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PubMed2026

Novel serum small extracellular vesicle miRNAs with multi-target RCA-CRISPR sensor for liver cancer detection.

BACKGROUND: Detecting liver cancer (LC) remains a significant challenge in clinical practice. Small extracellular vesicle (sEV) miRNAs show promise as non-invasive biomarkers for LC detection, yet their diagnostic potential remains largely unexplored. This study aimed to identify specific sEV miRNA signatures for LC detection and develop a novel synchronized multi-miRNA detection platform to enhance diagnostic efficiency and sensitivity. METHODS: High-throughput sequencing was conducted across four distinct cohorts: normal controls (NC), hepatitis B virus (HBV) patients, liver cirrhosis patients, and LC patients. This sequencing process identified miRNAs with differential expression, followed by RT-qPCR validation in serum sEV miRNAs from LC patients and NC. An innovative detection method, RCA-CRISPR, was introduced, combining rolling circle amplification (RCA) with CRISPR/Cas12a (RCA-CRISPR) for quick and sensitive miRNAs detection. RESULTS: Sequencing results showed a consistent elevation of hsa-miR-203b-5p, hsa-miR-4661-5p, and hsa-miR-219a-2-3p across all cohorts. RT-qPCR validations confirmed significant upregulation of these miRNAs in serum sEVs from LC patients, and the combined three-miRNA panel exhibited high diagnostic accuracy (p = 0.0003; AUC = 0.81). The RCA-CRISPR method demonstrated a detection limit of 3.12 pM for simultaneous multi-target miRNA detection, highlighting its exceptional sensitivity. CONCLUSIONS: Our study identifies hsa-miR-203b-5p, hsa-miR-4661-5p, and hsa-miR-219a-2-3p as promising sEV miRNA biomarkers for LC detection. The developed RCA-CRISPR sensor provides a robust tool for multi-miRNA analysis, potentially advancing non-invasive LC diagnostics. Future validation in larger, prospectively collected cohorts is essential to establish the clinical utility and performance of this biomarker panel and RCA-CRISPR sensor.

MicroRNAs↗

PubMed1990

Rapid inhibition of the c-jun proto-oncogene expression in avian oviduct by estrogen.

The c-jun proto-oncogene codes for an important component of the AP-1 transcription factor complex which regulates the expression of many genes. This paper demonstrates that the steady state c-jun mRNA level in the avian oviduct is markedly decreased to 50% of control values within 30 minutes after estrogen injection into the animals. In the avian liver, the level is rapidly increased over 10 fold. Nuclear run-off transcription analysis demonstrates that the decrease in the c-jun mRNA levels in the avian oviduct occurs at least in part at the level of transcription. These changes are reproducible in repeat experiments. This response in the avian oviduct is unique since 1) it is the first demonstration of a steroid effect on c-jun expression in any animal system, 2) the changes in c-jun mRNA occur much more rapidly than most steroid responsive genes, and 3) it is the rare demonstration of an estrogen inhibition of the expression of a gene. A role for the c-jun proto-oncogene as an early regulatory gene in the cascade model for steroid action is proposed.

Animals↗

PubMed1994

Differential steroid hormone induction of transcription from the mouse mammary tumor virus promoter.

The Mouse Mammary Tumor Virus (MMTV) contains sequences in its proximal promoter region to which both glucocorticoid and progesterone receptors can bind. In transient transfection experiments both hormones are able to stimulate transcription from reporter plasmids containing either native or consensus hormone response elements (glucocorticoid response element/progesterone response element). Previous experiments have demonstrated that the MMTV long terminal repeat is reproducibly assembled into a phased array of nucleosomes when stably introduced into cells. Stimulation by glucocorticoids of endogenous templates led to a rapid but transient increase in transcription initiation and mRNA accumulation that can be correlated with increased sensitivity to restriction enzymes. In contrast, experiments using progesterone or a truncated glucocorticoid receptor failed to elicit a similar increase in mRNA levels as dexamethasone from stable chromatin templates. In an attempt to understand this differential response, we have compared the responsiveness of the MMTV promoter to glucocorticoids and progesterone when it is organized in either stable chromatin or in transiently acquired plasmids. Our results demonstrate that the native chromatin structure prevents activation of this locus by progesterone, but permits stimulation by glucocorticoids.

Animals↗

PubMed1993

Monoamine oxidase B (MAO B) inhibitor therapy in Parkinson's disease: the degree and reversibility of human brain MAO B inhibition by Ro 19 6327.

The possibility of slowing the progression of Parkinson's disease (PD) with inhibitors of monoamine oxidase B (MAO B) has stimulated the development of new MAO B inhibitor drugs. Ro 19 6327 is a highly selective inhibitor of MAO B currently under clinical investigation. We used positron emission tomography (PET) and the MAO B tracer [11C]L-deprenyl to determine the degree and reversibility of human brain MAO B inhibition by Ro 19 6327 in six early Parkinson's disease patients who were treated with different doses of Ro 19 6327 (25 mg [n = 3], 50 mg [n = 2], and 100 mg [n = 1]; 0.34 to 1.4 mg/kg) every 12 hours for 1 week. Each patient had three PET scans to assess baseline MAO B activity, degree of trough inhibition, and reversibility. A control group of four elderly normal subjects was scanned twice to assess reproducibility of repeated measures. Four of the patients showed reduction of MAO B concentration to 1% to 7% of baseline on doses of 0.43 mg/kg or greater, and the remaining two at 0.34 mg/kg showed significant but incomplete inhibition (10% to 21% of baseline in the global region and in the thalamus, basal ganglia, and mesencephalon). Thus, 0.4 mg/kg or greater of Ro 19 6327 given every 12 hours is the minimum dose necessary to provide > 90% inhibition of brain MAO B in patients with early PD. Brain MAO B activity returned to baseline values by 36 hours after drug discontinuation.

Aged↗

PubMed1980

New head-positioning system for use with computed tomographic scanning.

A new head-positioning system has been developed to hold the head firmly during the performance of a computed tomograhic scan and to position the head reproducibly during repeat scans. This can be used to maintain the head in position during long scanning sessions, to relate a lesion to landmarks on scout views, to follow lesions in repeated scans over time, and to perform a variety of invasive procedures.

Head↗

PubMed2006

Determination of liquiritin, naringin, hesperidin, thymol, imperatorin, honokiol, isoimperatorin, and magnolol in the traditional Chinese medicinal preparation Huoxiang-zhengqi liquid using high-performance liquid chromatography.

High-performance liquid chromatography was employed to determine the contents of the eight marker components liquiritin, naringin, hesperidin, thymol, imperatorin, honokiol, isoimperatorin, and magnolol in the traditional Chinese medicinal preparation Huoxiang-zhengqi liquid. The separation was performed on a C(18) column by stepwise gradient elution with water-methanol-acetonitrile (0.01 min, 68:30:2; 20 min, 60:38:2; 50 min, 34:64:2; 65 min, 34:64:2; 75 min, 28:70:2; 85 min, 68:30:2) as the mobile phase at a flow rate of 1 ml/min, with UV detection at 283 nm. Eight regression equations showed good linear relationships between the peak area ratio of each marker to internal standard and amounts. The recoveries of the markers listed above were 97.4, 98.5, 97.4, 98.6, 97.8, 99.2, 97.0, and 97.5%, respectively. The repeatability and reproducibility (relative standard deviation) of the method were less than 2.2 and 3.0%, respectively.

Biphenyl Compounds↗

PubMed1982

Comparative study of effects of adrenaline, dobutamine and dopamine on systemic hemodynamics and renal blood flow in patients following open heart surgery.

In 10 patients following open heart surgery, adrenaline, dobutamine and dopamine were administered, and the changes in hemodynamic parameters and renal blood flow (RBF) were examined. RBF was determined by the local thermodilution method. Prior to the application of this method in clinical measurement, reliability of the method was checked using a model circuit. The correlation between the actual flow and flow obtained with this method was high (r = 0.999, p less than 0.005, n = 8). Reproducibility in repeated measurements was excellent, r = 0.997 (p less than 0.005, n = 8) in the model circuit and r = 0.985 (p less than 0.005, n = 89) in the clinical measurement. Adrenaline at rates of 0.02--0.08 microgram/kg/min showed a marked inotropic action without any significant change in RBF. With 0.04 microgram/kg/min of adrenaline, the RBF/CO (cardiac output) ratio declined significantly. We conclude that adrenaline is often effective in patients following open heart surgery, but renal vasoconstriction is the major disadvantage. After a 10-min administration of 2, 4 and 8 micrograms/kg/min of dobutamine, cardiac index (CI) and stroke volume index (SVI) showed a stepwise increase in accordance with an increase of dosage, and RBF also increased with CO. Consequently, no significant change in RBF/CO was found. Mean left atrial pressure (LAP) or mean pulmonary arterial wedge pressure (PAWP) decreased in 4 of 7 patients with 8.0 micrograms/kg/min of dobutamine. Thus, dobutamine is an excellent beta 1-adrenergic agonist with a weak alpha-action on both peripheral and renal vessels. With 2.0--2.5 micrograms/kg/min of dopamine, RBF increased by 15.5% (p less than 0.05). while no significant increase appeared in CI. With 4.0 micrograms/kg/min or more of dopamine, CI and SVI increased. With 16--20 micrograms/kg/min of dopamine, RBF increased by up to 44.8%. Significant increase of mean LAP or mean PAWP was observed with 8.0--10.0 micrograms/kg/min or more of dopamine. These findings indicate that the potential increase o LVEDP (left ventricular end-diastolic pressure) with 8--10 micrograms/kg/min or more of dopamine exerts a disadvantageous effect in patients following open heart surgery. However, the effect on the renal hemodynamics, especially with small doses of dopamine, is unique and not observed with adrenaline or dobutamine.

Adolescent↗

PubMed1998

Skeletal muscle deoxygenation during exercise assessed by near-infrared spectroscopy and its relation to expired gas analysis parameters.

The present study was performed to determine the relation between oxygenated hemoglobin (oxy-Hb) changes in working muscles and ventilatory parameters. Six active normal subjects, 21 sedentary normal subjects and 16 patients with heart failure performed an incremental exercise with expired gas analysis. Deoxygenation of the vastus lateralis muscle was monitored for oxy-Hb changes using near-infrared spectroscopy. Near the anaerobic threshold (AT), oxy-Hb started to decrease, forming the first inflection point (P1). Near the respiratory compensation point (RCP), the second inflection point (P2) was observed. Oxygen uptake at the AT, RCP, P1 and P2 decreased in magnitude first in the active normal subjects, then in sedentary normal subjects and finally in the heart failure patients. High correlation was demonstrated between AT and P1 (r=0.8, p<0.0005) and between RCP and P2 (r=0.9, p<0.0005). In 12 sedentary normal subjects who underwent repeat exercise, reproducibility was confirmed for both P1 and P2. Constant work rate exercises were performed in 5 sedentary normal subjects, and in all of them the oxy-Hb remained unchanged below the AT work rate, whereas oxy-Hb decreased above the AT work rate. Exercise capacity, with respect to both working muscle deoxygenation and ventilation, could be evaluated in detail by the concomitant use of near-infrared spectroscopy and expired gas analysis.

Adult↗

PubMed1993

Comparative effects of cimetidine and famotidine on the vagally stimulated acid secretion in the isolated mouse whole stomach.

We investigated the effects of cimetidine and famotidine on the acid secretory response to electrical vagal stimulation, bethanechol and histamine in the isolated mouse whole stomach preparation. The acid secretion elicited by electrical vagal stimulation at the position of the esophagus (10 Hz, 0.3 msec, 10 V for 5 min) was reproducible by repeated stimulation in each preparation, and it was abolished by tetrodotoxin, atropine and hexamethonium. This vagally stimulated acid secretion was abolished by cimetidine (3 mM), while it was only partly inhibited by famotidine (10-100 microM). Histamine (100 microM)-induced acid secretion was inhibited by cimetidine and famotidine, and the doses of these drugs required for complete inhibition were 3 mM and 10 microM, respectively. In contrast, bethanechol (10 microM)-induced acid secretion was slightly reduced by famotidine (1-100 microM), but markedly reduced by cimetidine (3 mM). In the guinea pig ileum, millimolar concentrations of cimetidine and famotidine shifted the dose-response curve of the contractile response to acetylcholine rightward. These findings suggest that the inhibitory effect of cimetidine on the vagally stimulated or bethanechol-induced acid secretion is elicited at least partly through mechanisms different from H2-antagonism.

Acetylcholine↗

PubMed1995

Pharmacological features of non-adrenergic non-cholinergic (NANC) relaxation induced by electrical vagal stimulation in isolated mouse stomach.

The non-adrenergic non-cholinergic (NANC) relaxatory response in mouse isolated whole stomach was investigated by electrical vagal stimulation (EVS) to clarify whether nitric oxide (NO) mediates vagal NANC transmission. The stomach was mounted in an organ bath, and the intragastric pressure was measured. Dual electrodes were placed on the esophagus. In the presence of atropine, propranolol and phentolamine, EVS induced a marked gastric relaxation. The response was frequency-dependent, and reproducible by repeated stimulation. The response was blocked by hexamethonium and NG-nitro-L-arginine (L-NNA), a NO synthase inhibitor, and significantly depressed by methylene blue, a soluble guanylate cyclase inhibitor, but not by hemoglobin, a radical trapping agent. The inhibitory effect of L-NNA was reversed by L-arginine, a substrate for NO synthase, but not by D-arginine. Exogenous NO caused a relaxation that was inhibited by hemoglobin and methylene blue, but not by L-NNA. The electrical field stimulation also elicited a gastric relaxation that was inhibited by L-NNA and methylene blue, but not by hexamethonium and hemoglobin. These results suggest that the inhibitory NANC response to EVS in the mouse stomach is largely mediated by release of NO, and it is exclusively due to stimulation of vagal preganglionic neurons.

Adult↗

PubMed2003

Elucidation with the protease alpha-chymotrypsin of the inhibitory modulating action of endogenous neuropeptide Y over sympathetic neurotransmission in rat vas deferens.

The physiological role of endogenous neuropeptide Y (NPY) in sympathetic neurotransmission was examined in rat and guinea pig vas deferens (VD), using alpha-chymotrypsin (alpha-CT). NPY-like immunoreactivity was detected in the longitudinal muscle layer of VD densely in rats but sparsely in guinea pigs, and it disappeared following surgical denervation. Under blockade of the prejunctional alpha(2)-adrenergic autoinhibition, alpha-CT potentiated the phasic contraction in rat, but not guinea pig, VD induced by trains of transmural nerve stimulation (TNS) in a frequency-dependent manner, which was reproducible during repeated applications and not affected by pretreatment with capsaicin. In contrast, alpha-CT did not potentiate the twitch response or contractions induced respectively by a single pulse TNS or by direct electrical stimulation to the smooth muscle. Exogenously applied NPY suppressed the twitch response, which was cancelled by alpha-CT, and excitatory junction potentials, although it affected neither spontaneous junction potentials nor the direct electrical stimulation-induced contraction. These observations provided further evidence to support that NPY is released endogenously by TNS at high frequency, acting prejunctionally to suppress sympathetic neurotransmission. Thus, the protease alpha-CT proved itself to be a useful tool to reveal a functional role of endogenously released peptides.

Animals↗

PubMed1999

[Transcranial Doppler ultrasonography: stereotactically guided examinations using magnetic resonance angiography].

The accurate localization of specific intracranial blood vessels is a major difficulty with transcranial Doppler sonography (TCD). It was the purpose of this study to develop a system enabling stereotactic navigation during a TCD examination on the basis of high-resolution three-dimensional magnetic resonance angiographic (MRA) data. During TCD, the examiner is provided--on a computer screen--with a projected view of the respective intracranial vessel anatomy. With the aid of an optoelectronic localization system, the spatial orientation and localization of the US probe is determined in real time, and correlated with the patient's MRA data using a dedicated stereotactic mask. Subsequently, the US beam and the points of insonation are displayed on the screen overlaid on the vessel anatomy. In this way the examiner gains real time control of the localization of the respective intracranial vessel insonated. Points of insonation can be stored and recalled for follow-up examinations. In addition to the successful verification of the system, it was shown that, in comparison with conventional TCD, stereotactic navigation distinctly improves the reproducibility of repeat TCD examinations.

Humans↗

PubMed2006

The structure of multi-neuron firing patterns in primate retina.

Current understanding of many neural circuits is limited by our ability to explore the vast number of potential interactions between different cells. We present a new approach that dramatically reduces the complexity of this problem. Large-scale multi-electrode recordings were used to measure electrical activity in nearly complete, regularly spaced mosaics of several hundred ON and OFF parasol retinal ganglion cells in macaque monkey retina. Parasol cells exhibited substantial pairwise correlations, as has been observed in other species, indicating functional connectivity. However, pairwise measurements alone are insufficient to determine the prevalence of multi-neuron firing patterns, which would be predicted from widely diverging common inputs and have been hypothesized to convey distinct visual messages to the brain. The number of possible multi-neuron firing patterns is far too large to study exhaustively, but this problem may be circumvented if two simple rules of connectivity can be established: (1) multi-cell firing patterns arise from multiple pairwise interactions, and (2) interactions are limited to adjacent cells in the mosaic. Using maximum entropy methods from statistical mechanics, we show that pairwise and adjacent interactions accurately accounted for the structure and prevalence of multi-neuron firing patterns, explaining approximately 98% of the departures from statistical independence in parasol cells and approximately 99% of the departures that were reproducible in repeated measurements. This approach provides a way to define limits on the complexity of network interactions and thus may be relevant for probing the function of many neural circuits.

Action Potentials↗

PubMed1998

Validation of serological assays for diagnosis of infectious diseases.

Assay validation is a series of the following interrelated processes: an experimental process: reagents and protocols are optimised by experimentation to detect the analyte with accuracy and precision, and to ensure repeatability and reproducibility in the assay. a relative process: its diagnostic sensitivity and diagnostic specificity are calculated relative to test results obtained from reference animal populations of known infection/exposure status. a conditional process: classification of animals in the target population as infected or uninfected is conditional upon how well the reference animal population used to validate the assay represents the population to which the assay will be applied (accurate predictions of the infection status of animals from test results and predictive values of positive and negative test results are conditional upon the estimated prevalence of disease/infection in the target population) an incremental process: confidence in the validity of an assay increases over time when use confirms that it is robust as demonstrated by accurate and precise results (the assay may also achieve increasing levels of validity as it is upgraded and extended by adding reference populations of known infection status) a continuous process: the assay remains valid only insofar as the assay continues to provide accurate and precise results as proved through statistical verification. Therefore, validation of diagnostic assays for infectious diseases does not end with a time-limited series of experiments based on a few reference samples. Rather, it is a process that also requires constant vigilance and maintenance, along with reassessment of its performance characteristics for each population of animals to which it is applied. It is certain that the current movement to develop and implement accreditation criteria for veterinary diagnostic laboratories may be of little worth unless there is some assurance that the assays conducted in such laboratories are properly validated. Fully accredited laboratories may generate highly reproducible test results, but the results may still misclassify animals as to their infection status due to an improper assay validation process. Therefore, assay validation is foundational to the core product of veterinary diagnostic laboratories--test results and their interpretation.

Animals↗

PubMed1981

The K+-sensitive hyperpolarization of rat skeletal muscle and metabolic inhibitors.

The electrogenic contribution of Na-pump to the resting potential was investigated in isolated "Na-rich" rat soleus muscles exposed to K-free medium for more than 4 hr at 37 degree C. When the resting potentials of fibres fell to around - 75 mV in the K-free medium, the addition of K+ to the bathing medium evoked a transient hyperpolarization. The K+-sensitive hyperpolarization was reproducible in repeated K+ challenged at 45 or 60 min cycles. The K+-sensitive hyperpolarization was completely abolished by cooling, by adding ouabain, or by substituting Li+ for Na+, and reduced by low and high pH. DNP and hypoxia had little effect on the K+-sensitive hyperpolarizations.

2,4-Dinitrophenol↗

PubMed2000

Helical and single-slice conventional CT versus electron beam CT for the quantification of coronary artery calcification.

OBJECTIVE: We compared electron beam CT with conventional CT to determine the best method for the assessment of the coronary calcium score. We used conventional CT to examine symptomatic and asymptomatic patients suspected of having coronary artery disease. SUBJECTS AND METHODS: One hundred sixty male patients underwent electron beam CT and helical CT with a pitch of 1 (n = 30) and 2 (n = 30) and using a single-slice mode with (n = 50) and without (n = 50) prospective ECG triggering. In another 50 patients, we determined reproducibility for repeated scanning using electron beam CT. For all images, we derived the calcium score according to the Agatston method. We performed regression analysis and determined mean variability. Mean variability was calculated as the ratio of the absolute difference to the mean of the corresponding calcium scores. RESULTS: The correlation coefficients for electron beam CT and all conventional CT modes were very high (range, 0.93-0.98). The mean variability was highest in the helical mode with a pitch of 2 (61.4%) and lowest for the single-slice mode with prospective ECG triggering (25.4%). For repeated electron beam CT, the correlation coefficient and mean variability were 0.99 and 22.1%, respectively. CONCLUSION: ECG-triggered single-slice conventional CT had the best agreement with electron beam CT calcium scores.

Calcinosis↗

PubMed1997

Endoscopy and otomicroscopy in the estimation of middle ear structures.

In order to study the variation within and between endoscopy and otomicroscopy a Gage repeatability and reproducibility design was created, with which the middle ears were studied of eight cadaver temporal bone blocks through the ear canal three times in random order using both methods. A Zeiss OPMT-1 operating microscope and Olympus endoscopes were used. The data were analysed in accordance with the analysis of variance principle, where the total variation was divided into different components. The anatomical areas were counted and registered via quadrants. There was a distinct difference between the results of the two methods in favour of the endoscopes. Furthermore, the variation between the methods and between the trials was analogous.

Aged↗

PubMed1990

WEHI 164 subclone 13 assay for TNF: sensitivity, specificity, and reliability.

Tumor necrosis factor alpha (TNF) is a peptide monokine involved in a number of immune reactions. To further understand the role of TNF in disease states it is critical to have an inexpensive, yet sensitive and specific assay. Additionally, the effects of prostaglandin E2 (PGE2), dexamethasone (dex), and cyclosporine A (CsA) on TNF gene expression have been studied, although little is known of the effects these compounds have on TNF containing samples. The aim of this study is to determine the sensitivity and specificity of a highly sensitive cell line to the actions of TNF, and to elucidate parameters which affect the stability of TNF in biological fluids. Dex and PGE2 at concentrations of 10(-5), 10(-7), and 10(-9) M, were shown not to effect the WEHI assay, and neither did CsA (10 ng/ml-1 ug/ml). The cells were not lysed by recombinant murine IL-1 alpha or beta, human recombinant IL-1 alpha or beta, human recombinant IL-2 or human recombinant IL-6 at concentrations ranging from 0.02 pg/ml to 1.0 ug/ml, or murine gamma-IFN from 100 pg/ml to 10 ng/ml. TNF containing samples with 1%-10% fetal calf serum maintained their cytolytic activity even after three freeze-thaw cycles. Serum samples did not lose any cytolytic activity with up to 11 cycles of freezing and thawing whereas, tissue culture media, containing TNF, lost significant activity with freeze-thawing. The WEHI assay has successfully detected cytolytic activity from lipopolysaccharide stimulated specimens from a number of different species. These data show the utility of this highly sensitive and specific assay. Furthermore, the WEHI assay showed a high degree of reproducibility in repeated assays.

Animals↗