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Rapid screening for plasmid DNA.

A procedure is described for demonstrating plasmid DNA and its molecular weight, based on rate zonal centrifugation of unlabelled DNA in neutral sucrose gradients containing a low concentration of ethidium bromide. Each DNA species is then visualized as a discrete fluorescent band when the centrifuge tube is illuminated with ultra-violet light. Plasmids exist as closed circular and as relaxed circular molecules, which sediment separately, but during preparation of lysates, closed circular molecules are nicked so that each plasmid forms only a single band of relaxed circles within the gradient.

Centrifugation, Density Gradient

A rapid screening method for the detection of monospecific antibodies against hemoglobins.

A simple procedure for the detection of monospecific antibody against C57BL/6 mouse hemoglobin that would not cross-react with DBA/2 mouse hemoglobin is described. The horse antiserum against C57BL/6 mouse hemoglobin is absorbed with DBA/2 mouse hemoglobin. The absorbed serum is then allowed to react with small amounts of DBA/2 hemoglobin-Sepharose and C57BL/6 hemoglobin-Sepharose in separate tubes, followed by reaction with fluorescein isothiocyanate-conjugated goat antihorse IgG. A strong fluorescence in the C57BL/6 immunoabsorbent and little or no fluorescence in the DBA/2 immunoabsorbent show the presence of monospecific antibody against C57BL/6 hemoglobin. The method has general applicability as antibodies against other hemoglobins and proteins can be detected.

Antibodies

Isolated heart perfusion techniques for rapid screening of myocardial preservation methods. Anoxia versus ischemia.

Isolated nonworking and working heart preparations are described and recent modifications that increase their reliability and scope are reviewed. Isolated perfused tissues are superior to other models for screening myocardial preservation techniques. The metabolic and functional differences between anoxia and ischemia are stressed, myocardial glycolysis is reviewed, and from basic studies potentially fruitful avenues for investigation of myocardial preservation techniques are outlined. Better application of available knowledge of myocardial metabolism could further reduce the risks of cardiac operation.

Animals

A rapid screening test for Cushing's syndrome.

A combination test consisting of two parts, a single dose of dexamethasone and a (1-24) ACTH injection, has been used for the diagnosis of Cushing's syndrome. The test was performed by administering 1 mg dexamethasone orally at 11 p.m. on the first day and 25 IU synthetic ACTH i.v. at 8 a.m. on the second day. Plasma cortisol was determined at 8 a.m. on the first and second day and 1-1/2 and 2 hours after the ACTH injection. The test was performed in 33 patients with Cushing's syndrome and in 114 controls without adrenal disease. After dexamethasone administration alone, cortisol values frequently overlapped between patients with Cushing's syndrome and controls. The validity of the test was considerably improved by adding ACTH stimulation. The test procedure is recommended as a screening test for Cushing's syndrome in ambulatory practice.

Adrenocorticotropic Hormone

Rapid screening and identification of genes involved in bacterial extracellular membrane vesicle production using a curvature-sensing peptide.

Bacteria secrete extracellular membrane vesicles (EMVs). Physiological functions and biotechnological applications of these lipid nanoparticles have been attracting significant attention. However, the details of the molecular basis of EMV biogenesis have not yet been fully elucidated. In our previous work, an N-terminus-substituted FAAV peptide labeled with nitrobenzoxadiazole (NBD; nFAAV5-NBD) was developed. This peptide can sense the curvature of a lipid bilayer and selectively bind to EMVs even in the presence of cells. Here, we applied nFAAV5-NBD to a genome-wide screening of hyper- and hypo-vesiculation transposon mutants of a Gram-negative bacterium, Shewanella vesiculosa HM13, to identify the genes involved in EMV production. We analyzed the transposon insertion sites in hyper- and hypo-vesiculation mutants and identified 16 and six genes, respectively, with a transposon inserted within or near them. Targeted gene-disrupted mutants of the identified genes showed that the lack of putative dipeptidyl carboxypeptidase, glutamate synthase β-subunit, LapG protease, metallohydrolase, RNA polymerase sigma-54 factor, inactive transglutaminase, PepSY domain-containing protein, and Rhs-family protein caused EMV overproduction. On the other hand, disruption of the genes encoding putative phosphoenolpyruvate synthase, d-hexose-6-phosphate epimerase, NAD-specific glutamate dehydrogenase, and sensory box histidine kinase/response regulator decreased EMV production. This study demonstrates the utility of a novel screening method using a curvature-sensing peptide for mutants with altered EMV productivity and provides information on the genes related to EMV production.IMPORTANCEConventional methods for isolation and quantification of extracellular membrane vesicles (EMVs) are generally time-consuming. nFAAV5-NBD can detect EMVs in the culture without separating EMVs from cells. In situ detection of EMVs using this peptide facilitated screening of the genes related to EMV production. We succeeded in identifying various genes associated with EMV production of Shewanella vesiculosa HM13, which would contribute to the elucidation of bacterial EMV formation mechanisms. Additionally, the hyper-vesiculating mutants obtained in this study would be valuable for EMV applications, such as secreting useful substances as EMV cargoes and producing artificially functionalized EMVs.

Shewanella

Rapid screening for significant bacteriuria using a Coulter Counter.

A Coulter Counter was evaluated for detecting significant bacteriuria. Simple urinary particle counts showed agreement in 883 of the 956 (92.4%) urine specimens examined compared to a standard culture procedure. They also provided a semiquantitative estimate of bacterial numbers in specimens diagnosed as infected by culture. Sample preparation before counting was minimal, and results were obtained within two to four minutes.

Bacteriological Techniques