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Results for “protocol optimization”

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At least 145 records · Page 8Linked to original sources
PubMed1991

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments.

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments were studied with goat anti-mouse and goat anti-human immunoglobulin G. Antibodies were immobilized on HW 65 polymeric support matrix activated with carbonyldiimidazole, hydrazide and iodoacetic acid. The most significant factors influencing the specific activity of stochastic coupling of antibodies are multi-site attachment, multiple orientations and steric hindrance imposed by crowding of antibody and the size of the antigen. In oriented immobilization the specific activity is affected only by steric hindrance. The specific activity of immunosorbents prepared by immobilization of F(ab') fragments can be improved to almost 100% by limiting the amount of protein immobilization and the size of the antigen. The present study shows the protocols for optimizing immobilized antibody performance.

Adipates↗
PubMed1992

Chemiluminescent DNA probes: evaluation and usefulness in forensic cases.

Five phosphatase-labelled oligonucleotide probes were evaluated in respect to their sensitivity, with the help of an optimized chemiluminescent protocol, for DNA-VNTR polymorphism determination. Their usefulness for the identification of biological traces is illustrated with casework examples.

Alkaline Phosphatase↗
PubMed1992

Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification.

Alu-PCR protocols were optimized for the generation of human DNA probes from yeast strains containing yeast artificial chromosomes (YACs) with human inserts between 100 and 800 kb in size. The resulting DNA probes were used in chromosome in situ suppression (CISS) hybridization experiments. Strong fluorescent signals on both chromatids indicated the localization of specific YAC clones, while two clearly distinguishable signals were observed in greater than or equal to 90% of diploid nuclei. Signal intensities were generally comparable to those observed using chromosome-specific alphoid DNA probes. This approach will facilitate the rapid mapping of YAC clones and their use in chromosome analysis at all stages of the cell cycle.

Base Sequence↗
PubMedDate not supplied

MRA-guided stereotactic radiation treatment planning for cerebral angiomas.

Stereotactic neurosurgical interventions are characterized by a high risk of rupturing intracranial vessels or damaging vital brain structures. By MRI and MRA, the anatomic information necessary for stereotactic treatment planning may be assessed with a single modality as a high-resolution digital image volume. The adequacy of MR as a modality for stereotactic guidance is demonstrated by the example of the radiosurgical treatment of cerebral angiomas. An optimized acquisition protocol, a stereotactic apparatus, a distortion correction and minimization method, and a 3D treatment planning workstation are elements of a proposed preoperative approach which in a clinical study met with good acceptance.

Algorithms↗
PubMed2026

The effect of tDCS on emotion-related risk-taking behavior and delay discounting in adults with ADHD.

INTRODUCTION: Adults with Attention Deficit Hyperactivity Disorder (ADHD) often engage in risky behaviors due to impaired decision-making processes. This study aims to investigate the effects of transcranial direct current stimulation (tDCS) over the dorsolateral prefrontal cortex (dlPFC) and ventromedial prefrontal cortex (vmPFC) on emotion-related risk-taking behavior and delay discounting in adults with ADHD. METHODS: Thirty adults with ADHD underwent three tDCS conditions, administered in a randomized order with at least one week between sessions: (1) left dlPFC anode/right vmPFC cathode, (2) left dlPFC cathode/right vmPFC anode, and (3) sham stimulation. In each session, participants completed the Delay Discounting Task (DDT) and the Modified Balloon Analogue Risk Task (mBART) under three emotional conditions (neutral, positive, and negative) which were induced using emotionally congruent photographs and sounds. Galvanic skin responses (GSR) were also recorded. In the DDT, both area under the curve (AUC) values and log-transformed discounting rates (log k) were calculated for small, medium, and large reward magnitudes (RM). Exploratory electric field modeling was also performed to characterize current distribution. RESULTS: The findings demonstrated task-specific effects of tDCS on decision-making. Although no overall tDCS effect was observed on DDT performance, significant tDCS × RM interactions emerged, particularly for smaller rewards. In contrast, exploratory analyses suggested that tDCS affected all mBART scores. Emotional condition did not influence consistently behavioral performance in either task, whereas both emotional stimulation and tDCS significantly affected GSR responses. However, exploratory electric field modeling indicated a broad prefrontal current distribution extending beyond the intended cortical targets. CONCLUSIONS: These findings suggest preliminary evidence that prefrontal tDCS can influence risk-related decision-making and autonomic responses in adults with ADHD. However, its effects on delay discounting appear to be context-dependent and limited to specific RMs. Future studies combining neuroimaging with individualized electric field modeling are needed to clarify the neural mechanisms underlying the observed effects of tDCS and to optimize stimulation protocols in adults with ADHD.

Humans↗
PubMed2026

Establishment of a cBSA-mediated miRNA delivery system in Camellia sinensis and functional validation of the Cs-miR163/CsSK1 module in cold stress response.

Cold stress severely limits tea (Camellia sinensis) yield and quality. MicroRNAs (miRNAs) are key post-transcriptional regulators of plant cold responses; however, in vivo functional validation in tea plants is hindered by the lack of efficient genetic transformation and nucleic acid delivery systems. In this study, a cationized bovine serum albumin (cBSA)-mediated miRNA delivery system was established in tea plants. The cold-responsive miRNA Cs-miR163 and its target gene CsSK1 (a negative regulator of cold tolerance) were used as a model. Direct cleavage of CsSK1 mRNA by Cs-miR163 was confirmed by 5' RLM-RACE and GUS transient expression assays, and enhanced cold tolerance was demonstrated in Arabidopsis overexpression lines. The cBSA preparation protocol was optimized, yielding stable cBSA/miRNA complexes with high protective capacity across temperatures of 15-35 °C and pH 4.5-7.2. Delivery parameters were systematically evaluated; optimal conditions were determined as 2 mg/mL cBSA with 10 nM miRNA and solution uptake into 3-cm cuttings for 5 days, enhancing miRNA delivery efficiency by approximately 48-fold. Transmission electron microscopy provided direct ultrastructural evidence that cBSA/miRNA nanocomplexes are internalized into tea plant cells via adsorptive-mediated endocytosis involving electrostatic membrane adsorption, membrane invagination, and cytoplasmic release. Under optimized conditions, cBSA-mediated delivery of Cs-miR163 silenced CsSK1 expression by approximately 72%, reduced relative electrolyte leakage and ROS accumulation, and markedly enhanced cold tolerance. The regulatory role of the Cs-miR163/CsSK1 module was clarified, and the established system provides a promising strategy for functional genomics in woody plants that warrants further testing in additional species and tissues.

Camellia sinensis↗
PubMed2025

Cell-type-specific DNA methylation dynamics in the prenatal and postnatal human cortex.

The human cortex undergoes extensive epigenetic remodeling during development, although the precise temporal and cell-type-specific dynamics of DNA methylation remain incompletely understood. In this study, we profiled genome-wide DNA methylation across human cortex tissue from donors aged 6 post-conception weeks to 108 years of age. We observed widespread, developmentally regulated changes in DNA methylation, with pronounced shifts occurring during early- and mid-gestation that were distinct from age-associated modifications in the postnatal cortex. Using fluorescence-activated nuclei sorting, we optimized a protocol for the isolation of SATB2-positive neuronal nuclei, enabling the identification of cell-type-specific DNA methylation trajectories in the developing cortex. Developmentally dynamic DNA methylation sites were significantly enriched near genes implicated in autism and schizophrenia, supporting a role for epigenetic dysregulation in neurodevelopmental conditions. Our findings underscore the prenatal period as a critical window of epigenomic plasticity in the brain with important implications for understanding the genetic basis of neurodevelopmental phenotypes.

Humans↗
PubMed1991

Clinical experience with technetium 99m teboroxime.

Technetium 99m teboroxime is a boronic acid adduct of technetium dioxime complex, a neutral lipophilic technetium-containing myocardial perfusion imaging agent with high myocardial extraction, rapid myocardial washout, and entero-hepatic excretion. Early "redistribution" occurs, and, based on preliminary animal data, appears to be due to differential washout alone since there is no reuptake of teboroxime into the heart; however, for practical purposes two injections of tracer, at stress and at rest, are used clinically. Due to the high photon flux of technetium, diagnostic images can be acquired in the narrow time window between the end of blood clearance and peak hepatic uptake. To begin an acquisition within 2 minutes of tracer injection, the patient must be moved rapidly from the treadmill to a camera that is set and ready to count. A complete set of planar images can be completed in less than 5 minutes. Optimal single photon emission computed tomography (SPECT) imaging requires a continuously counting single detector camera or a three-headed camera to complete acquisition in less than 10 minutes. The image quality is comparable to thallium 201 and the imaging time is a fraction of the time required to complete SPECT thallium scans. Both left ventricular function (ejection fraction) and planar imaging can be performed in less than 4 minutes, without moving the patient from the treadmill, using the Sim-400 (Scinticor, Milwaukee, WI), a portable high count-rate, multicrystal scintillation camera. Because teboroxime maintains high myocardial extraction at high myocardial flow, it may be uniquely suited to be used in combination with pharmacological stress, which does not require patient movement between stress and imaging. Work is in progress to further refine and optimize imaging protocols using teboroxime.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Measurement of lymphocyte proliferation: critical analysis of radioactive and photometric methods.

Different methods of lymphocyte proliferation are compared to identify a non-radioactive alternative to 3H-thymidine-test. The enzymatic assays evaluating the turnover of mitochondrial dehydrogenases (MTT-test) and lysosomal hexosaminidase (NAG-test) proved not sensitive enough to substitute for 3H-thymidine incorporation. The incorporation of the nucleotide analog 5-bromodeoxyuridine (BrdU) can be exploited using an ELISA-system (enzyme linked immunosorbent assay) employing a monoclonal anti-BrdU antibody to measure cell proliferation. An optimized test protocol of the BrdU-ELISA which fulfills the requirements for a sensitive and practicable non-radioactive alternative to 3H-thymidine-test is presented.

Autoradiography↗
PubMedDate not supplied

Combined in situ hybridization and immunocytochemistry in the assay of pharmacological effects on tyrosine hydroxylase mRNA concentration.

An assay for tyrosine hydroxylase (TH) mRNA by in situ hybridization in combination with immunocytochemistry (ICC) for TH on the same section is described. The in situ hybridization protocol was optimized for [35S]cRNA (complementary RNA, i.e. anti-sense strand) probe concentration and time of hybridization. The specificity of hybridization was measured by several critera. The advantage of measuring grain density versus grains per cell is discussed for quantitation of in situ autoradiography. Finally, the reserpine-induced increase in adrenal TH mRNA was used to validate quantitative aspects of the in situ hybridization technique by comparison with blot hybridization. In contrast to the adrenal, reserpine did not increase TH mRNA in substantia nigra (s. nigra) neurons as measured by either technique.

Animals↗
PubMed1989

Substrate specificity and variables affecting efficiency of mammalian flavin adenine dinucleotide synthetase.

Substrate specificity and product inhibition have been evaluated by using purified rat liver FAD synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2), obtained by an improved purification protocol with optimized flavin affinity chromatography. FMN analogues studied fall into three general classifications: those with substitution on the pyrimidinoid ring and nitrogen replacement, those with substitution on the benzenoid ring, and those with N(10) side chain modifications. Substitutions on the pyrimidinoid ring and replacement of nitrogens have the greatest influence on binding to enzyme and FAD formation. When the hydrogen-bonding capacity of the NH group at position 3 is blocked or removed by substitution, such FMN analogues do not act as substrates or inhibitors of the enzyme. Substitutions on the benzenoid ring by small groups seem to be tolerated, while larger groups inhibit binding. Length of the N(10) side chain is optimal with five carbons and has greatest affinity for the natural ribityl side chain. Affinity matrices show similar binding characteristics in that the N(3)-(carboxymethyl)riboflavin-agarose does not bind enzyme, while agaroses linked to the flavin N(10) side chain provide varying degrees of purification. The C = O group at position 2, the NH group at position 3, and a five-carbon side chain at the N(10) position seem to be most crucial for flavin substrate binding to enzyme. Nucleoside triphosphates other than ATP do not act as substrates or inhibitors when sufficient Mg2+ is present. Products of the reaction, FAD and PPi, act as inhibitors against both ATP and FMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1989

A limited sampling procedure for estimating adriamycin pharmacokinetics in cancer patients.

The aim of this study was to find a procedure allowing estimation of individual pharmacokinetic parameters for adriamycin with minimal cost and disturbance for the patient. Twenty-five patients with breast cancer were treated by short infusion of adriamycin at a dose of 12 mg m-2 week-1 (41 courses). Population characteristics were determined on 15 randomly chosen courses (10 patients, group I) in order to define two optimal sampling times (26 min and 24 h) and to perform Bayesian estimation on the remaining 26 courses (17 patients, group II). For patients of group II, Bayesian estimation (BE) associated with a reduced sub-optimal sampling protocol (20 min and 24 h) was compared with maximum likelihood estimation (MLE), the classical procedure. Regression analysis of clearance values obtained after BE versus MLE indicated a high correlation coefficient (r = 0.969) with the slope (a = 0.991 +/- 0.085) and the intercept (b = 2.271 +/- 4.810) close to 1 and 0 respectively. This original method is thus valid to measure accurately adriamycin clearance; it improves patient comfort and can be used routinely.

Adult↗
PubMed1992

Prior chemotherapy does not prevent effective mobilisation by G-CSF of peripheral blood progenitor cells.

In this study we demonstrate that the hemopoietic growth factor, G-CSF successfully mobilised progenitor cell populations into the peripheral blood in a population of patients despite intensive pretreatment with chemotherapy. Administration of G-CSF increased the numbers of peripheral blood progenitor cells (PBPC) by a median of 76-fold above basal levels. Maximal levels of PBPC were observed on days 5 and 6 after G-CSF treatment. In two patients a second cycle of G-CSF mobilised PBPC to levels comparable with those seen after the first cycle of G-CSF treatment. An earlier hemopoietic cell population (pre-CFC's) was also mobilised with levels increased up to 50-fold above basal levels. Using a standard mononuclear cell leukapheresis technique the PBPC were collected extremely efficiently (essentially 100%) and could be further successfully enriched by separation using a Ficoll gradient. For patients who underwent the optimal collection protocol (i.e. leukapheresis on days 5, 6 and 7) a total of 32 +/- 6 x 10(4) GM-CFC kg-1 were collected. The ability to mobilise PBPC using G-CSF alone and to successfully and efficiently harvest these cells has important implications for the future of transplantation and high dose chemotherapy procedures.

Adolescent↗
PubMed2026

Dynamic lysine acetylation and succinylation of platelet proteins regulates platelet storage lesion: mechanistic insights from multi-omics.

OBJECTIVES: Platelet storage lesion (PSL) severely impairs platelet function during storage, presenting a major hurdle in transfusion medicine; however, the dynamic interplay between global proteomic changes and post-translational modifications (PTMs) underlying these functional deteriorations remains insufficiently characterized. Here, we report the first comprehensive multi-omics analysis integrating global proteomics, acetylomics, and succinylomics to dissect the molecular dynamics during platelet storage. METHODS: We performed quantification of global proteomics, acetylome and succinylome based on TMT-labeled LC-MS/MS analysis, combined with antibody-affinity enrichment and purification. Dynamic molecular changes and functional transformation of platelet were also characterized under proper conditions stored for 1, 3, 5, 7 days, respectively. RESULTS: We systematically characterized 3,609 proteins, 1,308 acetylation sites, and 1,947 succinylation sites across multiple storage time points (D1, D3, D5, D7). We distinct temporal patterns of post-translational modifications, with succinylation showing more extensive coverage than acetylation in platelets. Pathway enrichment analysis revealed extensive metabolic reprogramming involving complement activation, energy metabolism, and cellular detoxification processes. The identification of specific motif patterns provided mechanistic insights into the functional specificity of these modifications. Random forest machine learning identified 20 core regulatory proteins representing critical nodes in PSL development. Furthermore, we employed real - time quantitative polymerase chain reaction (RT - QPCR) to measure the expression levels of key genes related to platelet function and PTM - associated pathways. CONCLUSION: By mapping the interplay between proteomic abundance shifts and PTM dynamics, this study provides a multidimensional understanding of PSL, establishing a foundational framework for optimizing storage protocols and enhancing transfusion safety.

Blood Platelets↗
PubMed1992

Review of respirator performance testing in the workplace: issues and concerns.

Performance capability of respirators has traditionally been evaluated by testing components of the respirator (e.g., filter efficiency), facepiece fit, total inward leakage, or some other measure of performance evaluated under laboratory conditions. In recent years, increased emphasis has been placed on development of test methods suitable for evaluating respirator performance in the workplace. The goal of such testing is to evaluate the level of protection provided by respirators in the work environment. The AIHA Respiratory Protection Committee believes that workplace testing of respirators has the potential to be an excellent tool for increasing knowledge about the effectiveness of respiratory protection. However, a number of technical issues remain to be addressed before optimal test protocols and data analysis methods can be defined. The progress made to date in workplace testing will be reviewed, and broader discussion about key elements that must be considered when developing guidelines for testing respirators in the workplace will be initiated.

Air Pollution, Indoor↗
PubMed1982

Mutagenicity of azo dyes in the Salmonella/activation test.

The detection of certain bis-azo biphenyl dyes (Trypan blue, Evans blue and Congo red) by a modification of the standard Ames test protocol is reported. Detection of a mutagenic response required both the inclusion of FMN in S-9 mix and use of a static pre-incubation step prior to addition of top-agar and pouring in the usual manner. Evidence is presented to substantiate the proposal that use of this protocol provides optimal conditions for the two stages of activation required for the production of mutagenic metabolites from these dyes. These are (i) initial flavin-mediated (hepatic) azo reduction resulting in the formation of proximate mutagenic metabolites followed by (ii) the oxidative metabolism by S-9 resulting in the formation of ultimately mutagenic products.

Animals↗
PubMed1985

Mouse cell clones for improved quantitation of carcinogen-induced altered differentiation.

Two new mouse epidermal cell lines have been isolated and characterized as target cells for three chemical carcinogens. The ability to grow these cells at low density (approximately 5 clonogenic cells/cm2) has permitted more precise quantitation of chemical carcinogen-induced changes in epidermal differentiation. The cell lines, designated 291 and 271c, retain the property previously observed in primary cultures of mouse epidermal cells, that is the regulation of terminal differentiation by extracellular Ca2+ ion. Altered response to extracellular Ca2+ after carcinogen treatment of these cells is the basis of the assay endpoint. Other normal properties demonstrated by these cells are keratin immunofluorescence patterns, ability to form cornified envelopes in response to Ca2+ and a lack of tumorigenicity. Both of the lines have high cloning efficiencies (up to 20%) and characteristic epidermal morphology. Their chromosome number, however, is near tetraploid. Dose response studies indicated an increase in colonies with altered response to Ca2+ proportional to the dose of three chemical carcinogens: DMBA 0.001-0.5 microgram/ml X 24 h, MCA 0.01-5 micrograms/ml X 24 h and MNNG 0.01-0.2 micrograms/ml X 1 h. The optimized assay protocol has provided a reproducible means of quantitating carcinogen-altered epidermal cells relative to carcinogen dose, and of isolating cell clones for studies of altered differentiation in carcinogenesis and chemotherapy.

Animals↗
PubMed1986

Suppression of B cell immunity to DNP by serotherapy with monoclonal anti-DNP antibodies. Effect of the treatment schedule on the magnitude of specific suppression and its duration.

The effect of prior administration of anti-DNP (N-[2,4-dinitrophenyl]-B-alanylglycylglycine) monoclonal antibodies on the humoral immune response of BALB/c mice was examined. One-time administration of a "cocktail" of two anti-DNP monoclonals resulted in suppression of the IgM anti-DNP response for one week after challenge but not longer. Maximal suppression of anti-DNP IgM plaque-forming cells was achieved by administration of antibody 1-2 weeks before challenge with DNP. Maximal suppression of serum IgM antibody was seen by administration of antibody 2-3 weeks before challenge with antigen. Following one-time administration of suppressive monoclonal antibody, the serum IgG antibody response to DNP was suppressed beginning 2 weeks after immunization and remained so for up to 241 days despite continual booster injections of antigen. Although most effective suppression of the humoral anti-DNP response was seen in animals receiving their single dose of suppressive antibody 2 weeks before first exposure to antigen, suppression of the IgG response was evident at all intervals examined up to 232 days in mice given monoclonal antibody between 0.1 day and 30 days before antigen, but not at earlier times. These findings suggest that regulatory networks, rather than the masking of antigenic determinants by passively administered antibody, play a role in antibody-mediated immunoregulation. They may be of use in designing strategies for optimizing immunosuppression protocols in clinical studies.

Animals↗