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Results for “protocol optimization”

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PubMed1983

The control issue in biofeedback training.

Well-delineated models and procedural descriptions are noticeably missing from the clinical biofeedback literature dealing with the training of physiological self-control. As a consequence, optimal protocols for the assessment and training of self-control of physiological functions have not eventuated. The present article reviews evidence suggesting that researchers have (a) confused self-control training, assessment, and measurement of tonic response levels, (b) neglected to determine the degree of acquired physiological self-control, and (c) failed to report the relationship between level of acquired physiological control and clinical outcome. The article also proposes an assessment and training paradigm that may be useful in reducing these methodological problems in future biofeedback research.

Arousal↗

PubMed1992

Survival of human oocytes cryopreserved with or without the cumulus in 1,2-propanediol.

BACKGROUND: Although cryopreservation of human preembryos has been carried out with success, the cryostorage of oocytes, which pose fewer controversial moral, ethical, and legal problems has been much less successful. Various attempts to cryopreserve human oocytes have been mostly unsuccessful and the search for an optimal protocol for oocyte cryopreservation remains elusive. We therefore undertook this study to determine the effect of oocyte cryostorage in 1,2-propanediol. METHOD: Mature human oocytes with or without their cumuli were cryopreserved in precooled 1,2-propanediol, then thawed and inseminated with sperms for in vitro fertilization. The outcome of insemination and subsequent embryonic development were also recorded and compared. RESULTS: Postthaw cryosurvival rate was significantly better when cryostorage was carried out with the oocyte cumulus intact as compared to those oocytes denuded of their cumuli (54 versus 27%, respectively; P < 0.05). Eight (44%) of 18 surviving postthaw oocytes with intact cumuli were fertilized normally, with cleavage in six, as compared to two (25%) and one, respectively, of those denuded of their cumulus prior to cryostorage. Development to the blastocyst stage was achieved in three embryos derived from oocytes with an intact cumulus at cryostorage. CONCLUSION: We conclude that 1,2-propanediol can be used with success in oocyte cryopreservation, although the issue of parthenogenecity is still to be resolved. Oocyte's with intact cumulus survive cryostorage better than those without it.

Adult↗

PubMed1987

Establishment of primary cell cultures: experiences with 155 cell strains.

Cell culture systems allow the examination of cell populations in a functional state. To simulate in vivo conditions as closely as possible freshly established cell strains are superior to permanent cell lines. Different aspects for the establishment of primary cell cultures obtained from various tissues are compared: Disintegration, culture media supplemented with basal additions, special supplements (growth factors, hormones), and attachment factors. The proliferation rates of the attained cell strains were evaluated by determination of cell doubling times. Procedures for how to obtain a relatively high plating efficiency (approx. 70% in our series of 219 attempts) of primary growth in vitro are described: (1) Mechanical disintegration is superior to enzymatic digestion. If mechanical treatment alone did not produce a sufficient number of viable cells, additional digestion with collagenase/dispase revealed a higher number of proliferating primary cultures than with trypsin. (2) Proliferation of cell cultures from normal and tumorous tissues of epithelial origin was superior in Leibovitz L 15 medium (58 of 87 (67%) cases). Cultures from mesenchymal tissues and tumors were found to have shortest cell doubling times in MEM and RPMI 1640 (16 of 23 (70%) cases). The media were supplemented with the basal additions indicated. (3) In approx. 30% of the cases special supplements like growth factors or hormones increased cell replication, although they were almost always not essential for cell growth. (4) Attachment factors only rarely contributed to the initiation of primary monolayer cultures. The application of various culture conditions does not lead to a protocol optimal for all tissues, for all probes of the same type of tumor, or for all tumor specimens of unique differentiation.

Cell Adhesion↗

PubMed1994

Polybrene/DMSO-assisted gene transfer. Generating stable transfectants with nanogram amounts of DNA.

Polybrene/DMSO-assisted gene transfer is a simple and versatile transfection strategy capable of producing high numbers of stable transfectants from adherent monolayer cultures with low (nanogram) quantities of exogenous DNA. The procedure involves two stages: adsorption and internalization. The former is mediated by polybrene (a polycation polymer) and favors the uniform coating of target cells with polybrene-DNA complexes. Following adsorption, the cells are permeabilized by a brief exposure to dimethyl sulfoxide (DMSO) to facilitate the uptake of DNA complexes. Diverse cell types can be exposed to a wide range of polybrene concentrations without adverse effects. By contrast, the key determinant of success is the DMSO permeabilization regime, which must be configured independently for each cell line. Protocols optimized for gene transfer in murine and human fibroblasts are presented along with a guide for the rapid optimization of the method. The advantages and limitations of the method are also discussed.

3T3 Cells↗

PubMed1994

Genetic transformation of plants by protoplast electroporation.

This article describes an optimized protocol for the electroporation of tobacco mesophyll protoplasts together with notes and data on the effects of various parameters and suggestions for work with protoplasts of other species. In this protocol, electroporation is achieved by means of electrical pulses from a high-voltage, capacitive-discharge unit. Procedures are described for measurement of protoplast viability with Evan's blue, the detection of transient expression of CAT and GUS gene plasmid constructs, and for the recovery of stable transformants based on selection for kanamycin resistance.

Cell Survival↗

PubMed2026

Liquid fermentation and an adapted cetyltrimethylammonium bromide (CTAB) method enable sequencing-grade DNA extraction from Phanerodontia australis.

High-quality genomic DNA extraction from basidiomycete fungi remains challenging due to polysaccharide co-purification and nuclease-mediated DNA degradation. We systematically compared 22 experimental conditions combining five DNA extraction methods (one Dellaporta-based method, three CTAB-based variants, and one commercial kit) with five mycelium preparation techniques (scalpel scraping, fungal suspension, silica bead homogenization, liquid fermentation, and liquid nitrogen treatment) for Phanerodontia australis (BRM62389). DNA quality was assessed by 1% agarose gel electrophoresis, NanoDrop spectrophotometry, and Qubit fluorimetry. The optimal protocol combined liquid-fermentation mycelium with an adapted CTAB method, yielding Qubit-quantified concentrations of 34.2 and 62.6 ng/µL (samples S21 and S22), 260/280 ratios of 2.06 and 2.05, 260/230 ratios of 1.88 and 1.85, and no detectable DNA degradation by agarose gel electrophoresis. This protocol enabled whole-genome sequencing, yielding a 37.62 Mb assembly with 98.5% completeness as assessed by BUSCO v5 (basidiomycota_odb10 lineage dataset, n = 1,764). Liquid fermentation is associated with the production of younger, actively growing hyphae with reduced cell wall thickness, and the adapted CTAB method effectively removed residual polysaccharides. This protocol provides a reliable, cost-effective approach for obtaining sequencing-grade DNA from P. australis.

Basidiomycota↗

PubMed1993

Immortalization of antigen selected B cells.

This paper reports the generation of monoclonal antibody producing hybridomas from a small number of antigen-specific B cells selected by panning on antigen-coated dishes and rosetting with antigen-coupled paramagnetic beads. Anti-HIV positive B cells from spleen could be recovered by panning with an efficiency of 5% and a purity of 24%. Immunobead selection of anti-HIV positive B cells from the same mice yielded a recovery of 17% and a purity of 7%. Various experimental conditions with respect to the selection of specific B cells were investigated, leading to an optimized protocol for the isolation of a limited subset of B cells. The selected cells retained their property to produce immunoglobulins and could be clonally expanded in the presence of human T cell supernatant and irradiated murine thymoma helper cells to generate sufficient cells for a mini-electrofusion with NS-1 myeloma cells. Up to 78 specific hybridomas could be generated from one anti-HIV positive B cell. An overall efficiency of specific B cell immortalization of up to 10% was obtained.

Animals↗

PubMed1994

Efficient transfer of antibodies into mammalian cells by electroporation.

An optimized protocol for electroporation is described which is suitable for introducing antibodies into mammalian cells. The method results in the uptake of detectable amounts of antibodies in 80% of the cells and in 40% large amounts are introduced. As an example, cell cycle activity (transition from the G1 to S phase) was inhibited by the introduction of monoclonal antibodies against G1-specific cyclin D1 into CV-1 and MCF7 cells. This specific antibody mediated inhibition of cellular function did not affect the viability of the cells since they recovered from the inhibition after some time. While approaching the efficiency of microinjection, the new protocol for electroporation of antibodies additionally permits treatment of the larger number of cells which are required for biochemical analyses.

Animals↗

PubMed1994

Effects of prostaglandin E2 on the micronucleus formation in mouse bone marrow cells by various mutagens.

The effects of prostaglandin E2 (PGE2), as a trigger of erythroid progenitor cells into the cell cycle, were studied on the induction of micronuclei by various mutagens; with mitomicin C (MMC) the optimal protocol was established. PGE2 itself did not induce any micronuclei in this experiment. The highest frequency of micronuclei and dose-response relationship between PGE2 doses and micronucleus frequency were observed 30 h after injection of MMC to mice administered PGE2 24 h previously. Sensitization by PGE2 pretreatment was also found for other mutagens, such as vincristine, 5-fluorouracil, benzo[a]pyrene, 1,1-dimethylhydrazine and 2-naphthylamine. These results support the hypothesis that accelerating the erythropoiesis increases the frequency of micronuclei induced by mutagens.

2-Naphthylamine↗

PubMed1989

Comprehensive polypeptide analysis of microdissected rat brain areas: combining 2-dimensional gel electrophoresis with 2-dimensional HPLC and immunoanalysis and sequencing procedures.

An improved methodology has been developed which allows resolution, identification, and quantitation of hundreds of proteins and neuropeptides from a single rat brain nucleus (5 mg wet wt.). After metabolic labelling, proteins (greater than about 15 kDa) are separated from peptides (less than about 10 kDa) by sonicating the tissue in an acidic peptide extraction buffer; after centrifugation, proteins are in the pellet, peptides in the supernatant. To quantitate peptide synthesis, peptides are resolved to purity by reverse-phase HPLC followed by ion exchange HPLC. Proteins are resolved with a two-dimensional (2-D) gel protocol optimized for neural tissue. To identify specific proteins by immunoanalysis, proteins are transferred to polyvinyl difluoride (Immobilon) and immunostained in the presence of Tween blocking buffer. After visualization with an avidin-biotin alkaline phosphatase procedure, the blot is post-stained with India ink to visualize the protein pattern context. To sequence spots, proteins are transferred to Immobilon, stained with Coomassie, and directly subjected to automated gas phase sequencing. The immunoblot procedure can detect less than 0.1 pmol protein, and the sequencing procedure can detect less than 10 pmol protein. After transfer enough material remains on the gels to allow subsequent autoradiography or silver stain. Quantitative analysis of 2-D gels is examined in a companion paper. These procedures should enhance the utility of 2-D gels in neurochemical studies.

Amino Acid Sequence↗

PubMed1991

Polymerase chain reaction (PCR) amplification for the detection of porcine parvovirus.

A polymerase chain reaction (PCR) amplification method was developed and evaluated to detect porcine parvovirus (PPV). A pair of 20-base primers and an oligonucleotide probe were derived from the DNA sequences common to two isolates of PPV, NADL-8 and NADL-2. The primers flanked 118-bp nucleotides within the region coding for the major structural protein VP2. After DNA amplification of PPV replicative form (RF), a 158-bp fragment was detected in agarose gels. This amplified fragment was shown to be specific for PPV DNA after Southern transfer and hybridization to a 20-base internal probe. The amplified fragment also contained a single EcoRI cleavage site. Various conditions, such as number of cycles and annealing temperature, were examined to optimize the conditions for detecting viral DNAs from infected cell cultures and swine fetal tissues. Four different isolates of PPV, NADL-8, NADL-2, KBSH and Kresse, and two other viruses, canine parvovirus (CPV) and pseudorabies virus (PRV), were included to determine specificity of amplification. Slot blot hybridization with a radiolabeled probe was used to evaluate the sensitivity of PCR amplification. The optimized protocol was specific for PPV detecting equally all four strains of PPV, but failing to amplify CPV or PRV sequences. The PCR method could detect at least 100 fg of viral replicative form (RF) DNA or the equivalent of 1 PFU of infectious virus. The applications of this method include routine detection of PPV in clinical samples and as a contaminant in mammalian cell lines.

Animals↗

PubMed1979

Time in chemo-immunotherapy models: an absolute or a relative variable? Lessons from the levamisole experience.

An analysis is presented of the findings from the available studies in several cancer models, where 2.5-20 mg/kg levamisole was given in combination with different types of cytostatic drugs. It appears that in chemo-immunotherapy experiments it may be preferable to measure time variables (increase in life span, timing of chemotherapy and of immunotherapy) as a per cent of the median survival time of the untreated controls instead of using time as an absolute measure (in days). As regards the study of levamisole in chemo-imnmunotherapy models, the increase in life span by the cytostatic alone seems to be the most important variable studied here. An "optimal" protocol for the evaluation of levamisole is proposed based upon all the findings of this analysis.

Animals↗

PubMed1992

Impaired response to hepatitis B vaccine in HIV infected children.

Eighteen human immunodeficiency virus (HIV) vertically infected children (HIV group) and 33 seroreverted children (SR group), who had completed hepatitis B vaccination (Engerix B, 20 micrograms dose) were studied. Four out of 18 (22%) HIV children failed to develop protective antibody levels (anti-HBs titres less than 10 mIU ml-1) compared with 1 out of 33 (3%) SR children (p less than 0.05). Magnitude of response among HIV children was significantly lower than among SR children. In HIV children the probability that anti-HBs titres persist above the protective levels was significantly lower than in the SR group at any time during the 24 month follow-up. These results show that HIV children have a suboptimal response to hepatitis B immunization and the protection is less durable. Further studies are needed to determine the optimal protocol for hepatitis B immunization in HIV children.

Child, Preschool↗

PubMed1983

Effects of strain, sex, route of administration and partial hepatectomy on the induction by chemical carcinogens of gamma-glutamyltranspeptidase foci in rat liver.

The incidence of gamma-glutamyltranspeptidase (GGT)-positive foci induced by 0.3 mmol/kg diethylnitrosamine (DENA) followed by promotion with 500 ppm sodium phenobarbital in drinking water and was the same in Fischer 344, Sprague-Dawley and Wistar-Lewis rats. There was no difference in the level of GGT-foci initiated by DENA, 7,12-dimethylbenz[a]-anthracene (DMBA), or 1,2-dimethylhydrazine (DMH) followed by promotion with phenobarbital with respect to sex or route of administration including gavage and intraperitoneal injection. Maximal stimulation by partial hepatectomy of DENA initiation of GGT-foci occurred when the DENA was administered 18 h after the operation. Our results indicate that the optimal protocol for the rat liver foci assay consists of using partial hepatectomized rats of 1 of the 3 strains and of either sex. The test substance should be administered by either gavage or intraperitoneal injection so that maximal DNA binding coincides with the maximal rate of DNA replication resulting from partial hepatectomy.

Animals↗

PubMed1991

Hybridization methods for DNA sequencing.

I have conducted a general analysis of the practicability of using oligonucleotide hybridization to sequence DNA. Any DNA sequence may be sequenced by hybridization with a complete panel of oligonucleotides. However, sequencing DNA segments over 2 kb long requires an unrealistic number of hybridization reactions. The optimal protocol is to hybridize 7-mer or 8-mer mixed oligonucleotide probes to immobilized DNA fragments 80 bp long: should this prove impractical, hybridization of labeled 270-bp fragments to immobilized mixed 10-mers is a potential alternative. Both protocols require no more experiments to sequence large regions of DNA than conventional m13-based sequencing and are much easier to automate, thus reducing the requirements for skilled personnel. In the ideal case, hybridization sequencing reduces the number of experiments required to sequence megabase DNA by 90%.

Base Sequence↗

PubMed2026

Non-tobacco nicotine dependence and postoperative complications after total ankle arthroplasty: A propensity-matched cohort study.

BACKGROUND: The clinical impact of non-tobacco nicotine dependence (NTND) is poorly defined. This study evaluated the association between NTND and complications following total ankle arthroplasty (TAA). METHODS: A retrospective cohort study using the TriNetX Research Network was performed. Adults undergoing primary TAA (Current Procedural Terminology [CPT] 27702) between 2010 and 2025 were included. Patients were categorized into NTND (International Classification of Diseases, Tenth Revision [ICD-10]: F17, excluding tobacco-specific codes) and nonsmoker cohorts. Propensity score matching (1:1) yielded 939 NTND patients and 939 controls. Ninety-day medical and wound complications and ≥ 2-year mechanical outcomes were assessed. RESULTS: NTND patients had higher rates of 90-day readmission (11.7% vs 6.5%; OR 1.9), wound disruption (4.3% vs 2.6%; OR 1.7), surgical site infection (3.1% vs 1.6%; OR 2.0), and sepsis (2.4% vs 1.1%; OR 2.3). At a minimum 2-year follow-up, NTND was not associated with increased risk of mechanical complications. CONCLUSION: These findings challenge the assumption that smokeless nicotine products are benign in the perioperative setting and support incorporating NTND screening and cessation counseling into preoperative optimization protocols. Future prospective studies are warranted to further characterize the dose-dependent effects of non-tobacco nicotine exposure and to evaluate the impact of perioperative cessation strategies on outcomes following TAA. LEVEL OF EVIDENCE: IV.

Humans↗

PubMed2026

Clinical applications of digital twin technology in In Vitro Fertilisation.

BACKGROUND: Digital twin technology, originating from aerospace and manufacturing industries, has emerged as a transformative tool in healthcare. In vitro fertilisation (IVF) faces persistent challenges including suboptimal embryo selection, unpredictable treatment outcomes, and limited personalisation of protocols. Despite advances in assisted reproductive technology, existing literature exhibits fragmentation: artificial intelligence applications in embryo selection, ovarian stimulation, and endometrial assessment have been developed independently without systematic integration into comprehensive treatment frameworks. Digital twin technology offers unprecedented opportunities to create virtual replicas of biological systems, enabling real-time monitoring, predictive modelling, and personalised treatment strategies. AIM: This narrative review aims to critically examine the current applications of digital twin technology in IVF, evaluate its potential benefits and limitations, synthesize existing evidence into an integrative conceptual model, and identify future directions for implementation in reproductive medicine. METHOD: A comprehensive narrative review was conducted using PubMed, Scopus, Web of Science, and IEEE Xplore databases. A narrative review approach was selected over systematic review to accommodate the heterogeneity of evidence types in this emerging field, including theoretical frameworks, simulation studies, and proof-of-concept implementations that would be excluded from systematic reviews. Search terms included "digital twin," "IVF," "in vitro fertilisation," "assisted reproductive technology," "embryo selection," and "predictive modelling." Studies published between 2015 and 2025 were included, focusing on original research articles, systematic reviews, and proof-of-concept studies describing digital twin applications in reproductive medicine. RESULTS: Digital twin technology in IVF demonstrates significant potential across multiple domains including embryo development simulation, ovarian response prediction, endometrial receptivity modelling, and personalised stimulation protocols. Current applications integrate artificial intelligence, machine learning algorithms, time-lapse imaging, and omics data to create comprehensive virtual models. Early evidence suggests improvements in embryo selection accuracy, ovarian response prediction, and treatment protocol optimization, though large-scale randomized controlled trials remain limited. Implementation challenges include data integration complexity, computational requirements, regulatory considerations, and validation requirements. CONCLUSION: Digital twin technology represents a paradigm shift in IVF practice, offering personalised, predictive, and precision medicine approaches. This review synthesizes existing evidence to propose an integrative conceptual model for digital twin implementation across the IVF treatment spectrum, identifies critical knowledge gaps, and establishes research priorities to advance clinical translation. Despite current limitations, continued advancement promises improved success rates and patient outcomes.

Humans↗

PubMed2026

Effect of adding umbilical cord blood derived stem cells to haploidentical stem cell transplant (haplo-cord) on post-transplant survival and graft-versus-host disease in patients with hematological malignancies: A systematic review and meta-analysis.

BACKGROUND AND OBJECTIVES: Haploidentical stem cell transplantation (haplo-SCT) carries a substantial risk of graft-versus-host disease (GvHD), whereas umbilical cord blood (UCB) transplantation offers lower GvHD risk but slower engraftment. The haplo-cord approach combines both graft sources, aiming to mitigate GvHD while ensuring timely engraftment. This meta-analysis compares haplo-cord transplantation with haplo-SCT alone for the treatment of hematological malignancies. METHODS: Four electronic databases and two clinical trial registries were systematically searched. Effect sizes from eligible studies were pooled using odds ratios (ORs) for dichotomous outcomes and hazard ratios (HRs) for time-to-event outcomes. RESULTS: Twelve studies met the inclusion criteria. Haplo-cord was associated with a statistically significant reduction in chronic GvHD (OR = 0.62, 95%-CI: 0.42-0.93), while no significant difference was observed for grade II-IV acute GvHD (OR = 0.75, 95%-CI: 0.52-1.09). Survival outcomes favored haplo-cord, with lower HRs for overall survival (HR = 0.68, 95%-CI: 0.53-0.86) and event-free survival (HR = 0.61, 95%-CI: 0.52-0.72), while non-relapse mortality was not significant. Relapse at 3 years was significantly lower with haplo-cord (OR = 0.54, 95%-CI: 0.35-0.82). Haplo-cord also demonstrated higher day-30 engraftment, along with lower relapse-related and GvHD-related mortality, while CMV and EBV viremia showed no difference between groups. CD34 selection in the haplo graft significantly influenced effect sizes and heterogeneity in both subgroup analyses and meta-regression for acute GvHD. CONCLUSION: Haplo-cord transplantation improves GvHD outcomes, survival, and relapse risk compared with haplo-SCT alone. However, whether protocol optimization, possibly via CD34 selection, confers additional benefit remains uncertain and requires confirmation in future studies.

Humans↗