator terahertz metamaterial dielectric sensors by the introduction of etched trenches into their inductive-capacitive gap area, both through finite element simulations and in experiments performed using terahertz time-domain spectroscopy. The enhanced sensitivity is demonstrated by observation of an increased frequency shift in response to overlaid dielectric material of thicknesses up to 18 µm deposited on to the sensor surface. We show that sensitivity to the dielectric is enhanced by a factor of up to ∼2.7 times by the incorporation of locally etched trenches with a depth of ∼3.4 µm, for example, and discuss the effect of the etching on the electrical properties of the sensors. Our experimental findings are in good agreement with simulations of the sensors obtained using finite element methods.

Tracking sperm in three-dimensions.

Sperm motility, crucial for fertilization, has been mostly studied in two dimensions (2D) by recording their swimming trajectories near a flat surface. However, spermatozoa swim in three-dimensions (3D) to find eggs, with their speed being the main impediment to track them under realistic conditions. Here, we describe a novel method allowing 3D tracking and analysis of the trajectories of multiple free-swimming sperm. The system uses a piezo-electric device displacing a large focal distance objective mounted on a microscope to acquire 70 image stacks per second, each stack composed of 60 images that span a depth of 100 microm. With this method, 3D paths of multiple sperm in the same field could be visualized simultaneously during 1 s. Within the same sample we found that surface-confined sperm swam 25% slower, produced 3-fold fewer circular revolutions per second, and had trajectories of 134% greater radius of curvature than those sperm swimming freely in 3D.

Transcription inhibition induced by modified triple helix-forming oligonucleotides: a quantitative assay for evaluation in cells.

Oligonucleotides can bind to double-stranded DNA in a sequence-specific manner to form triple helices. Uniformly modified, pyrimidine-rich oligodeoxyribonuclotides containing internucleosidic N3'-P5' phosphoramidate linkages are known to form very stable triplexes with their DNA target. Psoralen-conjugated triple helix-forming oligonucleotides (Pso-TFOs) can additionally be photo-induced to become irreversibly bound to their targeted DNA sequence. Here, we have examined the ability of various 15-mer phosphoramidate TFOs targeted to the HIV-1 polypurine tract (PPT) sequence to prevent transcription elongation in cell cultures; the PPT sequence has been cloned in the transcribed region of a reporter firefly luciferase gene (luc) and transient expression experiments performed. We show that the level of transcription inhibition of the reporter gene in cells perfectly correlates with the amount of covalent triplex at the PPT site. The efficacy of non-covalent triplexes (either omitting the irradiation step with the psoralen conjugate, or using the unsubstituted oligonucleotide) has been studied in our expression system; the oligonucleotides were introduced into living cells by cationic lipid-mediated delivery or directly into the cell nucleus by microinjection. This experimental approach allowed us to evaluate the intrinsic activity of triplexes as transcriptional inhibitors; transcription elongation was inhibited in cells in a sequence-dependent and concentration-dependent manner. This experimental system is convenient for quantitative and fast evaluation of new chemistries of antigene oligonucleotides as inhibitors of gene expression in cells and in vivo.

Real-time imaging of gene expression in single living cells.

Recent advances in reporter gene technologies are now allowing us to image gene transcription at the single cell level, using either fluorescence or luminescence microscopy. Here, the basis of these techniques is outlined and their advantages and disadvantages in various biological systems are discussed.

Dynamic changes in prolactin promoter activation in individual living lactotrophic cells.

The firefly luciferase gene has become widely used as a convenient reporter for studies of gene promoter regulation. Very recently, the development of ultralow-light imaging cameras has enabled the quantitative digital imaging of light signals resulting from luciferase activation in the presence of luciferin substrate. We have applied this technology to the study of PRL promoter activation in individual pituitary tumor cells to study the temporal and spatial characteristics of the expression of a well-characterized pituitary hormone gene. Rat pituitary GH3 cells were transfected by lipofection with a luciferase reporter gene linked to 5000 bp from the human PRL gene 5'-flanking region. A series of stably transfected cell clones were generated, and one of these was chosen for detailed study on the basis of appropriate regulation of high-level luciferase expression by a series of known stimuli including TRH, forskolin, the calcium channel agonist Bay K8644, and basic fibroblast growth factor (bFGF). These cells were subjected to direct imaging of luciferase activity using a Hamamatsu photon-counting camera linked to a Zeiss Axiovert microscope with an Argus-50 image processor. Cells were exposed to 1 mM luciferin, and images were integrated over 30-min periods for up to 72 h. The total photon count over a given field settled to steady levels within 10 h and then remained constant for over 55 h. Addition of forskolin, TRH, or bFGF increased the total photon count of fields of 20-100 cells by 2- to 4-fold consistent with previous data from transient expression assays using the human PRL promoter. Individual cells, on the other hand, showed marked marked temporal and spatial heterogeneity and variability of luciferase expression when studied at 3-h intervals. Unstimulated cells showed variable luciferase expression with up to 40-fold excursions in photon counts per single cell area within 12-h periods. Stimulation of cells with either TRH, forskolin, or bFGF resulted in smooth increases in photon output over fields of 20-100 cells, but again individual cell responses differed widely, with some cells showing slow progressive rises in photon output, others showing phasic or transient responses, and yet others showing no response. In conclusion, we found a surprising degree of heterogeneity and temporal variability in the level of gene expression in individual living pituitary tumor cells over long periods of time, with markedly divergent responses to hormonal or intracellular stimulation. The use of stably transfected clonal cell lines with extended periods of reporter gene imaging offers a valuable insight into control of gene expression in living cells in real time.

Effectiveness and duration of intramuscular antimotion sickness medications.

Motion sickness inhibits gastric motility, making the oral route ineffective for medications. The intramuscular route is an effective alternative. The rotating chair was used to produce the M 111 level of motion sickness on the Graybiel Symptom Scale. The intramuscular medications given 30 minutes before rotation were compared with placebo (saline, 1 mL) for effectiveness and duration in increasing the number of tolerated head movements. Average placebo number of head movements was 294. Promethazine 25 mg increased head movements by 78% (P < .05), with a duration of 12 hours. Scopolamine 0.2 mg increased head movements by 91% (P < .05), with a duration of 4 hours. The effect of caffeine 250 mg and ephedrine 25 mg was not significant. When combined with scopolamine, ephedrine produced an 32% additive effect. Scopolamine 0.08 mg, 0.1 mg, and 0.2 mg and also promethazine 12.5 mg and 25 mg were significant (P < .05). Promethazine appears to be the drug of choice for intramuscular use because of a longer duration and a high level of effectiveness. Scopolamine was of high effectiveness, but had a duration of 4 hours. It was eight times as potent by the intramuscular as by the oral route.

Effects of motion sickness and antimotion sickness drugs on gastric function.

This study examined the effects of motion sickness and antimotion sickness drugs on gastric emptying (GE). Drugs were tested in normal and motion sick subjects. To induce motion sickness, subjects performed head movements while seated in a rotating chair. Gastric emptying of liquid (300 mL) was determined by nuclear medicine techniques, whereas gastric electrical activity, the electrogastrogram (EGG), was monitored from surface (cutaneous) electrodes positioned over the abdominal area. Gastric emptying was severely inhibited at the peak of motion sickness symptoms, but returned to normal 15 minutes later when symptoms abated. In normal (non-motion sick) subjects intramuscular (IM) scopolamine (0.1 mg) and IM promethazine (25 mg) inhibited GE, whereas erythromycin ethylsuccinate (EES) suspension (200 mg) given orally increased GE. When administered to motion sick subjects, IM scopolamine and IM promethazine added slightly, but not significantly, to the inhibition of GE already present. Oral EES did not significantly alter GE in motion sick subjects. Although EGG frequency remained within normal limits (approximately 2.5-3.5 cpm) after liquid ingestion in both normal and motion sick subjects, EGG amplitude was differentially affected in the two groups. Electrogastrogram amplitude increased twofold to fourfold after liquid ingestion in normal, but not in motion sick subjects. The results suggest that (1) maximal inhibition of GE is coincident with peak motion sickness symptoms, (2) both IM scopolamine and IM promethazine inhibit GE in normal subjects, but do not add significantly to the inhibition of GE already established during motion sickness, (3) orally administered erythromycin enhances GE in normal, but not in motion sickness subjects, and (4) the normal stimulatory effect of liquid ingestion on gastric motility does not occur in motion sick subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Prokinetic effects of erythromycin after antimotion sickness drugs.

Motion sickness and the antimotion sickness drugs scopolamine (SCP) and promethazine (PMZ) inhibit gastric emptying (GE). This study was conducted to determine if erythromycin would exert its well-known prokinetic effects in normal and motion-sick subjects given antimotion sickness drugs. Fifteen fasted volunteers (11 males, 4 females) participated in the study. In control tests, 8 subjects were given intramuscular (i.m.) saline (SAL, 0.5 ml), SCP (0.1 mg), or PMZ (25 mg). GE of liquid (300 ml) containing 1 mCi of Tc 99m diethylenetriaminepentaacetic acid (DTPA) was measured by sequential gastric scintigraphy 30 minutes after i.m. treatments. In other tests, GE was measured in 8 subjects after each i.m. treatment, followed 10 minutes later by 200 mg of erythromycin ethylsuccinate (ESS) suspension given orally. In a third group of tests, 7 subjects received an i.m. treatment, oral EES 10 minutes later, and were then brought to an advanced level of motion sickness short of vomiting. To induce motion sickness, blindfolded subjects made timed head movements while seated in a rotating chair. GE was measured immediately after rotation. GE half-life, rate constant, area under the curve (AUC), and lag time were calculated using conventional mathematical methods for analyzing exponential rate processes. GE parameters calculated for normal and motion-sick subjects given antimotion sickness drugs and EES were compared with those from subjects given i.m. treatments (control) only. In normal subjects, EES significantly (p < 0.05) increased the GE rate constant for all i.m. treatments and reduced the AUC for SAL, SCP, and PMZ by 49% (p < 0.05), 44% (p < 0.05), and 69% (p < 0.01), respectively. In motion-sick subjects, lag time was significantly (p < 0.05) increased, and the rate constant and AUC values were unchanged from control for all i.m. treatments. The authors conclude that oral EES reverses the gastrostatic actions of the antimotion sickness drugs but does not affect the inhibition of gastric emptying associated with motion sickness. The results suggest that motion sickness and antimotion sickness drugs reduce GE through different mechanisms.

Habituation and motion sickness.

The vestibular, cerebellar, and reticular systems are central in importance, in motion sickness and habituation, to the effects of motion. Nuclear medicine single photon emission computed tomography (SPECT) studies of cerebral blood flow and power spectral electroencephalographic recordings during motion sickness were used to determine alterations in the central nervous system. The rotating chair with and without visual stimulation was used to study the rate of habituation and the effect of antimotion sickness medications on this rate. An increase of theta waves over the frontal cortex indicated a decreased activation of the higher centers during motion sickness. Motion sickness also produces an increase of blood flow in the central cerebellum that has connections to the reticular system. This increase in cerebellar activity is relayed to the reticular system whereby neural recruitment builds up to trigger the vomiting center, producing motion sickness. Habituation may be a conditioned compensatory activation of the reticular neurons that prevents this disruption of normal activation. The rate of habituation when motion sickness was prevented by scopolamine was slowed, indicating that, if the central nervous system is not challenged by disruption of normal activation, it does not produce the compensatory reactions that result in habituation.

Role of vein patch angioplasty in isolated operations for profunda femoris stenosis and disabling claudication.

This report has described the treatment of disabling claudication by correction of profunda femoris artery stenosis. Vein patch angioplasty and endarterectomy with primary closure were the procedures used. An overall success rate of 71 percent was noted with no significant complications, however, the angioplasty group had a higher likelihood of success (83 percent versus 40 percent). They also were noted to have a significantly improved ankle-arm Doppler index, with a mean follow-up of 21 months. Possible explanations for these observations have been considered and technical recommendations discussed.

The effect of intravenous nutrition on muscle mass and exercise capacity in perioperative patients.

This study was undertaken to compare the efficacy of four different types of perioperative intravenous nutritional support. Fifty-five patients undergoing routine major surgery were studied. They were prospectively assigned to one of four study groups. Group 1 received formal total parenteral nutrition (90 g amino acids, 3,000 calories as glucose, per day); Group 2, 100 g glucose per day; Group 3, 90 g amino acids per day; and Group 4, peripheral parenteral nutrition (90 g amino acids plus 1,600 calories, 60 percent as fat per day). Group 1 was maintained on therapy for 3 weeks and the other groups for 8 days. Nitrogen balance, maintenance of body cell mass, serum albumin levels, and maintenance of exercise capacity were measured. Patients receiving peripheral parenteral nutrition maintained their nutritional parameters, as did those receiving total parenteral nutrition. These infusions were both markedly superior to those receiving glucose alone or those receiving amino acids alone. Nitrogen balance was not correlated with maintenance of function, but maintenance of body cell mass was correlated with maintenance of exercise capacity (r = 0.66, p less than or equal to 0.01). We conclude that perioperative peripheral parenteral nutrition, in contradistinction to hypocaloric infusions of glucose or amino acids, is capable of maintaining postoperative muscle mass and function close to preoperative levels after major surgery, and in situations of relatively mild surgical stress, approaches the efficacy of total parenteral nutrition in this regard. A significant correlation exists between changes in body cell mass determined from isotope dilution and changes in the exercise capacity of large muscle masses.

Relative value of glucose and amino acids in preserving exercise capacity in the postoperative period.

The value of crystalline amino acids compared with glucose in maintaining functional muscle mass (maximum exercise capacity) in the perioperative period was studied. Twelve surgical patients received 100 g of glucose (Group 1) for 7 to 10 days perioperatively, and 12 (Group 2) received 90 g of crystalline amino acids for a similar period. Maximum exercise capacity, nitrogen balance, and serum albumin were studied. The use of amino acids instead of glucose spared nitrogen. Net nitrogen loss was 64.7 +/- 6.7 g in Group 1 compared with 34.7 +/- 4 g in Group 2 (p less than or equal to 0.001). Exercise capacity decreased 13.8 +/- 4.5 percent in Group 1 and 13.3 +/- 2.9 percent in group 2. The serum albumin level decreased by 0.30 +/- 0.2 g/100 ml in Group 1 compared with 0.34 +/- 0.15 g/100 ml in Group 2. These differences were not significant. Changes in serum albumin were correlated with changes in exercise capacity (r = 0.7, p less than or equal to 0.002), but neither was significantly correlated with nitrogen loss. We concluded that the use of amino acids instead of glucose during moderate periods of semi-starvation associated with moderate trauma will not influence loss of exercise capacity significantly, although some nitrogen will be spared; patients undergoing moderately severe surgical procedures accompanied by moderate periods of semistarvation will lose approximately 14 percent of their exercise capacity; and loss of exercise capacity is not correlated with loss of nitrogen under these conditions but is loosely correlated with changes in serum albumin levels.

Tissue nitrogen and potassium variation in trauma, starvation, and realimentation.

Total body or exchangeable potassium is used as an important indicator of body cell mass in the study of body composition. Body composition studies have been used extensively in the study of nutrition but recent work has questioned the validity of using changes in total body potassium as a measure of protein or nitrogen variation. To investigate the relationship between tissue nitrogen and potassium during nutritional manipulation 382 tissue samples from 100 surgical patients were analyzed by Kjeldahl analysis for nitrogen content and flame photometric analysis for potassium content. Nitrogen was related to potassium in parenchymous or cellular tissues by the relationship N (mg/g) = 14.7 + 0.17 K (microEq/g). The tissue content of the two elements was highly correlated (r = 0.80, p less than or equal to 0.001). For skeletal muscle a similar relationship existed N (mg/g) = 16.6 + 0.15 K (microEq/g) (r = 0.76, p less than or equal to 0.001). These relationships held for all nutritional states and degrees of trauma and nutritional manipulation. In sequential parenchymous tissue samples obtained from 15 subjects delta N/delta K = 0.17 +/- 0.03 mg/microEq. Nonparenchymous or acellular tissue nitrogen and potassium were poorly correlated. The potassium content was very low in these tissues. Exchangeable potassium was a valid indicator of parenchymous tissue nitrogen and as such a legitimate measure of nutritional status.

Antimotion-sickness efficacy of scopolamine 12 and 72 hours after transdermal administration.

The antimotion sickness remedy, transdermal therapeutic system-scopolamine, administered in this experiment was scheduled to deliver 1.0 mg of scopolamine over a period of 3 d, and this paper compares its efficacy 12 and 72 h after administration. In a double-blind study, six male college students were individually exposed to a standardized provocative test in a slow rotation room after six apparently identical treatments comprising four placebos and two medications. Efficacy was categorized as beneficial, inconsequential, or detrimental. None of the responses was detrimental. Following the first administration of the therapeutic system, there were four beneficial responses after 12 h but none was beneficial after 72 h. Following the second treatment regimen, there were four beneficial responses after 12 h and three beneficial responses after 72 h. Great individual differences were demonstrated, two subjects accounting for six beneficial responses and two accounting for only one beneficial response. The difference in efficacy after 12 and 72 h has practical and theoretical significance.

Experimental motion sickness: efficacy of transdermal scopolamine plus ephedrine.

A double-blind, placebo-controlled study compared the efficacy of transdermal therapeutic system-scopolamine administered alone and combined with ephedrine sulfate given orally in doses of 12.5, 25, and 50 mg. Eight normal male students were exposed to stressful accelerations in a slow-rotation room after receiving 10 apparently identical treatments comprising the four drugs and six placebos. Efficacy of the drug was defined in terms of the placebo range and categorized as beneficial, inconsequential, or detrimental. None of the effects was detrimental. Overall beneficial effects were 60% for transdermal therapeutic system-scopolamine (plus placebo) and 57% for the three transdermal therapeutic system-scopolamine plus ephedrine combinations.

Electrogastrograms during motion sickness in fasted and fed subjects.

Seven human volunteers were subjected to stressful Coriolis stimulation (rotating chair) either during the fasted state or following the ingestion of yogurt (6 oz). Subjects tested after yogurt reached a Malaise-III (M-III) endpoint of motion sickness after significantly (p less than 0.01) fewer head movements than subjects tested in the fasted state. Surface electrogastrogram (EGG) recordings at M-III were similar for both dietary states and consisted of a brief period of tachygastria, followed by a period of low amplitude EGG waves. Ingestion of yogurt enhanced susceptibility to motion sickness but did not affect the associated pattern of EGG.

Mechanisms of antimotion sickness drugs.

UNLABELLED: Eight subjects, male and female, were rotated using the step method to progressively increase the speed of rotation (+2 rpm) after every 40 head movements to a maximum of 35 rpm. The end-point for motion sickness was the Graybiel Malaise III total of symptoms short of frank nausea. The drug treatments were placebo, scopolamine 0.6 mg and 1 mg, scopolamine 0.6 mg/d-amphetamine 10 mg, scopolamine 1 mg/d-amphetamine 10 mg and amphetamine 10 mg. RESULTS: Scopolamine increased tolerated head movements over placebo level by +81, scopolamine 1 mg + 183, d-amphetamine + 118, scopolamine 0.6/d-amphetamine + 165, and scopolamine 1 mg/d-amphetamine 10 mg + 201. DISCUSSION: The drugs effective in preventing motion sickness are divided into those with central acetylcholine blocking activity and those which enhance norepinephrine activity. A combination of both of these actions produces the most effective antimotion sickness medications. CONCLUSIONS: The balance between the acetylcholine and norepinephrine activity in the CNS appears to be responsible for motion sickness.

The effect of antimotion sickness drugs on habituation to motion.

The effect of antimotion sickness drugs on habituation was studied. Subjects were rotated once a day for 5 d to the malaise III end-point after receiving placebo, 1 mg scopolamine, 10 mg d-amphetamine, or the combination of 0.6 mg scopolamine with 5 mg of d-amphetamine. The placebo scores had a Spearman coefficient of correlation of 0.88 with the initial untreated tests. This demonstrated a high reliability for the M-III end-point and that little habituation resulted from the test design. The combination of 0.6 scopolamine with 5 mg amphetamine produced the fastest rate of habituation closely followed by the dose of 1 mg scopolamine. 10 mg of d-amphetamine also produced an increase in habituation over placebo scores. When the medications were discontinued on day 5 a rebound in sensitivity to vestibular stimulation occurred with scopolamine and scopolamine with d-amphetamine. The increased habituation appears to be due to the greater exposure to vestibular stimulation permitted by the medications.