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At least 19 recordsLinked to original sources
PubMed2025

Dual RNA isolation from blood: an optimized protocol for host and bacterial RNA purification for dual RNA-sequencing analysis in whole blood sepsis samples.

Dual RNA-sequencing (dual RNA-seq) holds significant promise for deciphering bacterial virulence mechanisms during systemic infections. However, its application in sepsis research is hindered by technical challenges, including a low bacterial burden in blood and limited sample volumes and RNA yield from vulnerable populations, such as neonates. We developed an optimized protocol [dual RNA isolation from blood (DRIB)] for simultaneous stabilization, isolation and purification of high-quality host leukocyte and bacterial RNA from low-volume whole blood samples (0.5 ml). This protocol is compatible with clinical sample collection workflows and high-throughput RNA sequencing. The feasibility of DRIB for dual RNA-seq was validated using a pilot cohort of clinical adult sepsis samples, enabling the investigation of host-bacterial gene expression during sepsis. The DRIB protocol yielded 2.10-6.91 µg of total RNA per clinical sample in our pilot cohort. Dual-species ribosomal RNA (rRNA) depletion and RNA-seq generated 16.6-24.8 million filtered reads per sample, with 63±7% of reads uniquely mapped to host or bacterial sequences. Host genes accounted for 51-68% (8.4-10.9 million) reads, while 0.5-6.7% (79,496-789,808 reads) mapped to bacterial genomes. Bioinformatic analysis revealed that both shared and individual transcriptional patterns were identified in host and bacterial responses, including pathways related to immune metabolism and metal-ion binding. Our optimized DRIB protocol and RNA-seq pipeline effectively captured both host and bacterial RNA transcription in clinical sepsis samples. Expanding this approach to larger cohorts and varying disease timepoints will provide crucial new insights into host-bacterial gene co-expression dynamics in sepsis progression and outcomes.

Humans↗
PubMed2026

Trans-Mitochondrial Cybrid Generation from mtDNA Patient Platelets: An Efficient Protocol Optimizing Colony Selection and Functional Validation.

Trans-mitochondrial cybrid cell line generation represents the gold-standard method for determining pathogenicity by enabling biochemical analyses of a specific mitochondrial DNA (mtDNA) variant of interest at high and low percentages (heteroplasmy levels) within an otherwise identical mtDNA and nuclear genome background. Historically, the cybrid generation process has been tedious and poorly efficient. Here, we describe a highly efficient and effective protocol for generating trans-mitochondrial cybrid cell lines by fusing human platelets with a standard osteosarcoma 143B cell line to provide an isogenic nuclear background depleted of mtDNA (Rho0 cells). Cell isolates capture a given mtDNA genome of interest to establish stable cell lines harboring different degrees of heteroplasmy, or to compare divergent effects of distinct mitochondrial haplogroups. Because cybrids from mitochondrial patients may be more difficult to establish with standard protocols, this current methodology focuses on isolating mtDNA variants where the electron transport chain activity is affected. We here demonstrate that colony selection techniques reduce time and improve the yield of generating high-level heteroplasmy mtDNA mutant cybrid lines. A case study is provided of cybrid generation for a variant of unknown significance in MT-ND1, m.3985G>A (p.E227K). We analyze the efficiency of the cybrid generation process using this protocol and run functional studies performed by high-resolution respirometry. High-level heteroplasmy MT-ND1 m.3985G>A cybrid mutants generated by this protocol are shown to have impaired complex I-dependent mitochondrial respiration relative to wild-type control, demonstrating m.3985G>A is likely pathogenic.

Humans↗
PubMed1989

The effect on fertilization of exposure of mouse oocytes to dimethyl sulfoxide: an optimal protocol.

Mouse oocytes, with or without an intact cumulus mass, were exposed to various concentrations of dimethyl sulfoxide (DMSO) at different temperatures for different periods of time and using different protocols of DMSO addition and removal. The effect of these procedures on the chymotrypsin sensitivity of the zona pellucida and the fertilizability of the oocytes was then assessed. Some procedures were found to affect adversely both the zona pellucida and the cumulus mass, resulting in reductions in the fertilization rate. As a result of both these and previously reported experiments (1-3), an optimal schedule is proposed for the handling of mouse oocytes during cryopreservation, namely, to equilibrate cumulus-intact oocytes in 1.5 M DMSO precooled to 4 degrees C prior to freezing, to remove DMSO at 4 degrees C after thawing prior to restoring the oocytes to 37 degrees C, to loosen or remove the cumulus cells, and then to hold oocytes at 37 degrees C for at least 1 hr to allow recovery of the spindle prior to insemination.

Animals↗
PubMed1992

Optimal protocol and trajectory visualization for conformational searches of peptides and proteins.

Conformational searches by molecular dynamics and different types of Monte Carlo or build-up methods usually aim to find the lowest-energy conformation. However, this is often misleading, as the energy functions used in conformational calculations are imprecise. For instance, though positions of local minima defined by the repulsive part of the Lennard-Jones potential are usually altered only slightly by functional modification, the relative depths of the minima could change significantly. Thus, the purpose of conformational searches and, correspondingly, performance criteria should be reformulated and appropriate methods found to extract different local minima from the search trajectory and allow visualization in the search space. Attempts at convergence to the lowest-energy structure should be replaced with efforts to visit a maximum number of different local energy minima with energies within a certain range. We use this quantitative criterion consistently to evaluate performances of different search procedures. To utilize information generated in the course of simulation, a "stack" of low energy conformations is created and stored. It keeps track of variables and visit numbers for the best representatives of different conformational families. To visualize the search, projection of multidimensional walks onto a principal plane defined by a set of reference structures is used. With Met-enkephalin as a structural example and a Monte Carlo procedure combined with energy minimization (MCM) as a basic search method, we analyzed the influence on search efficiency of different characteristics as temperature schedules, the step size for variable modification, constrained random step and response mechanisms to search difficulties. Simulated annealing MCM had comparable efficiency with MCM at constant and elevated temperature (about 600 K). Constraining the randomized choice of side-chain chi angles to optimal values (rotamers) on every MCM step did not improve, but rather worsened, the search efficiency. Two low-energy Met-enkephalin conformations with parallel Tyr1 and Phe4 rings, a gamma-turn around the Gly2 residue, and Phe4 and Met5 side-chains forming together a compact hydrophobic cluster were found and are suggested as possible structural candidates for interaction with a receptor or a membrane.

Amino Acid Sequence↗
PubMed1988

Surveillance for stage I non-seminomatous germ cell tumours of the testis: the optimal protocol has not yet been defined.

Forty-six patients with clinical stage I testicular non-seminomatous germ cell tumours were followed up according to a protocol of active surveillance between 1979 and 1987. The median follow-up time was 40+ months. Thirteen patients (28%) relapsed, predominantly in retroperitoneum and/or lung. Ten of these relapses (76%) occurred within 8 months of orchiectomy. Relapses occurred in 7/35 T1 tumours and 5/10 T2 to T4 tumours. No correlation was detected between the histological type and relapse rate. Three late relapses were diagnosed at 23, 29 and 36 months. Eleven of the relapsed patients remain in prolonged complete remission after PVB chemotherapy +/- surgery; one patient, who initially refused treatment at the time of relapse, has died. Another relapsed with predominant elements of rhabdomyosarcoma intermingled with malignant teratoma in a bone metastasis. He had a partial response to PVB chemotherapy but subsequently died. Thirty-four patients (74%) did not undergo lymphography (LG) and had a higher relapse rate (11/34) than those who had LG (2/12); this was not a statistically significant difference in this small series. The policy of active surveillance is not yet the "state of the art" and should be under constant scrutiny with respect to safety and practice.

Adult↗
PubMed1991

Genetic transformation of various species of Enterococcus by electroporation.

A transformation system for Enterococcus faecalis was developed which uses untreated (i.e., non-protoplasted) cells and the electroporation technique. The optimized protocol resulted in transformation efficiencies of up to 4 x 10(6) transformants per microgram of plasmid DNA. All strains of E. faecalis tested could be transformed by this method, albeit with differing transformation efficiencies. Using the protocol optimized for E. faecalis we successfully transformed Enterococcus faecium, E. hirae, E. malodoratus and E. mundtii.

Cell Division↗
PubMed1991

Factors influencing transient expression in cytotoxic T cells following DEAE dextran-mediated gene transfer.

A number of transfection protocols have been tested for the introduction of exogenous DNA into cytotoxic T cells. These included electroporation, lipofection, calcium phosphate coprecipitation, polybrene-assisted gene transfer, and DEAE dextran-mediated transfer. Only the latter gave significant and reproducible transfection efficiencies coupled with low toxicity. The DEAE dextran protocol was optimized for the transfection of a transcription reporter construct pRSVcat into a cloned cytotoxic cell line. Among the parameters investigated were cell density, amount of input DNA, concentration of DEAE dextran, DNA adsorption time, temperature, use of permeabilization and expression facilitators, and recovery time. The optimized protocol was then used to demonstrate the presence of cis-acting regulatory regions in the 5'-flanking sequences of two cytotoxic cell-specific serine protease genes and, in addition, was shown to be applicable to other cloned T-cell lines.

Adsorption↗
PubMed1991

Asthma in the emergency department: impact of a protocol on optimizing therapy.

The objective of this study was to evaluate the impact of a simple educational intervention on the prescribing habits of internal medicine residents in the treatment of acute asthma in a busy emergency department (ED). Prescribing habits for 16 residents were documented for 4 months. The first 2 months served as a control period during which eight residents managed asthma patients without the benefit of any specific educational intervention beyond standard department protocols. A total of 129 patients treated by the residents during this initial phase were assessed. During the second 2-month period, a 10-minute verbal presentation and explicit written treatment protocol were provided to another eight residents, and their treatment of 83 patients was covertly evaluated. Increased prescribing of desired therapy was significantly improved in every area except that of prescribing an inhaled steroid metered dose inhaler for use as a discharge medication. The 10-minute verbal presentation given in conjunction with a three-page handout was found to be highly effective for eliciting improvement in treatment practices during short clinical rotations. The duration of this effect beyond each rotation is unknown. This educational intervention should be presented by the ED medical director, clinical pharmacist, or other appropriate clinician in virtually any ED as quality of patient care can be dramatically improved.

Acute Disease↗
PubMed2026

Comparative evaluation of three high-molecular-weight DNA extraction kits for Oxford Nanopore sequencing of Clostridioides difficile and Clostridium perfringens.

UNLABELLED: Clostridioides difficile and Clostridium perfringens are Gram-positive, spore-forming anaerobic pathogens affecting humans and animals, for which genomic data have been mainly generated using short-read or hybrid sequencing approaches. In this study, we evaluated three commercial non-bead-beating DNA extraction kits designed for high-molecular-weight DNA recovery for Oxford Nanopore long-read whole-genome sequencing of two C. difficile and two C. perfringens strains, including one reference strain and one clinical or environmental isolate per species. Based on sequencing performance and kit ease of use, one kit was selected for additional sequencing of plasmid-carrying strains of both species. All three kits allowed correct identification of sequence types, toxin-encoding genes, and antimicrobial resistance determinants, confirming their suitability for clinical and epidemiological applications. However, the BT MasterPure Kit provided the highest DNA concentrations, longest fragment sizes, and superior read lengths and N50 values, particularly for C. difficile, achieving >100× coverage and enabling reliable circularization of chromosomes and plasmids, including a C. difficile metronidazole resistance plasmid and C. perfringens plasmids carrying toxin and antibiotic resistance genes. The other kits produced slightly lower DNA yields, resulting in shorter reads and reduced genome coverage for C. difficile, highlighting the challenge of extracting high-quality DNA from Gram-positive, spore-forming bacteria. Overall, this study provides practical guidance for selecting DNA extraction protocols optimized for Oxford Nanopore sequencing of C. difficile and C. perfringens, supporting high-quality genome assemblies and plasmid characterization and facilitating the routine genomic surveillance of clinically relevant spore-forming pathogens. IMPORTANCE: High-quality genomic data are essential for accurate characterization of Clostridioides difficile and Clostridium perfringens, two clinically and epidemiologically important Gram-positive, spore-forming pathogens. However, long-read sequencing performance can be strongly influenced by the choice of DNA extraction method, particularly for organisms with robust cell walls, where commonly used methods can lead to fragmented DNA. In this work, DNA of four strains was extracted using three commercial high-molecular-weight DNA extraction kits and sequenced using Oxford Nanopore Technologies. The best-performing kit was also evaluated using three additional strains known to harbor plasmids in order to assess its plasmid recovery efficiency. The results demonstrated successful plasmid recovery, circularization, and characterization. DNA extraction protocols optimized for Oxford Nanopore sequencing enable the rapid and cost-effective characterization of C. difficile and C. perfringens for genomic surveillance or outbreak investigations.

Clostridioides difficile↗
PubMed1991

Routine programmed electrical stimulation in survivors of acute myocardial infarction for prediction of spontaneous ventricular tachyarrhythmias during follow-up: results, optimal stimulation protocol and cost-effective screening.

Of 3,286 consecutive patients treated for acute myocardial infarction, electrophysiologic testing was performed in 1,209 survivors (37%) free of significant complications at the time of hospital discharge to determine their risk of spontaneous ventricular tachyarrhythmias during follow-up. Sustained monomorphic ventricular tachycardia was inducible by programmed electrical stimulation in 75 (6.2%). Antiarrhythmic therapy was not routinely prescribed regardless of the test results. During the 1st year of follow-up, 14 infarct survivors (19%) with inducible ventricular tachycardia experienced spontaneous ventricular tachycardia or fibrillation in the absence of new ischemia compared with 34 (2.9%) of those without inducible ventricular tachycardia (p less than 0.0005). During the extended follow-up period (median 28 months) of those with inducible ventricular tachycardia, 19 (25%) had a spontaneous electrical event; 37% of these first events were fatal. These results suggest that the most cost-effective strategy for predicting arrhythmia will be obtained by restricting electrophysiologic testing to infarct survivors whose left ventricular ejection fraction is less than 40% and using a stimulation protocol containing four extrastimuli. Electrophysiologic testing is the single best predictor of spontaneous ventricular tachyarrhythmias during follow-up in infarct survivors. The majority (94%) with a negative test benefit from the more reliable reassurance that all is well, whereas the 25% risk of electrical events in those with inducible ventricular tachycardia justifies a prospective trial of effective prophylactic antiarrhythmic interventions.

Anti-Arrhythmia Agents↗
PubMed1990

The suppressive effect of an antibody to the alpha beta cell receptor in rat adjuvant arthritis: studies on optimal treatment protocols.

An antibody (R73) to the alpha beta T cell receptor (TCR) was able to dramatically suppress adjuvant arthritis (AA) in rats. The efficacy of R73 treatment was investigated with regard to antibody dosage, injection route and timing of injections. R73 was equally effective in reducing joint swelling throughout a wide dose range (80 to 2000 microgram/dose) when given on day 15, 18, and 21 after arthritis induction. Both i.p. and i.v. injection were able to suppress the pre-existing joint swelling to the same extent. However, R73 was only effective when given before or at the peak of joint swelling which occurred between day 18 to 24. Synovial membrane hyperplasia, mononuclear cell infiltration as well as cartilage and bone destruction were markedly reduced after therapy. The effect of R73 was associated with depletion of alpha beta+ T cells from the circulation even at low antibody doses. Only few alpha beta+ T cells were found in the pannus tissue. Late treatment on day 27, 30 and 33 after arthritis induction did not influence clinical scoring of the disease and histological examination thereafter did not show any difference between treated animals and controls. We conclude that antibody therapy directed at the TCR seems to be very effective even in pre-existing autoimmune diseases if the relevant T cell population is affected. However, inflammation and joint destruction may reach a state at which anti-TCR treatment is no longer effective.

Animals↗
PubMed1988

Optimization of MR protocols: a statistical decision analysis approach.

A new method of optimizing MRI data acquisition protocols is presented. Tissues are modeled with probability density functions (PDFs) of tissue parameter values (such as T1, T2). The imaging data acquisition process is modeled as a mapping from a tissue parameter space to a signal strength space. Tissue parameter PDFs are mapped to signal strength PDFs for each tissue in a clinical problem. The efficacy of an MRI protocol is evaluated using the methods of statistical decision analysis applied to the signal strength PDFs, including the propagation of noise. This procedure evaluates the ability to discriminate different tissues based on the signal strengths produced with the protocol. The model can incorporate an arbitrary number of tissues, parameters, and pulse sequences in the protocol. The multivariate nature of MRI and the observed broad distribution of tissue parameter values makes this model more appropriate for optimizing data acquisition protocols than methods which maximize the signal-difference-to-noise ratio between discrete values of the tissue parameters. It is shown that these two methods may calculate different optimal protocols. The method can be used to optimize data acquisition for quantitative computer-based tissue classification, as well as imaging. Data acquisition and image processing philosophies are discussed in light of the method.

Computer Simulation↗
PubMed1991

Is nuclear medicine cost-effective?

Clearly, there is currently no consensus on the cost-effectiveness of nuclear medicine--or in fact any other aspect of medicine. It is hoped that common sense prevails in clinical medicine today. An appropriate case history and physical examination may negate the need for any additional investigation. From the perspective of the capital cost of equipment and supply costs, ultrasound is clearly the most cost-effective diagnostic imaging modality. But while it is useful, it does not always provide definitive answers, and other modalities must be used to arrive at a diagnosis. In comparison, the capital cost of general radiology equipment and nuclear medicine equipment is relatively equal. Radiology has more operating costs per case than nuclear medicine and requires a lower staffing component per given volume of examinations. In any given diagnostic imaging procedure, the practitioner and imagist must maintain a dialogue to ascertain the appropriateness of the study and to use the available resources in the most effective manner. This is even more imperative when CT scanning and MRI are included in the equation. The development of an investigative protocol that makes the most efficient use of the various imaging modalities without compromising the quality of care makes sense for the patient, the physician and the insurance provider. It is unreasonable to expect the physician to be aware of the optimal protocol for the diagnostic workup of every patient. The guidance of the imaging department is required to maximize the efficient use of the available facilities. A critical and exhaustive appraisal of the medical literature may be required to determine the optimal diagnostic protocol.(ABSTRACT TRUNCATED AT 250 WORDS)

Capital Expenditures↗
PubMed2025

ATAC-seq in Emerging Model Organisms: Challenges and Strategies.

The Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) is a versatile and widely utilized method for identifying potential regulatory regions, such as promoters and enhancers, within a genome. ATAC-seq has been successfully applied to a wide range of established and emerging model organisms. However, implementing this method in emerging model systems, such as arthropods, can be challenging due to several factors that influence data quality. These factors include the availability of a sufficient amount and quality of tissue or cells, the need for species- and tissue-specific protocol optimization, the completeness and accuracy of the reference genome, and the quality of the genome annotation. In this article, we emphasize the key steps in the ATAC-seq protocol that, based on our experience, have the greatest impact on data quality when adapting this method for emerging model organisms. Specifically, we discuss the importance of nuclei isolation, the incubation conditions of the Tn5 transposase, and PCR amplification of the library. Furthermore, we outline essential quality checkpoints during the bioinformatic analysis of ATAC-seq data to assist in assessing data integrity and consistency. Given that many emerging model organisms may not be readily available in laboratory cultures, we also emphasize the importance of evaluating how different preservation methods affect ATAC-seq data quality. Based on examples in one spider and one ant species, we demonstrate that replication and thorough quality controls at all steps of the protocol and data analysis are essential to assess the usability of ATAC-seq data. Our data highlights the importance of isolating the right number of intact nuclei, as well as ensuring optimal amplification conditions during library preparation to obtain good-quality sequence data for downstream analyses. We recommend using fresh tissue samples if possible because we show that direct cryopreservation of the tissue may affect chromatin integrity. This effect could be avoided or reduced by preserving the homogenate in cell culture medium. Overall, we explain the ATAC-seq protocol and downstream analyses in detail and give step-by-step advice to researchers who are new to the field and want to implement this method. With careful planning and validation, ATAC-seq can reveal the regulatory landscape of a genome and aid in identifying elements that govern gene expression.

Animals↗
PubMed1987

Critical features of bacterial mutation assays.

This review discusses features of the standard protocols currently used in bacterial mutation assays which can have a critical effect upon the test outcome. Such features if not strictly controlled may affect the results qualitatively as well as quantitatively. These include the dose-intervals of the test compound used; the number of bacterial cells exposed to the test compound; the phase of growth such cells are in during exposure; and the length of time that the cells are exposed prior to the addition of soft agar (in the Salmonella/plate incorporation assay). The following possible modulating effects will also be discussed: the nature of the solvent used; the nature and quantity of the exogenous metabolizing system (usually liver S9-fraction) and the quantity of amino-acid (e.g. histidine) within the test system being that either deliberately added or present unavoidably within the test sample (e.g. biological fluids). If bacterial mutagenicity assays are to realize their full potential for the detection of genotoxic carcinogens, the use of rigid protocols should be discouraged. Where possible, consideration of the compound's structure should lead to the employment of the optimal protocol for the detection of genotoxic carcinogens within that chemical class. The relative speed and low cost of bacterial assays should be exploited in this way to avoid the generation of false negative results during the primary screening of novel compounds.

Animals↗