Two rapid assays for screening of patulin biodegradation

Artículo sobre distintos ensayos para comprobar la biodegradación de la patulinaThe mycotoxin patulin is produced by the blue mould pathogen Penicillium expansum in rotting apples during postharvest storage. Patulin is toxic to a wide range of organisms, including humans, animals, fungi and bacteria. Wash water from apple packing and processing houses often harbours patulin and fungal spores, which can contaminate the environment. Ubiquitous epiphytic yeasts, such as Rhodosporidium kratochvilovae strain LS11 which is a biocontrol agent of P. expansum in apples, have the capacity to resist the toxicity of patulin and to biodegrade it. Two non-toxic products are formed. One is desoxypatulinic acid. The aim of the work was to develop rapid, high-throughput bioassays for monitoring patulin degradation in multiple samples. Escherichia coli was highly sensitive to patulin, but insensitive to desoxypatulinic acid. This was utilized to develop a detection test for patulin, replacing time-consuming thin layer chromatography or high-performance liquid chromatography. Two assays for patulin degradation were developed, one in liquid medium and the other in semi-solid medium. Both assays allow the contemporary screening of a large number of samples. The liquid medium assay utilizes 96-well microtiter plates and was optimized for using a minimum of patulin. The semisolid medium assay has the added advantage of slowing down the biodegradation, which allows the study and isolation of transient degradation products. The two assays are complementary and have several areas of utilization, from screening a bank of microorganisms for biodegradation ability to the study of biodegradation pathways

Rapid and label-free identification of single leukemia cells from blood in a high-density microfluidic trapping array by fluorescence lifetime imaging microscopy.

The rapid screening and isolation of single leukemia cells from blood has become critical for early leukemia detection and tumor heterogeneity interrogation. However, due to the size overlap between leukemia cells and the more abundant white blood cells (WBCs), the isolation and identification of leukemia cells individually from peripheral blood is extremely challenging and often requires immunolabeling or cytogenetic assays. Here we present a rapid and label-free single leukemia cell identification platform that combines: (1) high-throughput size-based separation of hemocytes via a single-cell trapping array, and (2) leukemia cell identification through phasor approach and fluorescence lifetime imaging microscopy (phasor-FLIM), to quantify changes between free/bound nicotinamide adenine dinucleotide (NADH) as an indirect measurement of metabolic alteration in living cells. The microfluidic trapping array designed with 1600 highly-packed addressable single-cell traps can simultaneously filter out red blood cells (RBCs) and trap WBCs/leukemia cells, and is compatible with low-magnification imaging and fast-speed fluorescence screening. The trapped single leukemia cells, e.g., THP-1, Jurkat and K562 cells, are distinguished from WBCs in the phasor-FLIM lifetime map, as they exhibit significant shift towards shorter fluorescence lifetime and a higher ratio of free/bound NADH compared to WBCs, because of their glycolysis-dominant metabolism for rapid proliferation. Based on a multiparametric scheme comparing the eight parameter-spectra of the phasor-FLIM signatures, spiked leukemia cells are quantitatively distinguished from normal WBCs with an area-under-the-curve (AUC) value of 1.00. Different leukemia cell lines are also quantitatively distinguished from each other with AUC values higher than 0.95, demonstrating high sensitivity and specificity for single cell analysis. The presented platform is the first to enable high-density size-based single-cell trapping simultaneously with RBC filtering and rapid label-free individual-leukemia-cell screening through non-invasive metabolic imaging. Compared to conventional biomolecular diagnostics techniques, phasor-FLIM based single-cell screening is label-free, cell-friendly, robust, and has the potential to screen blood in clinical volumes through parallelization

Automated mass spectrometer/analysis system: A concept

System performs rapid multiple analyses of entire compound classes or individual compounds on small amounts of sample and reagent. Method will allow screening of large populations for metabolic disorders and establishment of effective-but-safe levels of therapeutic drugs in body fluids and tissues

Establishing a rapid and effective method for screening salt tolerance in soybean

Chlorine (Cl) toxicity has been recognized as a constraint for soybean production. Although the use of a Cl-tolerant crop easily solves the problem, current screening methodologies for Cl tolerance are often ineffective because of inadequate means of detecting and measuring plant response to salinity. In order to facilitate the evaluation process and selection of Cl-tolerant genotypes, a study was conducted to develop a rapid and effective method for screening Cl tolerance in soybean. Seeds of five soybean cultivars, each representing either the includer or excluder genotype to salt stress, were grown in a greenhouse in two different growing media (potting mix or sandy loam) with four different concentrations of sodium chloride (NaCl) solutions. Visual symptoms of Cl toxicity were rated on a 1 to 6 scale (1 as healthy and 6 as dead), and the score was compared with relative shoot/ root dry weight and Cl concentration in shoot/root to corroborate the accuracy of the visual ratings. Reduced dry weight was associated with higher Cl concentrations in both root and shoot tissues. The optimal NaCl concentration for screening was determined as 120 mM NaCl since it effectively differentiated excluders from includers. There were negative, significant correlations between relative shoot dry weight and Cl concentration in shoot tissue (r = -0.91 p = 0.05), and Cl concentration in shoot was also significantly correlated with visual rating score (r = 0.79 p = 0.05). The presented methodology is simple, rapid, and effective for screening for salt tolerance in soybean

Improved design of electrophoretic equipment for rapid sickle-cell-anemia screening

Effective mass screening may be accomplished by modifying existing electrophoretic equipment in conjunction with multisample applicator used with cellulose-acetate-matrix test paper. Using this method, approximately 20 to 25 samples can undergo electrophoresis in 5 to 6 minutes

Molecular analysis of PKU-associated PAH mutations: a fast and simple genotyping test

Abstract: Neonatal screening for phenylketonuria (PKU, OMIM: 261600) was introduced at the end of the 1960s. We developed a rapid and simple molecular test for the most frequent phenylalanine hydroxylase (PAH, Gene ID: 5053) mutations. Using this method to detect the 18 most frequent mutations, it is possible to achieve a 75% detection rate in Italian population. The variants selected also reach a high detection rate in other populations, for example, 70% in southern Germany, 68% in western Germany, 76% in Denmark, 68% in Sweden, 63% in Poland, and 60% in Bulgaria. We successfully applied this confirmation test in neonatal screening for hyperphenylalaninemias using dried blood spots and obtained the genotype in approximately 48 h. The method was found to be suitable as second tier test in neonatal screening for hyperphenylalaninemias in neonates with a positive screening test. This test can also be useful for carrier screening because it can bypass the entire coding sequence and intron–exon boundaries sequencing, thereby overcoming the questions that this approach implies, such as new variant interpretations