A simple procedure for the isolation of fungal DNA for dot blot analysis

A rapid method for the detection of transforming sequences in a fungal strain would be advantageous when trying to determine if unselected sequences are present. Colony hybridization protocols for filamentous fungi have been developed (Stohl and Lambowitz, 1983. Anal. Biochem. 134:82-85; Paietta and Marzluf, 1984. Neurospora Newsletter 31:40) and a modified method thereof was described (McClung and Dunlap, 1988. Fungal Genetics Newsletter 35:26-27). In this report a simple method for the isolation of fungal DNA from a single transformed colony suitable for dot blot analysis is described. If the original transformant colony is not too small it is sufficient to extract the DNA of one half of that colony. That means that no subculturing is necessary and the results can be available within 24 h starting from the transformant colony

Characterization of a mutation in a strain of Penicillium camembertii affecting the production of cyclopiazonic acid

Penicillium camembertii is a filamentous fungus used for the production of mold- fermented white cheese. It is a domesticated form of Penicillium commune, especially adapted to the food environment (Pitt et al. 1986. Food Microbiol. 3:363-371). Despite its use as food starter culture, P. camembertii is able to produce cyclopiazonic acid (CA), a secondary metabolite toxic to animals and humans (Holzapfel 1968. Tetrahedron 24:2101- 2119); Le Bars 1979. Appl. Environ. Microbiol. 38:1052-1055). The synthesis of CA starts from the amino acid tryptophan with the condensation of acetyl-CoA and isoprenoids, especially dimethyl allylpyrophosphate (Holzapfel 1980. In: P.S. Steyn, The biosynthesis of mycotoxins, Academic Press). Acetyl-CoA is also the direct precursor of the isoprenoid

Understanding the effect of postharvest tomato temperatures on two toxigenic Alternaria spp. strains: growth, mycotoxins and cell-wall integrity-related gene expression

BackgroundTomato fruits are susceptible to Alternaria spp. spoilage. A correct postharvest management is necessary to prevent mould growth and mycotoxin accumulation, being the temperature one of the main factors. The effect of different postharvest temperatures (5, 12, 25 and 35 °C) on growth, mycotoxin production and a stress-related gene expression by two Alternaria spp. was assessed. ResultsGrowth rates decreased rapidly when temperature was higher than the optimum (25 °C), while a gradual reduction was detected at lower temperatures. Tenuazonic acid (TeA) was strongly synthesised at all temperatures evaluated, with a maximum between 12 and 25 °C. Alternariol monomethyl ether (AME) was produced only at the two lowest temperatures; with a peak at 12 °C. Regarding the expression of the stress-related RHO1 gene, during active fungal growth both Alternaria spp. showed more copies of the gene as temperature increased. At the stationary phase, the RHO1 gene expression was significantly higher at 12 °C, coinciding with AME highest accumulation. ConclusionChanges on temperatures related to different postharvest stages of tomato fruits markedly affect toxigenic Alternaria spp. The highest levels of both mycotoxins were recorded at 12 °C, a common storage temperature for tomato fruit. Additionally, an association between alternariols biosynthesis and the cell wall integrity pathway was noticed in relation to temperature, suggesting that temperature may act as stressor stimulating the RHO1 gene expression, which in turn triggers this mycotoxin synthesis. These results will be useful in developing new strategies to efficiently control Alternaria spoilage in tomato fruit and by-products.Fil: Da Cruz Cabral, Lucía Mariana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Universidad de Extremadura; EspañaFil: Rodríguez, Alicia. Universidad de Extremadura; EspañaFil: Delgado, Josué. Universidad de Extremadura; EspañaFil: Patriarca, Andrea Rosana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentin

Automation and data processing with the immucor Galileo (R) system in a university blood bank

Background: The implementation of automated techniques improves the workflow and quality of immuno-hematological results. The workflows of our university blood bank were reviewed during the implementation of an automated immunohematological testing system. Methods: Work impact of blood grouping and subgrouping, cross- matching and antibody search using the Immucor Galileo system was compared to the previous used standard manual and semi- automated methods. Results: The redesign of our workflow did not achieve a significant reduction of the specimen's working process time, the operator's time however was reduced by 23%. Corresponding results were achieved for blood grouping, Rhesus typing, antibody screen and for autocontrol when changing from two semi- automated to the Galileo system. Because of the higher sensitivity of the Immucor antibody detection system, the rate of the initial positive antibody screens rose from 4 to 6% Conclusion: The Immucor Galileo system automates routine blood bank testing with high reliability, specificity and higher sensitivity compared to our previous used standard manual and semi- automated methods

VEGF isoforms and their expression after a single episode of hypoxia or repeated fluctuations between hyperoxia and hypoxia: Relevance to clinical ROP

Purpose—Fluctuations in oxygen are associated with the development of severe retinopathy of prematurity (ROP) in humans. However, the causal relationships between oxygen variability and severe ROP remain unknown. We investigated whether isoforms of vascular endothelial growth factor (VEGF) were differentially stimulated by hypoxia and by repeated fluctuations between hypoxia and hyperoxia, and whether isoforms were differentially expressed in association with intravitreous neovascularization. We also determined whether pigment epithelium-derived factor (PEDF) was dysregulated by oxygen fluctuations perhaps contributing to a delay in normal retinal vascular development. Methods—We used the 50/10 oxygen-induced retinopathy (50/10 OIR) model that exposes newborn rat pups to repeated cycles of 24 h of 50% oxygen alternating with 24 h of 10% oxygen to cause a condition similar to human ROP. Animals were euthanized at postnatal day 2 (P2; after one cycle of 50/10% oxygen), P7 (after 3.5 cycles of 50/10% oxygen), and P14 (after seven cycles of 50/10% oxygen). Room air raised control rat pups were also exposed to a single episode of 24 h of hypoxia at P7 and P14 and assayed immediately afterwards. Retinal VEGF isoforms and PEDF were measured by RT-PCR. Total VEGF protein was measured by ELISA. Results—We found that repeated cycles of hyperoxia and hypoxia caused greater expression of VEGF protein compared to control than did a single cycle of hyperoxia and hypoxia. VEGF164 mRNA had a greater fold change over control after repeated oxygen fluctuations than after a single episode of hypoxia. However, the other isoforms, VEGF188 and VEGF120, were expressed to a similar degree regardless of whether the stimulus was a single episode of hypoxia or repeated fluctuations in oxygen. VEGF164 was the predominant isoform expressed at the time of maximal intravitreous neovascularization. Retinal PEDF expression was elevated in pups in the 50/10 OIR model compared to control at P7, immediately after 50% oxygen. PEDF expression in the experimental group was similar to control at P18, when intravitreous neovascularization occurred. Conclusions—Repeated fluctuations in oxygen results in a greater expression of the pathologic isoform, VEGF164, than does hypoxia alone. However, the other isoforms were upregulated to an equivalent degree over control by repeated fluctuations in oxygen or a single episode of hypoxia. Total VEGF protein was increased to a greater degree by repeated fluctuations in oxygen compared to a single cycle of oxygen. PEDF was increased over control early in the 50/10 OIR model and may play a role in the observed delay in retinal vascularization. These findings provide insight into the effect of repeated oxygen fluctuations on the development of severe ROP in preterm infant

Performance of a new pulse contour method for continuous cardiac output monitoring: validation in critically ill patients

Background A new calibrated pulse wave analysis method (VolumeView™/EV1000™, Edwards Lifesciences, Irvine, CA, USA) has been developed to continuously monitor cardiac output (CO). The aim of this study was to compare the performance of the VolumeView method, and of the PiCCO2™ pulse contour method (Pulsion Medical Systems, Munich, Germany), with reference transpulmonary thermodilution (TPTD) CO measurements. Methods This was a prospective, multicentre observational study performed in the surgical and interdisciplinary intensive care units of four tertiary hospitals. Seventy-two critically ill patients were monitored with a central venous catheter, and a thermistor-tipped femoral arterial VolumeView™ catheter connected to the EV1000™ monitor. After initial calibration by TPTD CO was continuously assessed using the VolumeView-CCO software (CCOVolumeView) during a 72 h period. TPTD was performed in order to obtain reference CO values (COREF). TPTD and arterial wave signals were transmitted to a PiCCO2™ monitor in order to obtain CCOPiCCO values. CCOVolumeView and CCOPiCCO were recorded over a 5 min interval before assessment of COTPTD. Bland-Altman analysis, %errors, and concordance (trend analysis) were calculated. Results A total of 338 matched sets of data were available for comparison. Bias for CCOVolumeView−COREF was −0.07 litre min−1 and for CCOPiCCO-COREF +0.03 litre min−1. Corresponding limits of agreement were 2.00 and 2.48 litre min−1 (P<0.01), %errors 29 and 37%, respectively. Trending capabilities were comparable for both techniques. Conclusions The performance of the new VolumeView™-CCO method is as reliable as the PiCCO2™-CCO pulse wave analysis in critically ill patients. However, an improved precision was observed with the VolumeView™ technique. Clinicaltrials.gov identifier NCT0140504

Exogenous leukemia inhibitory factor (LIF) attenuates retinal vascularization reducing cell proliferation not apoptosis

To study the effect of leukemia inhibitory factor (LIF) on rat retinal vascular development, Sprague–Dawley rats at postnatal age 3 days (p3) were given intraperitoneal (IP) LIF and analysis performed at p6 (p3/6). p7 rats were given intravitreous (IV) LIF and analysis performed at p9 (p7/9). Control animals were PBS injected. At the time of analysis retinal flatmounts were prepared and stained with Griffonia lectin and activated caspase-3. The retinal peripheral avascular area was measured and number of apoptotic cells counted. In vitro, human retinal microvascular endothelial cells (RMVECs) were cultured in media containing LIF, with and without neutralizing antibody to LIF. Cells were stained with activated caspase-3 and apoptotic cells counted. Proliferation was measured by counting cell numbers, and cell cycle stage was determined using propidium iodide staining and FACS analysis. LIF injected either IP or IV had no effect on body weight or total retina area, but significantly increased the peripheral retinal avascular area. In both IP and IV injected groups there was no difference in the number of apoptotic cells between PBS-or LIF-injected groups; although in the p7/9 retinas, both injected groups had significantly more apoptotic cells than the non-injected group. In vitro, there was no effect of LIF on RMVEC apoptosis; however, cell counts were significantly lower in the LIF-treated group. Antibody to LIF restored the cell counts to untreated levels. LIF reduced the number of cells in S phase. LIF attenuates retinal vascular development in vivo through growth arrest, and not apoptosis, of endothelial cells