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At least 811 records · Page 45Linked to original sources
PubMed2006

Simultaneous determination of 6 beta-blockers, 3 calcium-channel antagonists, 4 angiotensin-II antagonists and 1 antiarrhythmic drug in post-mortem whole blood by automated solid phase extraction and liquid chromatography mass spectrometry. Method development and robustness testing by experimental design.

A method for the simultaneous determination of the beta-blockers atenolol, sotalol, metoprolol, bisoprolol, propranolol and carvedilol, the calcium-channel antagonists diltiazem, amlodipine and verapamil, the angiotensin-II antagonists losartan, irbesartan, valsartan and telmisartan, and the antiarrhythmic drug flecainide, in whole blood samples from forensic autopsies was developed. Sample clean-up was achieved by precipitation and solid phase extraction (SPE) with a mixed-mode column. Quantification was performed by reversed phase high performance liquid chromatography with positive electrospray ionization mass spectrometric detection (HPLC-MS). The method has been developed and robustness tested by systematically searching for satisfactory conditions using experimental designs including factorial and response surface designs. With the exception of amlodipine, the concentration limit of quantification (cLOQ) covered low therapeutic concentration levels for all the compounds. Within assay precisions and accuracies (bias) were 3.4-21% RSD and from -24 to 21% for the concentration range 1.00-5.00 microM, respectively. Between assay precisions were 4.4-28% RSD for the concentration range from 0.1 to 5 microM and recoveries varied from 9 to 103%. The method is used for determination of cardiovascular drugs in post-mortem whole blood samples from forensic autopsy cases.

Adrenergic beta-Antagonists↗
PubMed2026

Mitigating pH-induced instability in deruxtecan-based ADCs: an onboard-mixing icIEF approach for robust charge heterogeneity characterization.

Accurate charge variant analysis of antibody-drug conjugates (ADCs) is essential for understanding product heterogeneity and ensuring quality control. However, Deruxtecan (DXd)-based ADCs present a unique analytical challenge due to the intrinsic instability of the payload, where the lactone ring readily undergoes hydrolysis under alkaline conditions, resulting in time-dependent shifts in charge distribution during imaged capillary isoelectric focusing (icIEF). In this study, we describe the development of an onboard-mixing icIEF method designed to minimize pH-induced degradation during sample preparation. By separating ADC samples from carrier ampholytes (CAs) prior to injection and enabling real-time mixing within the instrument, this approach effectively suppresses premature lactone ring opening and stabilizes charge variant profiles. Comparative studies between conventional premixing and onboard-mixing approach demonstrated that the latter significantly enhances reproducibility, particularly for acidic variants that are highly sensitive to structural conversion. Comprehensive method validation confirmed excellent precision, linearity, and sensitivity, with consistent performance across run-to-run and intra-day analyses. The results underscore the importance of controlling microenvironmental pH exposure in the analysis of chemically instable ADCs. The proposed onboard-mixing strategy provides a robust and efficient solution for icIEF-based characterization, reducing analytical artifacts while simplifying method development. This approach is broadly applicable to ADCs and other biotherapeutics containing pH-sensitive functional groups.

Hydrogen-Ion Concentration↗
PubMed2005

Alcohol-based hand disinfection: a more robust hand-hygiene method in an intensive care unit.

This study involved observation of hand-hygiene behaviour and evaluation of the effect of alcohol-based hand disinfection and handwashing with plain liquid soap on microbial flora. The study was performed in a combined medical and surgical intensive care unit. We demonstrated a crude compliance of hand hygiene of 50.4%, which was only performed adequately in 20.8% of cases. Of this group, handwashing and hand-disinfection procedures were performed properly 34.0% and 71.6% of the time, respectively. Hand samples for bacteriological examinations with the glove juice method demonstrated that whilst handwashing was sensitive to the way in which hand hygiene was performed, alcohol-based hand disinfection was less sensitive to such performance. Our study demonstrated that alcohol-based hand disinfection is a robust hand-hygiene method with many advantages in a practical setting. It is very feasible for use in hospital wards.

Adult↗
PubMed2004

A robust human T-cell culture method suitable for monitoring CD8+ and CD4+ T-cell responses from cancer clinical trial samples.

Many tumor antigenic determinants have been identified and included in cancer clinical trials. Due to low T-cell frequencies even after vaccination, few T-cell responses can be revealed ex vivo without in vitro stimulation. Various expansion protocols have been employed for this purpose and the outcomes tend to be quite variable, partly due to the high complexity involved in the protocols. Here we systematically studied various common culture conditions including sera, cytokines and feeders and describe a reliable "bulk" culture method that is robust, simpler and more economical. We demonstrated that fetal calf serum (FCS) supported T-cell proliferation better than multiple commercially available pooled human AB sera. IL-2 is critical in our cultures, but IL-7, IL-15 and anti-CTLA-4 in combination with IL-2 did not further enhance T-cell expansion. We typically achieve more than a 40-fold expansion within a 10-day culture period for antigen-specific T cells measured by HLA-peptide tetramer before and after culture. This method was not only validated by multiple operators as a standard operating procedure for monitoring T-cell responses but was also successfully used for discovering novel CD8+ and CD4+ T cells specific to previously unknown epitopes from the NY-ESO-1 tumor antigen.

Animals↗
PubMed2004

CrELISA: a fast and robust enzyme-linked immunosorbent assay bypassing the need for purification of recombinant protein.

A multitude of antigens has been recently identified by screening of cDNA expression libraries derived from human tumors with autologous sera. Using a phage autoantibody assay and small panels of sera derived from cancer patients or controls it has been shown that some of these antigens display cancer-associated autoantibody responses. The diagnostic and prognostic significance of these potentially cancer-related autoantibodies remains unclear until large-scale assays are developed and serological data are available for hundreds of cancer patients and controls. The major bottleneck for the development of large-scale assays are the cloning, expression and the purification of each of the respective antigens. Due to these limitations and despite the potential clinical relevance large-scale autoantibody tests are established for only a few of these tumor antigens. Here we describe an enzyme-linked immunosorbent assay, Crude lysate ELISA (CrELISA), suitable for antigens identified by expression screening based on crude lysates of antigen-expressing bacteria. This assay permits sensitive and specific autoantibody seroscreening without the need of laborious and time-consuming cloning, expression and purification of recombinant proteins. CrELISA is robust and provides a versatile high throughput procedure for the rapid evaluation of multiple antigens in large-scale serology.

Antibodies, Neoplasm↗
PubMed2006

A robust method for production of MHC tetramers with small molecule fluorophores.

Tetramers of major histocompatibility complex molecules (MHC) are now well-established reagents for the detection of antigen-specific T cells by flow cytometry. MHC tetramers are prepared by mixing enzymatically biotinylated MHC molecules with commercial preparations of streptavidin, usually conjugated to a fluorescent phycobiliprotein such as phycoerythrin (PE) or allophycocyanin (APC). While data obtained with MHC tetramers prepared with small molecule fluorophores has been reported, considerable lot-to-lot variation among conventional streptavidin conjugates to small molecules prevents routine preparation of such reagents. We now report robust preparation of MHC tetramers with small molecule fluorophores, using a recombinant mutant of streptavidin incorporating a carboxy-terminal cysteine in each of the four identical subunits that is conjugated to maleimide derivatives of any of several small molecule fluorophores. These reagents significantly expand the versatility of the MHC tetramer methodology.

Flow Cytometry↗
PubMed2005

The transition state for folding of a peripheral subunit-binding domain contains robust and ionic-strength dependent characteristics.

The denaturant dependencies of the folding and unfolding kinetics were used to characterize the structure of the transition state for folding of E3BD, a peripheral subunit-binding domain. For the majority of E3BD mutants, the Phi-values calculated at 298 K from the analysis of chevron plots were in good agreement with those previously determined at 325 K using Arrhenius analysis. This agreement further demonstrates the general robustness of Phi-value analyses, since different experiments, methods of denaturation and thermodynamic assumptions were used to determine each set of Phi(F) values. The structure of the transition state for folding was grossly conserved at 298 K and 325 K, with residues in Helix I playing a lesser role in folding than those located in the 3(10) helix, disordered loop and Helix II. However, the energetic contributions of a cluster of basic residues close to the N-terminus and Helix I, which are an integral part of the ligand-binding site, were susceptible to ionic strength effects because of electrostatic strain in native and transition states of E3BD at low ionic strength. We found no evidence of the downhill folding previously proposed for E3BD, even though the conditions employed in this study significantly increased the energetic bias towards the native state.

Bacterial Proteins↗
PubMed2026

Towards a Robust cell-free DNA Isolation Protocol for NGS Applications in a Clinical Molecular Diagnostics Setting.

Cell-free DNA (cfDNA), released from apoptotic and necrotic cells into body fluids, is a non-invasive source of genetic information for disease prediction, diagnosis, and monitoring. However, its low abundance makes cfDNA highly susceptible to various pre-analytical influences, potentially increasing high molecular weight (HMW) or genomic DNA (gDNA) compromising downstream cfDNA analyses. This study evaluated the impact of different cfDNA-stabilizing blood collection tubes (BCT; Cell-Free DNA BCT, Streck; S-Monovette cfDNA Exact, Sarstedt) stored at room temperature for 1, 5, or 10 days, prior to plasma isolation using different isolation methods (magnetic bead-based or silica column-based) on cfDNA stability and yield. DNA quantity and quality were assessed by fluorometric quantification, automated fragment analysis, and gene-specific quantitative PCR. Streck-based workflows maintained stable cfDNA yields and characteristic mononucleosomal fragmentation profiles across all storage times. In contrast, Sarstedt tubes showed reduced cfDNA concentrations after 5 days and a pronounced increase at 10 Days, accompanied by high-molecular weight DNA patterns consistent with white-blood cells (WBC) lysis. These trends were largely independent of the extraction method. Overall, the results demonstrate that blood collection tube chemistry critically influences cfDNA integrity during delayed processing. Streck tubes, particularly when combined with silica column-based isolation method, provided the most robust and reproducible workflow for routine molecular diagnostics, whereas Sarstedt tubes produced physiologically implausible results after extended storage.

blood collection tubes↗
PubMed2004

The dynamics of spatial behavior: how can robust smoothing techniques help?

A variety of setups and paradigms are used in the neurosciences for automatically tracking the location of an animal in an experiment and for extracting features of interest out of it. Many of these features, however, are critically sensitive to the unavoidable noise and artifacts of tracking. Here, we examine the relevant properties of several smoothing methods and suggest a combination of methods for retrieving locations and velocities and recognizing arrests from time series of coordinates of an animal's center of gravity. We accomplish these by using robust nonparametric methods, such as Running Median (RM) and locally weighted regression methods. The smoothed data may, subsequently, be segmented to obtain discrete behavioral units with proven ethological relevance. New parameters such as the length, duration, maximal speed, and acceleration of these units provide a wealth of measures for, e.g., mouse behavioral phenotyping, studies on spatial orientation in vertebrates and invertebrates, and studies on rodent hippocampal function. This methodology may have implications for many tests of spatial behavior.

Algorithms↗
PubMed2006

Statistically robust detection of spontaneous, non-stereotypical neural signals.

Neural signals of interest are often temporally spontaneous and non-stereotypical in waveform. Detecting such signals is difficult, since one cannot use time-locking or simple template-matching techniques. We have sought a statistical method for automatically estimating the baseline in these conditions, and subsequently detecting the occurrence of neural signals. One could consider the signals as outliers in the distribution of neural activity and thus separate them from the baseline with median-based techniques. However, we found that baseline estimators that rely on the median are problematic. They introduce progressively greater estimation errors as the neural signal's duration, amplitude or frequency increases. Therefore, we tested several mode-based algorithms, taking advantage of the most probable state of the neural activity being the baseline. We found that certain mode-based algorithms perform baseline estimation well, with low susceptibility to changes in event duration, amplitude or frequency. Once the baseline is properly established, its median absolute deviation (MAD) can be determined. One can then use it to detect spontaneous signals robustly as outliers from the noise distribution. We also demonstrate how the choice of detection threshold in terms of MADs can be used to bias against false positives, without creating too many false negatives or vice versa.

Action Potentials↗
PubMed2004

Development of a robust procedure for assessing powder flow using a commercial avalanche testing instrument.

The objectives of this work were to develop a robust procedure for assessing powder flow using a commercial avalanche testing instrument and to define the limits of its performance. To achieve this a series of powdered pharmaceutical excipients with a wide range of flow properties was characterized using such an instrument (Aeroflow, TSI Inc., St. Paul, MN, USA). The experimental conditions (e.g., sample size, rotation speed) were rationally selected and systematically evaluated so that an optimal standard-operating-procedure could be identified. To evaluate the inherent variability of the proposed methodology samples were tested at multiple sites, using different instruments and operators. The ranking of the flow properties of the powders obtained was also compared with that obtained using a conventional shear-cell test. As a result of these experiments a quick, simple, and rugged procedure for determining the flow properties of pharmaceutical powders in their dilated state was developed. This procedure gave comparable results when performed at four different testing sites and was able to reproducibly rank the flow properties of a series of common pharmaceutical excipient powders. The limits of the test method to discriminate between different powder samples were determined, and a positive correlation with the results of a benchmark method (the simplified shear cell) was obtained.

Chemistry, Pharmaceutical↗
PubMed2006

Augmentation of near infrared diffuse reflectance and transmittance spectral data for the development of robust PLSBC models for classifying double blind clinical trial tablets.

The water content of clinical trial tablets can be different between and within different tablet batches, depending on the relative humidity conditions during their production, packaging, storage and analysis. These water variations lead to important spectral variations in the near infrared spectral region which can lead to a wrong identification if the classification model was based on unrepresentative data towards the water content. As model development for clinical trial studies needs to be extremely fast - within one working day - with generally only one batch available, the principle of data augmentation has to be applied to render more robust classification models. Therefore, tablets available for constructing the model are being processed in order to increase or decrease their water content and to make them more representative for tablets to be tested in the future. The inclusion of a deliberate water variation is the most efficient way to develop a model, for which no additional model redevelopment will be required to pass the system suitability tests and to obtain a correct identification.

Chemistry, Pharmaceutical↗
PubMed2005

Verbal and physical non-aggressive agitated behaviors in elderly persons with dementia: robustness of syndromes.

OBJECTIVE: More than a decade ago, different syndromes of agitation were identified in elderly nursing home residents, and it was found that these syndromes correlate with medical, cognitive, and psychosocial functioning. The present study was conducted to examine the robustness of two major syndromes, verbal agitation and physical non-aggressive agitation, as assessed via direct observations. METHODS: Study participants were 175 elderly persons with dementia recruited from 11 nursing home facilities in Maryland. Observations of the participants' behavior were conducted using the agitated behaviors mapping instrument. RESULTS: The profiles that emerged for physically agitated residents and for verbally agitated residents were remarkably similar to those originally reported. Specifically, verbally agitated behaviors correlated with female gender, with cognitive decline, poor performance of activities of daily living, impaired social functioning, and signs of depressed affect. Physically non-aggressive agitated behaviors correlated with cognitive impairment and with fewer concurrent medical diagnoses. CONCLUSIONS: Examining correlates of different syndromes of agitated behaviors may provide researchers with valuable information that can be used for in-depth analysis of both the characterization and etiology of agitation, thus paving the way for the development of interventions that target particular types of problem behaviors.

Activities of Daily Living↗
PubMed2005

A robust characterization of retinoic acid response elements based on a comparison of sites in three species.

The availability of high-throughput genomic sequencing has allowed us to construct a more robust characterization of retinoic acid response elements than was possible in the past. We located human, mouse, and rat homologs for each of 51 well-documented, conserved retinoic acid response elements. Mathematical and statistical analyses of these 153 sites, 78 of which are new, shows that 92% of response elements have direct-repeat symmetry, but that only 76% exhibit canonical spacing attributes. While the familiar '(a/g)g(g/t)tca' hexamer motif is upheld, the more relaxed sequence, '(a/g)g(g/t)(g/t)(g/c)a', represents a 10% consensus. Sites are as likely to be on the coding strand as on the non-coding strand, and 86% of them are in upstream locations. From a statistical point of view, DR1 elements are fundamentally different from DR2 and DR5 elements, but this is only evident in the 5' hexamer. While there is considerable variation in core positions, and while no nucleotide can be considered forbidden at any position, variation among species at a fixed locus appears surprisingly constrained once a functional site has been attained.

Animals↗
PubMed2004

Absence of robust ischemic preconditioning by five 1-minute total umbilical cord occlusions in fetal sheep.

OBJECTIVE: To determine to what extent a series of five 1-minute total umbilical cord occlusions, intended to induce ischemic preconditioning (IP), affects the physiologic responses to a 10-minute total umbilical cord occlusion (damaging insult [DI]) 1 hour later and provides cardio- and neuroprotection. METHODS: In 14 chronically catheterized late gestation fetal sheep (127-131 days' gestation), we performed a 10-minute total umbilical cord occlusion (DI), preceded by a series of five 1-minute total cord occlusions with 2-minute intervals (5CO, n = 7) or sham occlusions (n = 7) 1 hour prior to DI. RESULTS: The 5CO induced a reduction in the arterial partial pressure of oxygen (Po(2)) from 21 +/-1 to 14 +/-3 Torr, arterial O(2) content from 6.9 +/- 0.4 to 3.1 +/- 0.7 vol%, and increases in the partial pressure of carbon dioxide (Pco(2)) from 46 +/- 2 to 58 +/- 3 Torr, and [H(+)] from 43 +/- 1 to 54 +/- 2 nM. 5CO reduced fetal heart rate from 178 +/- 6 to 151 +/- 6 beats per minute (bpm), and increased arterial pressure from 45 +/- 1 to 57 +/- 2 mmHg, cerebral blood flow (CBF) from 100 +/- 3 to 129 +/- 10%, and cerebral heat production (H(brain)) from 25 +/- 2 to 29 +/- 1% degrees C. The responses to DI were not significantly different between the groups without and with 5CO; values for Po(2) were 5.6 +/- 1.5 and 5.8 +/- 1.9 Torr, O(2) content 0.6 +/- 0.1 and 0.8 +/- 0.1 vol%, lactate 10.7 +/- 0.7 and 10.8 +/- 0.7 mM, fetal heart rate 97 +/- 5 and 87 +/- 8 bpm, mean arterial pressure 22 +/- 3 and 21 +/- 2 mmHg, CBF 50 +/- 10 and 36 +/- 5%, and H(brain) 7.0 +/- 1.4 and 5.9 +/- 1.1% degrees C, respectively, except for Pco(2) (126 +/- 4 and 112 +/- 2 Torr) and [H(+)] (126 +/- 3 and 114 +/- 3 nM). Histologic proof of cardio- or neuroprotection by 5CO could not be obtained because five fetuses died before they were to be killed at day 3 after the experiment; two fetuses in the 5CO group demonstrated major histologic damage of myocardium and brain. CONCLUSION: In the late gestation fetal sheep, a series of five 1-minute total umbilical cord occlusions did not result in major changes in physiologic responses to a hypoxic-ischemic DI 1 hour later. In addition, the procedure did not result in robust cardio- and neuroprotection, in contrast to IP reported in adults.

Animals↗
PubMed2006

Motor potential profile and a robust method for extracting it from time series of motor positions.

Molecular motors are small, and, as a result, motor operation is dominated by high-viscous friction and large thermal fluctuations from the surrounding fluid environment. The small size has hindered, in many ways, the studies of physical mechanisms of molecular motors. For a macroscopic motor, it is possible to observe/record experimentally the internal operation details of the motor. This is not yet possible for molecular motors. The chemical reaction in a molecular motor has many occupancy states, each having a different effect on the motor motion. The overall effect of the chemical reaction on the motor motion can be characterized by the motor potential profile. The potential profile reveals how the motor force changes with position in a motor step, which may lead to insights into how the chemical reaction is coupled to force generation. In this article, we propose a mathematical formulation and a robust method for constructing motor potential profiles from time series of motor positions measured in single molecule experiments. Numerical examples based on simulated data are shown to demonstrate the method. Interestingly, it is the small size of molecular motors (negligible inertia) that makes it possible to recover the potential profile from time series of motor positions. For a macroscopic motor, the variation of driving force within a cycle is smoothed out by the large inertia.

Animals↗
PubMed2006

Selection for mutational robustness in finite populations.

We investigate the evolutionary dynamics of a finite population of RNA sequences replicating on a neutral network. Despite the lack of differential fitness between viable sequences, we observe typical properties of adaptive evolution, such as increase of mean fitness over time and punctuated-equilibrium transitions, after initial mutation-selection balance has been reached. We find that a product of population size and mutation rate of approximately 30 or larger is sufficient to generate selection pressure for mutational robustness, even if the population size is orders of magnitude smaller than the neutral network on which the population resides. Our results show that quasispecies effects and neutral drift can occur concurrently, and that the relative importance of each is determined by the product of population size and mutation rate.

Animals↗
PubMed2006

Robust measures of three-dimensional vascular tortuosity based on the minimum curvature of approximating polynomial spline fits to the vessel mid-line.

The clinical recognition of abnormal vascular tortuosity is important in the diagnosis of many diseases. This paper presents a novel approach to the quantification of vascular tortuosity, using robust metrics based on unit speed parameterizations of three-dimensional (3D) curvature. The use of approximating polynomial spline-fitting obviates the need for arbitrary filtering of mid-line data which is necessary with other tortuosity indices. The metrics were tested using both two-dimensional and three-dimensional synthesized images that mimicked clinically significant pathologies: two of the three metrics were scale invariant, additive, and produced tortuosity values tailored to be independent of the resolution of the imaging system. Our methodology is designed to explicitly handle the challenge of noisy data, and is largely tolerant of the inaccuracies in the mid-line extraction. While all the proposed metrics are sensitive to gently curved vessels, the rms curvature of the smoothest path was more effective in recognizing abnormalities involving high-frequency coiling such as occurs in malignant tumors. We have also indicated how values from two projection images, such as acquired in biplane angiography, can be combined to give an accurate approximation of the 3D value of this metric. Our proposed methodology is well-suited to automated detection and measurement, which are a prerequisite for clinical implementation.

Algorithms↗