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Selective photo-reduction of N-nitroamines combined with micellar electrokinetic chromatography and laser fluorimetric detection.

N-nitroso compounds (NOC) are potent carcinogens. Reliable methods for the analysis of volatile carcinogenic NOC are well established; however selective and sensitive methods for routine analysis of thermally unstable, ionic or non-volatile NOC are still needed. For this purpose, a method based on micellar electrokinetic chromatography (MEKC) with laser induced fluorescence (LIF) detection is described for the simultaneous determination of a broad range of N-nitroso compounds. In this procedure, the nitroso group is photolytically cleaved from the NOC to yield the corresponding amine. The amines are then derivatized with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), identified and quantified using MEKC-LIF. For the standard mixture of NOC, this method has good sensitivity and a large dynamic range. The detection limit provided by the method is 9 ppb for N-nitrosopyrrolidine.

4-Chloro-7-nitrobenzofurazan↗

A comparison between real-time quantitative PCR and DNA hybridization for quantitation of male DNA following myoblast transplantation.

The transplantation of muscle precursor cells (myoblasts) is a potential therapy for Duchenne muscular dystrophy. A commonly used method to detect cell survival is quantitation of the Y chromosome following transplantation of male donor cells into female hosts. This article presents a direct comparison between real-time quantitative PCR (Q-PCR) and the DNA hybridization (slot-blot) technique for quantitation of Y chromosome DNA. Q-PCR has a significantly greater linear quantitation range and is up to 40-fold more sensitive at low concentrations of male DNA, detecting as little as 1 ng of male DNA in each female tibialis anterior (TA) muscle. At high male DNA concentrations, accurate quantitation by Q-PCR is 2.5 times higher than the maximum possible with slot-blot. In conclusion, Q-PCR has a higher dynamic range and is more efficient than slot-blot analysis for the detection of donor cell engraftment in a transsexual transplantation model.

Animals↗

Optical plankton analyser: a flow cytometer for plankton analysis, I: Design considerations.

The design criteria for a flow cytometer (FCM) for the analysis of field samples of phytoplankton are described. The criteria are based on the occurrence of a wide variety of particle sizes in field samples, normally at low concentrations. The instrument should be able to analyse cells and colonies from 0.5 to 500 microns diameter and of over 2,000 microns length. A minimum flow rate of 4 microliters.s-1 was calculated from natural plankton concentrations. Commercially available FCMs are not suited to measure this range of sizes at this rate. Further limitations of standard FCMs are uneven illumination or incomplete processing of long signals. In addition, long filamentous colonies can break into small fragments caused by too high acceleration in the standard flow cuvette. Recognition of these limitations is of importance for the flow cytometry of phytoplankton. The new design was developed to avoid these limitations. A dynamic range 5 to 6 decades could be accomplished by a combination of logarithmic amplifiers, a slit-shaped focal spot, and a pulse integration system that can process long pulses. Multilaser capability to identify different phytoplankton species, a low fluid shear cuvette, and a trigger gate-extension for inhomogeneously fluorescent algal filaments were included in the design.

Electronics↗

Determination of trace silver by solid substrate-room temperature phosphorescence quenching method based on double catalytic system of meta-nitrophenyfluorone-polyoxyethylene-chromium-potassium bromate-beta-cyclodextrin.

A new solid substrate-room temperature phosphorescence (SS-RTP) quenching method for the determination of trace silver has been established. It is based on the fact that when using Mg(2+) as ion perturber and beta-CD as surfactant, the system of meta-nitrophenyfluorone (R)-polyoxyethylene-Cr(III) can emit strong and stable room temperature phosphorescence signal on filter paper whose surface is modified by polyvinyl alcohol (PVA)-H(3)BO(3)-NaOH. Ag(I) can catalyze KBrO(3) oxidizing R-PEO-Cr(III) system which causes the quenching of SS-RTP. The reducing value of phosphorescence intensity (DeltaI(p)) is directly proportional to the concentration of Ag(I) in the range of 3.2-160 ag spot(-1) (corresponding concentration: 2.43 fg ml(-1), the sample volume: 0.40 microl spot(-1)) with a detection limit (LD) of 0.97 ag spot(-1). The regression equation of working curve can be expressed as DeltaI(p)=13.92+0.3089m(Ag)+ (ag spot(-1)) (r=0.9983, n=6). This method has many advantages, such as a wide linear dynamic range, high sensitivity, good repeatability and selectivity. It has been applied to the determination of trace silver in real samples with satisfactory results. What is more, the mechanism of SS-RTP quenching method based on Ag(I) catalyzing KBrO(3) oxidizing meta-nitrophenyfluorone has also been discussed.

Catalysis↗

Analyzing protein kinase dynamics in living cells with FRET reporters.

Genetically encoded reporters based on fluorescence resonance energy transfer (FRET) are being developed for analyzing spatiotemporal dynamics of kinase activities in living cells, as the activities of this class of enzymes are often dynamically regulated and spatially compartmentalized within specific signaling context. Here we describe a general modular design and engineering strategies for the development of activity reporters for kinases of interest, using A-kinase activity reporter (AKAR) as an illustrative example. Discussed here are basic structure of such reporters, design considerations, reporter gene construction, cellular and in vitro characterization. Strategies for improving specificity, dynamic range or sensitivity, reversibility and integrity of the reporter as well as basic methods for live-cell time-lapse imaging using these reporters are summarized. Discussion of using this approach in the study of MAPK cascades is also provided. These FRET-based kinase activity reporters, along with analogous probes based on alternative designs, provide real-time tracking of kinase dynamics with subcellular resolution, which should complement other methods and offer great opportunities to delineate the molecular mechanisms underlying the complex regulation of kinases.

Animals↗

Auditory-nerve integrity after middle-fossa acoustic-tumor removal.

We sought to investigate the functional integrity of the auditory nerve in patients with postoperative hearing loss after middle cranial fossa acoustic-tumor removal in a case-series descriptive study. The study setting was a tertiary referral center, a private otologic practice. The study population comprised seven patients who underwent a middle-cranial-fossa approach for unilateral acoustic-tumor resection and sustained postoperative anacusis with an anatomically intact auditory nerve. Four were men and three women; they ranged in age from 30 to 60 years; all underwent surgery between 1990 and 1994 and agreed to return to the center to participate in the study during 1995. Diagnostic electrical promontory stimulation was used to determine the functional integrity of the auditory nerve. Our main outcome measures were the presence or absence of discrete tone perception, electrical threshold, maximum acceptable level and dynamic range, gap detection and temporal difference limen during electrical promontory stimulation. Three of the seven patients demonstrated positive responses to electrical promontory testing (e.g., discrete tone perception). All three were able to perform the gap-detection and temporal difference limen tests. None of the preoperative characteristics was related to performance on promontory stimulation testing. We conclude that the middle cranial fossa approach permits anatomic--and, in some cases, functional auditory-nerve preservation. These data suggest that auditory rehabilitation in some patients who sustain anacusis after the middle cranial fossa approach to acoustic tumors, as in neurofibromatosis 2, may be provided with cochlear implantation.

Adult↗

Absolute quantification of the model biomarker prostate-specific antigen in serum by LC-Ms/MS using protein cleavage and isotope dilution mass spectrometry.

Protein cleavage-isotope dilution mass spectrometry (PC-IDMS) can be used to quantify proteins, with an isotope-labeled analogue of the peptide fragment used as an internal standard. Here, we investigate use of a standard LC-MS/MS platform for quantifying a model biomarker directly from serum by this technique. We synthesized a peptide (IVGGWECEK) identical to the N-terminal tryptic fragment of PSA but with each glycine containing two 13C atoms and one 15N atom. PSA-free human serum was denatured with urea followed by the introduction of PSA standard and the stable isotope labeled internal standard peptide. The sample was then proteolyzed with trypsin and subjected to quantification using LC-MS/ MS on a triple quadrupole mass spectrometer. A linear least squares calibration curve made from five different concentrations of PSA added to serum and digested (each made in triplicate and randomly injected three times) had a mean slope of 0.973 (SE = 0.023), intercept of -0.003 (SE = 0.022), and R2 of 0.971. Recovery of calibrators ranged from 70 to 85% with a mean run-to-run CV of 13% and a mean within-run CV of 5.7%. PC-IDMS is a promising technique for quantifying proteins covering a broad range of applications from standardizing immunoassays to monitoring post-translational modifications to quantifying newly discovered biomarkers prior to the development and implementation of an immunoassay, just to name a few. Issues surrounding the application of PC-IDMS for the absolute quantification of proteins include selection of a proteolytic fragment for quantification that can be cleaved and isolated reproducibly over a broad dynamic range, stable isotope labeled synthetic peptide standards that give consistent results, and LC-MS/MS methods that provide adequate sensitivity and reproducibility without creating impractical analysis times. The results presented here show that absolute quantification can be performed on the model biomarker PSA introduced into denatured serum when analyzed by LC-MS/MS. However, concerns still exist regarding sensitivity compared to existing immunoassays as well as the reproducibility of PC-IDMS performed in different matrixes.

Humans↗

Determination of carbon dioxide detection thresholds in trained rats.

Concentration-response functions for the detection of CO2 were established for six rats. The animals were tested in a wind tunnel apparatus and trained using standard operant techniques and a discrete trials, go, no-go successive discrimination paradigm. The primary conclusion to be drawn from the performance measurements is that, at least under carefully controlled conditions, rats can detect physiologic concentrations of CO2 (0-4%). Minimum detectable concentrations fell within the range of 0.04-1.7% CO2. The concentration-response function describing the detectability of CO2 for the six rats was divided into an upper and lower limb at a concentration (5%) that was approximately equal to the end expiratory CO2 levels for the rat (4.88%). High levels of performance were observed for concentrations above this point, while those below it (0.02-2.5%) represented the dynamic range of detectability. Based on a 65% performance criterion, the average threshold performance for six rats was 0.52%. Possible interpretations of these data are discussed.

Animals↗

Simultaneous analysis of eight human Th1/Th2 cytokines using microarrays.

The adaptive immune system induces T cells to change from a naive phenotype to a Th1/Th2 phenotype each of which produce characteristic types of cytokines. Knowledge of whether a specific immune response is Th1 or Th2 is a useful indicator for diseases with basis in immune function disorder. An assay that can rapidly analyze multiple cytokines indicative of these two cell types from small sample quantities can be an extremely useful research and diagnostic tool. Silanized glass slides were printed with multiple arrays of capture antibodies to detect eight different cytokines involved in the Th1/Th2 response along with control proteins for assessing assay performance. Arrays were developed by sequential addition of known antigen amounts, detector antibodies and a fluorescent detection system followed by imaging and quantification. These arrays were used to determine the specificity, sensitivity and reproducibility of the assay and the performance compared with conventional ELISA. This multiplexed assay is able to measure human Th1/Th2 cytokines in sample volumes lower than 20 microl. The assay sensitivity for the eight cytokines range from 0.3 microg/l for IL-4 to 6.4 microg/l for IL-5 which are either comparable to or higher than those reported for conventional ELISA or bead-based multiplex ELISA methods. This assay can be automated to measure expression levels of multiple Th1/Th2 cytokines simultaneously from tens to hundreds of biological samples. This assay platform is more sensitive and has a larger dynamic range as compared to a conventional ELISA in addition to significantly reducing the time and cost of assay. This platform provides a versatile system to rapidly quantify a wide variety of proteins in a multiplex format.

Cytokines↗

Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase.

We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-resolved fluorescence quenching (TruPoint). High sensitivity was obtained using fluorescent europium chelates as labels, internally quenched by suitable quenchers and released upon enzymatic reaction. This approach allows robust and sensitive monitoring of low enzyme activities. The assay technology and the choice of donor-acceptor pairs were evaluated in three different enzymatic assays, a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades. All the assays produced an increasing signal, were sensitive, and had a good dynamic range. There were significant differences in optimized quenchers for each of the assays depending on the size, flexibility, and rigidity of the substrates. Also, clear differences in the energy-transfer reactions, their requirements for spectral overlapping, ionic interactions, and energy-transfer distances were found. Each of the enzymatic assays was briefly tested in a high-throughput screening environment by analyzing signal dynamics and statistical relevance as Z' factors.

Caspases↗

Are phantoms useful for predicting the potential of dose reduction in full-field digital mammography?

A phantom study was performed in full-field digital mammography to investigate the opportunity and the magnitude of a possible dose reduction that would leave the image quality above the accepted thresholds associated with some classical phantoms. This preliminary work is intended to lay the groundwork for a future clinical study on the impact of dose reduction on clinical results. Three different mammography phantoms (ACR RMI 156, CIRS 11A and CDMAM 3.4) were imaged by a full-field digital mammography unit (GE Senographe 2000D) at different dose levels. Images were rated by three observers with softcopy reading and scoring methods specific to each phantom. Different types of data analysis were applied to the ACR (American College of Radiology) and the other two phantoms, respectively. With reference to the minimum acceptance score in screen/film accreditation programmes, the ACR phantom showed that about 45% dose reduction could be applied, while keeping the phantom scores above that threshold. A relative comparison was done for CIRS and CDMAM, for which no threshold is defined. CIRS scoring remained close to the reference level down to 40% dose reduction, the inter- and intra-observer variability being the main source of uncertainty. Contrast-detail curves provided by CDMAM overlapped down to 50% dose reduction, at least for object contrast values ranging between 30% and 3%. This multi-phantom study shows the potential of further reducing the dose in full-field digital mammography beyond the current values. A common dose reduction factor around 50% seems acceptable for all phantoms. However, caution is required before extrapolating the results for clinical use, given the limitations of these widely used phantoms, mainly related to their limited dynamic range and uniform background.

Equipment Failure Analysis↗

Gait changes in children with spastic diplegia after selective dorsal rhizotomy.

Twenty-six ambulatory children underwent preoperative and 1-year postoperative assessments after selective dorsal rhizotomy. These included spasticity, passive range of motion, tone, three-dimensional motion analysis, and electromyography. Independent and dependent ambulators were evaluated separately. A decrease in spasticity was found in all lower extremity muscle groups. An increase in passive range of motion was found only at the hip for both independent and dependent ambulators. Gait changes included increases in velocity and stride length in the independent ambulators. An improvement in hip extension during stance was found in the dependent ambulators only; however, an increase in knee extension and dorsiflexion in stance were seen in both groups. Selective dorsal rhizotomy improves both passive and dynamic range of motion in children with spastic diplegia.

Cerebral Palsy↗

Development of a rapid ferricyanide mediated assay for biochemical oxygen demand using a mixed microbial consortium.

Ferricyanide-mediated (FM) microbial reactions were used for the rapid determination of the biochemical oxygen demand (BOD) of a range of synthetic and real wastewater samples. Four single-species microbial seeds and a synthetically prepared microbial consortium were compared. In all cases, the microbial consortium exhibited a greater extent and rate of biodegradation compared to the individual microbial seeds. Markedly improved correlation to the standard BOD5 method was also noted for the microbial consortium (compared to the single-species seeds). A linear dynamic range up to 200 mg BOD5 L(-1) was observed, which is considerably greater than the linear range of the standard BOD5 assay and most other rapid BOD assays reported. In addition, biodegradation efficiencies comparable to the 5-day BOD5 assay (and much greater than other rapid BOD assays) were observed in 3 h. A highly significant correlation (R = 0.935, p = 0.000, n = 30) between the FM-BOD method and the standard BOD5 method was found for a wide diversity of real wastewater samples. The results indicate that the FM-BOD assay is a promising, rapid, alternative to the standard 5-day BOD5 assay.

Bacteria↗

Simple setup for nanosecond time-resolved spectroscopic measurements by a digital storage oscilloscope.

An application of a digital storage oscilloscope for nanosecond time-resolved spectroscopic measurements is demonstrated in the range from the single-photon region to the multi-photon region. In comparison to the time-correlated single photon counting (TCSPC) method, the measurement setup can be greatly simplified by averaging the signals measured by the oscilloscope. Moreover, the multi-photon events of the fluorescence emissions can be tracked by this simple setup although there still exist some disadvantages in the dynamic range of the signal due to radio frequency noise, and the temporal response of the photo-multiplier tube. This method can simplify time-resolved optical measurements in the nanosecond range, such as fluorescence decay and time-of-flight measurements of diffusing light. Thus, this simple method will be applicable in many clinical and industrial uses.

Analog-Digital Conversion↗

Evaluation of nano-liquid chromatography-tandem mass spectrometry in a column switching setup for the absolute quantification of peptides in the picomolar range.

A standard nanospray-liquid chromatography-tandem mass spectrometry system in a column switching setup for the absolute quantification of leucine-enkephalin was evaluated. Analytes were loaded on a C18 trapping column and back-flushed in the 75 microm analytical column. Quantification was performed with a triple quadrupole instrument. Validation results show that it is feasible, with a conventional nano-LC system in the column switching setup, to quantify peptides as low as 500 amol on column (50 pmol/L). Weighted linear regression analysis proves a good linearity in a dynamic range of almost three orders of magnitude. Nevertheless, robustness remains a key issue in nano-LC-MS/MS.

Chromatography, Liquid↗

Recombinant Kv1.3 potassium channels stabilize tonic firing of cultured rat hippocampal neurons.

We transfected cultured hippocampal neurons with the cDNA of the voltage-gated K+ channel Kv1.3 to investigate the mechanisms by which a specific ion channel influences excitability. In transfected neurons under voltage clamp we observed an additional outward current that was blocked selectively by margatoxin. Under current-clamp conditions, Kv1.3-expressing neurons fired tonically over a wide range of stimulation intensity. In non-transfected neurons, or in Kv1.3-expressing cells blocked with margatoxin, only a few action potentials were elicited before a stationary depolarized state was reached. We attribute the specific effect of Kv1.3 to its particularly slow deactivation near the resting potential. A computational model showed that a continuous outwards current arises in Kv1.3-expressing neurons during the interspike intervals. It expands the dynamic range so that these neurons still fire tonically at stimulus current intensities at which non-transfected cells have already been driven into a stationary depolarized state.

Action Potentials↗

Implantation of the lateral cochlear wall for auditory nerve stimulation.

Although cochlear implants now regularly achieve gratifying results, traditional intrascalar implants have certain limitations. Extraluminal implants may offset some of these problems by accessing neurons subserving a wider tonotopic range, avoiding intracochlear insertion trauma, and offering alternatives when cochlear obliteration is present. We have investigated the utility of a lateral cochlear wall implant in a normal-hearing cat model with implants at the middle and basal turns, and found successful activation of the auditory nerve at thresholds of 28.1 and 40.6 microA, respectively. No adventitial stimulation of the facial nerve was noted within the dynamic range. Maximum responsiveness was observed with implants of the middle turn of the cochlea, an area that is not reliably approached with current intrascalar implants. These observations support and extend prior observations of the feasibility of extraluminal stimulation of the auditory nerve.

Animals↗

Trunk kinematics and the influence on valgus knee stress in persons with high sacral level myelomeningocele.

Historically, trunk movement has been thought to be reactionary to lower body motions. The excessive trunk movement in patients with myelomeningocele may be a primary mechanism during ambulation. The purpose of this study was to quantify three-dimensional trunk movement in patients with myelomeningocele. Patients walked with ankle-foot orthoses (AFOs), both with and without crutches. Data were collected in reference to global and pelvic coordinates. Patients who walked with AFOs and crutches showed less trunk dynamic range of motion than patients without crutches. Between the two patient groups, there was a significant difference in trunk obliquity but not in trunk tilt or rotation. There is a good correlation between maximum trunk obliquity and coronal plane valgus knee stress. Overall, coronal plane valgus knee stress is multifactorial and trunk motion and external tibial torsion are major contributors. However, when using trunk kinematics to describe dynamic motion, both global and pelvic coordinate systems are important reference frames.

Adolescent↗