Use of CAS-agar plate modified to study the effect of different variables on the siderophore production by Aspergillus

AIMS: To evaluate the suitability of chrome azurol S (CAS) agar plate assay as a quantitative methodology for siderophore production. METHODS AND RESULTS: Aspergillus species (A. flavus, A. niger, A. tamarii) were inoculated in the CAS-agar plates and the siderophores production was determined and expressed as CAS-reaction rate (mm per day). All the species showed positive CAS reaction with different rates depending on culture conditions and A. flavus showed the highest CAS-reaction rate. The siderophore production in solid medium expressed as CAS-reaction rate was correlated with siderophore production in liquid medium. CONCLUSIONS: The use of CAS-agar plate assay was modified and the evaluation of CAS reaction in mm per day made it possible to study and quantify the effect of several variables on the siderophore production by Aspergillus fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe the CAS-agar plate assay as a quantitative methodology, which make it possible to select and evaluate the siderophore production by several microorganisms (fungi and bacteria) according to different culture conditions.     

Gas chromatography/mass spectrometry assays for the determination of debrisoquine and sparteine metabolites in microsomal fractions of rat liver.

Debrisoquine and sparteine are prototype substrates of a genetic deficiency in cytochrome P450-dependent drug metabolism. Sensitive assays of in vitro oxidation of sparteine and debrisoquine are required for evaluation of this polymorphism. The activities were measured by quantitative analysis of 2-dehydrosparteine and 4-hydroxydebrisoquine production, respectively, using capillary column gas chromatography coupled with mass selective ion detection. With a single extraction, separation of parent drug, metabolite, and a suitable internal standard was readily achievable. Time-dependent production of both metabolites could be detected from as little as 40 micrograms of microsomal protein. Both activities showed a maximal activity with a 240-min incubation period. The ability to simultaneously quantify the parent drug and its metabolite suggests it would also be useful for evaluation of in vivo metabolism.     

Quantitative urease test for Helicobacter pylori infection in patients with gastric and duodenal ulcer

The monitoring for the presence of H. pylori carrier state in a group of patients with gastric and duodenal ulcer was carried out with subsequent determination of the relationship between the intensity of the urease activity of the bioptic specimen of the mucous membrane and the severity of the course of the disease. For this purpose we developed the scheme for the evaluation of the severity of the disease with quantitative criteria. The data obtained in this work showed no correlation between the severity of this disease and usease activity. Still the method of the quantitative determination of the urease activity of the bioptic specimen made it possible to evaluate the rate of the production of hydroxyl anions by a given H. pylori strain and thus quickly establish the presence of carrier state.     

Evaluation of two CD-ROMs from a series on cell biology

Two CD-ROMs from a series dealing with various major aspects of cell biology are evaluated in this paper using quantitative and qualitative approaches. The findings delimit similarities and differences of the two CD-ROMs and shed light on how the programs could be used in the learning process and how they should not be. The overall impression, as well as the graphical and technical features, received a predominantly good rating. The defined target groups were reached (e.g., students in secondary schools), different learning approaches were supported (e.g., discovery and autonomous learning), the CD-ROMs' usability was assessed as being easy and intuitive, and the majority of the evaluators were satisfied with the level of interactivity. Navigational problems encountered in CD-ROM 1 were overcome by a successful implementation of new navigational functions in CD-ROM 2. Most students found the CD-ROM to be a suitable complement to, or an extension of, their lessons. We conclude that many, but not all of the requirements for the various stages of the learning process could be satisfied with the existing CD-ROMs. The requirements not met are discussed to obtain insights that could help to improve the production of multimedia learning material. The use of quantitative and qualitative approaches in the evaluation of learning modules is discussed, as the study began by collecting and analyzing anecdotal reviews and was then extended to include a qualitative evaluation.     

Carbohydrate quantitative digestion and absorption in ruminants: from feed starch and fibre to nutrients available for tissues

Carbohydrates are the main source of energy in ruminants. Their site, extent and kinetics of digestion highly impact the amount and profile of nutrients delivered to peripheral tissues, and the responses of the animal, i.e. ingestion, efficiency of production, N and methane excretion, quality of products and welfare. Development of multi-objective feed evaluation systems thus requires a more integrated quantitative knowledge on carbohydrate digestion and yield of terminal products, as well as on their metabolism by splanchnic tissues. The objective of this paper is to review (i) quantitative knowledge on fibre, starch and sugar digestion, volatile fatty acids (VFA) and glucose production and splanchnic metabolism and (ii) modelling approaches which aim at representing and/or predicting nutrient fluxes in the digestive tract, portal and hepatic drainage. It shows that the representation of carbohydrate digestion and VFA yield is relatively homogeneous among models. Although published quantitative comparisons of these models are scarce, they stress that prediction of fibre digestion and VFA yield and composition is still not good enough for use in feed formulation, whereas prediction of microbial N yield and ruminal starch digestion seems to be more satisfactory. Uncertainties on VFA stoichiometric coefficients and absorption rates may partly explain the poor predictions of VFA. Hardly any mechanistic models have been developed on portal-drained viscera (PDV) metabolism whereas a few exist for liver metabolism. A qualitative comparison of these models is presented. Most are focused on dairy cows and their level of aggregation in the representation of nutrient fluxes and metabolism highly differs depending on their objectives. Quantitative comparison of these models is still lacking. However, recent advances have been achieved with the empirical prediction of VFA and glucose production and fluxes through PDV and liver based on the current INRA feed evaluation system. These advances are presented. They illustrate that empirical prediction of ruminal VFA and intestinal glucose production can be evaluated by comparison with measured net portal net fluxes. We also illustrate the potential synergy between empirical and mechanistic modelling. It is concluded that concomitant empirical and mechanistic approach may likely help to progress towards development of multi-objective feed evaluation systems based on nutrient fluxes.     

A new evaluation method for quantifying PI3K activity by HTRF assay

PIK3CA, coding a catalytic subunit of PI3K p110α, is frequently mutated in cancer. In previous studies, p110α with hotspot mutations such as E545K and H1047R were shown to be gain-of-function mutations. However, quantitative evaluation of these mutants was not well established. Recently, a new method for measuring PI3K activity using homogeneous time-resolved fluorescence (HTRF) has been developed. Using this method, we constructed a quantitative evaluation system for PI3K activity. Serial dilutions of standard PIP3 were subjected to the PI3K-HTRF assay in order to establish a regression line for calibration. The recombinant FLAG-tagged p110α proteins were engineered together with a regulatory subunit p85α in human embryonic kidney 293T cells. Anti-FLAG-Ig immunoprecipitates were then subjected to the assay, which enabled us to quantitatively evaluate the activities of hotspot mutants of p110α. We believe this method will also be applicable to the evaluation of p110α having uncharacterized mutations found in cancer.     

Real-time quantification of the proliferative state in astrocytomas

OBJECTIVE: To evaluate proliferative activity in a set of gliomas and to compare the quantitative data obtained by a real-time processor with the labelling index (LI) and mitotic index (MI). STUDY DESIGN: Ki-67 immunostaining was performed on paraffin-embedded specimens from 42 cases of glioblastomas, 17 cases of anaplastic astrocytomas and 14 cases of low grade astrocytomas. Nuclear positivity was calculated as LI and by a real-time image processor for quantitative evaluation. MI was also calculated at 10 high-power fields. The data obtained from glioblastomas were compared with those from anaplastic and low grade astrocytomas. To all the data was applied the Pearson test to verify the correlation between counting and quantitative values and between proliferative markers and survival. RESULTS: A positive trend from low grade astrocytomas to glioblastomas was found for Ki-67 (LI and quantitative values) and MI, with highly significant differences between the three grades of gliomas considered. A good correlation between LI and quantitative values of Ki-67 was found. Very little relationship resulted between survival and Ki-67 LI. No relationship was found between survival and quantitative values of Ki-67. CONCLUSION: Ki-67 allowed effective separation of astrocytic tumors with different grades of malignancy. Quantitative evaluation of color information by means of a real-time processor proved to be a useful, objective and fast way to obtain readings, useful for grading purposes but not for prognostic evaluation.     

Effect of leptin gene polymorphism on the breeding value of milk production traits in Iranian Holstein

New molecular techniques focused on genome analysis, open new possibilities for more accurate evaluation of economiclly important traits in farm animals. Milk production traits are typical quantitative characteristics controlled by a number of genes. Mutations in their sequences may alter animal performance as well as their breeding values. In this study, we investigated the effect of Kpn2I restriction fragment length polymorphisms in the leptin gene, on bull breeding values for milk yield, fat, and protein yield, and their percentage. In order to test for an association between the leptin single-nucleotide polymorphism in exon 2 and milk productivity, we genotyped 134 Iranian Holstein bulls. Breeding values for milk-related traits (milk yield, fat, and protein yield and percentage) were estimated using the BLUP based on an animal model. The effect of the genotypes of Kpn2I polymorphism on the breeding values for milk-related traits was examined using least square methods. The T allele frequency was 0.425. Genotypes were distributed according to the Hardy-Weinberg equilibrium. Bulls with TT genotype had higher milk, fat and protein yield compared with TC and CC bulls (P < 0.05). Bulls with CC genotype had higher protein percentage compared with TT and TC bulls (P < 0.05). The association between leptin polymorphism with milk production traits suggests that this marker may be useful for selection based on molecular information.     

Influence of batch or fed-batch growth on Staphylococcus epidermidis biofilm formation

AIMS: To make a quantitative evaluation of the differences in biofilm formation by Staphylococcus epidermidis using batch and fed-batch growth systems and to correlate this with production of the major biofilm polysaccharide, poly-N-acetyl glucosamine (PNAG). METHODS AND RESULTS: Dry weight measurements of biofilms formed in batch and fed-batch conditions were compared with haemagglutination titres, which measure the amount of PNAG produced. Strains grown in batch systems developed less biofilm than when grown in fed-batch systems. A good correlation was found between the amount of biofilm formed in fed-batch systems and the haemagglutination titres. CONCLUSIONS: Differences in biofilm formation and PNAG production by S. epidermidis are dependent on the availability of nutrients, with higher availability correlating with more biofilm and PNAG production. SIGNIFICANCE OF AND IMPACT OF THE STUDY: Comparisons of the formation of biofilms by S. epidermidis are dependent on choosing an appropriate biofilm growth system. Comparability or disparity of conclusions among different investigations will be strongly influenced by which mode S. epidermidis biofilms are formed.     

Human immunodeficiency disease: impairment of cellular interactions leading to abnormal mediator production in mixed lymphocyte culture reaction.

Leukocyte migration inhibitory factor (LMIF) production in mixed lymphocyte culture (MLC) reactions is the result of cellular interactions based on two separate phenomena: the capacity of lymphocytes to stimulate in MLC, and the capacity of lymphocytes to respond in MLC. Puromycin-treated lymphocytes are capable of stimulating allogeneic cells for LMIF production, but are unable to respond with synthesis of LMIF (one-way MLC-LMIF test). We have studied the stimulating and responding capacity of lymphocytes from patients with different immunodeficiency syndromes in a one-way MLC-LMIF assay. Lymphocytes from patients known to have qualitative and quantitative defects of T cell or B cell functions (Hodgkin's disease, mycosis fungoides, thymoma, chronic lymphatic leukemia) were found to respond poorly as measured by mediator production although their stimulating fuction was frequently retained. Patients with advanced solid tumors often had both MLC-stimulating and responding functions depressed. There was no apparent correlation between mitogen response and MLC-induced LMIF responses or between MLC proliferative response (as measured by thymidine incorporation) and mediator production. Studying of stimulatory and responding capacity of lymphocytes in the MLC-LMIF assay provides a new tool for assessing immunocompetence and allows for in vitro evaluation of cellular interactions that may play an important role in vivo.     

Conjugation in starforming Rhizobium lupini

Summary ARhizobium strain which exhibits high efficiency of starformation has been isolated from the root nodules ofLupinus luteus. Mutants of this strain were induced by repeated treatment of the wild type with nitrous acid. The mutants are marked with one or two auxotrophic characters and with different colors (carotenoid production). Two-, three-, and four-point crosses were performed. The maximum recombination rate is around 10%. A quantitative evaluation of the results of the crosses indicates the existence of a circular linkage map.The recombination is the result of a conjugation between cells during which most probably always the whole chromosome is transferred.The research was supported by NSF Grant. No. GB-4287 and NJH Grant No. 1 PO 1 GM 13234.     

Relation between in vitro production of ascosonchine and virulence of strains of the potential mycoherbicide Ascochyta sonchi: a method for its quantification in complex samples

The potential of the fungus Ascochyta sonchi as a mycoherbicide for the biocontrol of the perennial weeds Sonchus arvensis and Cirsium arvense that occur throughout temperate regions of the world is under evaluation. Ascosonchine, a newly discovered enol tautomer of 4-pyridylpyruvic acid with potential herbicidal properties, is the main phytotoxin produced by this fungus. A simple and sensitive method has been developed for the rapid quantitative analysis of ascosonchine based on HPLC with UV detection. The toxin content in culture filtrates of different strains of A. sonchi was measured. The strains tested produced up to 2.7 mg/L when grown in static conditions. Toxin production was compared with the virulence on the host plant of each strain to determine if the most virulent strains could be simply selected by choosing the best toxin producers. The results obtained do not support this approach. The same HPLC method was also applied to quantify toxin production under different fungal growth conditions, in order to achieve the highest toxin production. The most productive strain synthesised more than 8 mg/L when grown for 8 weeks in static conditions.     

Production of plant sesquiterpenes in Saccharomyces cerevisiae: effect of ERG9 repression on sesquiterpene biosynthesis

The yeast Saccharomyces cerevisiae was chosen as a microbial host for heterologous biosynthesis of three different plant sesquiterpenes, namely valencene, cubebol, and patchoulol. The volatility and low solubility of the sesquiterpenes were major practical problems for quantification of the excreted sesquiterpenes. In situ separation of sesquiterpenes in a two-phase fermentation using dodecane as the secondary phase was therefore performed in order to enable quantitative evaluation of different strains. In order to enhance the availability of the precursor for synthesis of sesquiterpenes, farnesyl diphosphate (FPP), the ERG9 gene which is responsible for conversion of FPP to squalene was downregulated by replacing the native ERG9 promoter with the regulatable MET3 promoter combined with addition of 2 mM methionine to the medium. This strategy led to a reduced ergosterol content of the cells and accumulation of FPP derived compounds like target sesquiterpenes and farnesol. Adjustment of the methionine level during fermentations prevented relieving MET3 promoter repression and resulted in further improved sesquiterpene production. Thus, the final titer of patchoulol and farnesol in the ERG9 downregulated strain reached 16.9 and 20.2 mg/L, respectively. The results obtained in this study revealed the great potential of yeast as a cell factory for production of sesquiterpenes.     

Weathering of Basalts by Aspergillus sp. FS-4 Strain: Glass Compositions are Prone to Weathering

Abstract

Mechanisms underlying microbe-induced dissolution of basalt and its effect on glass composition remain unclear. Two powdered basalts, with a glass composition of 27?vol% (Basalt-T) and 18?vol% (Basalt-F), respectively, were inoculated with Aspergillus sp. FS-4 in Czapek medium. Changes in pH, siderophores and main dissolved elements were examined. Basalt-F led to a greater decrease in pH and higher production of siderophores than Basalt-T mainly due to the fungi activity. Element dissolution was 2 to 3 fold higher in Basalt-T than in Basalt-F, and the dissolution was non-stoichiometric for both basalts. FS-4 appears important for basalt weathering. Composition of geologic material had a considerable impact on weathering. Materials with a higher glass percentage are prone to weathering. Further quantitative evaluation is required.     

Quantitative analysis of age-associated accumulation of mitochondrial DNA with deletion in human hearts

We have demonstrated that myocardial mitochondrial DNA (mtDNA) with a 7,436 base-pair deletion existed in all subjects that were over 70 years old. Since each mitochondrion has two or three copies of its own DNA, quantitative analysis is required for the evaluation of the role of mtDNA with deletions in the age-related deterioration of cardiac performance. For this purpose, the kinetic polymerase chain reaction (PCR) method developed in our laboratory was used in this investigation to determine myocardial mtDNA with this 7,436 base-pair deletion in human cadavers of various ages. The mtDNA population with this deletion increased exponentially with age [log f (% of deleted mtDNA) = -3.136 + 0.0454 x age, r = 0.95, P less than 0.01)], and was estimated at 3% and 9% in subjects of age 80 and 90, respectively. The deleted portion encodes 7 subunits of the mitochondrial ATP production system, and a population of mtDNA with this deletion over a certain threshold might induce a significant deterioration of cardiac energy metabolism. Cardiac function is known to deteriorate with age, and an increase in the population of mtDNA with deletion is likely to be an important contributing factor to aged heart (presbycardia).     

Use of a focal infectivity assay for testing susceptibility of HIV to antiviral agents

A highly sensitive and quantitative focal immunoassay has been developed for detecting the human immunodeficiency virus (HIV). The assay can be used to measure cell-free virus or the production of HIV by virus-infected cells. Both laboratory-adapted strains of HIV and patient isolates can be studied with this assay. In this communication, we demonstrate the utility of this assay for measuring the effects of anti-HIV agents on viral isolates. We show that the anti-viral effects of such diverse agents as azidothymidine, interferon-alpha, immunotoxins, soluble CD4 and antibody can be accurately quantified. This assay may be used in the discovery and evaluation of new anti-HIV therapies or may be adapted for use in testing the sensitivity of patient isolates to standard therapeutic agents.     

中国绿豆应用型核心样本农艺性状的分析

为提高种质资源在育种中的利用效率,建立了我国绿豆（Vigna radiata）应用型核心样本。该样本既包括了资源库中具有特异性状的种质和曾经在生产上大面积种植的品种,也包括了在育种中使用频繁的亲本及苗头品系等。农艺性状变异分析表明,该核心样本具有丰富的表型变异,是绿豆种质资源的代表性样本。聚类分析可将核心样本分为4大类,但类别内种质与其地理来源相关性不明显。不同来源表型数据的分析发现,不同性状间的一致性存在差异。但产量相关性状的表现均与当前育种目标相接近,说明该核心样本具有较高的实用性。     

Comprehensive comparison of iTRAQ and label-free LC-based quantitative proteomics approaches using two Chlamydomonas reinhardtii strains of interest for biofuels engineering

Comprehensive comparisons of quantitative proteomics techniques are rare in the literature, yet they are crucially important for optimal selection of approaches and methodologies that are ideal for a given proteomics initiative. In this study, two LC-based quantitative proteomics approaches--iTRAQ and label-free--were implemented using the LTQ-Orbitrap Velos platform. For this comparison, the model used was the total protein content from two Chlamydomonas reinhardtii strains in the context of alternative biofuels production. The strain comparison includes sta6 (a starch-less mutant of cw15) that produces twice as many lipid bodies (LB) containing triacylglycerols (TAGs) as its parental strain cw15 (a cell wall-deficient C. reinhardtii strain) under nitrogen starvation. Internal standard addition was used to rigorously assess the quantitation accuracy and precision of each method. Results from iTRAQ-4plex labeling using HCD (higher energy collision-induced dissociation) fragmentation were compared to those obtained using a label-free approach based on the peak area of intact peptides and collision-induced dissociation. The accuracy and precision, number of identified/quantified proteins and statistically significant protein differences detected, as well as efficiency of these two quantitative proteomics methods were evaluated and compared. Four technical and three biological replicates of each strain were performed to assess both the technical and biological variation of both approaches. A total of 896 and 639 proteins were identified with high confidence, and 329 and 124 proteins were quantified significantly with label-free and iTRAQ, respectively, using biological replicates. The results showed that both iTRAQ labeling and label-free methods provide high quality quantitative and qualitative data using nano-LC coupled with the LTQ-Orbitrap Velos mass spectrometer, but the selection of the optimal approach is dependent on experimental design and the biological question to be addressed. The functional categorization of the differential proteins between cw15 and sta6 reveals already known but also new mechanisms likely responsible for the production of lipids in sta6 and sets the baseline for future studies aimed at engineering these strains for high oil production.     

Genetic dissection of the maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) MAMP response

Key message

Loci associated with variation in maize responses to two microbe-associated molecular patterns (MAMPs) were identified. MAMP responses were correlated. No relationship between MAMP responses and quantitative disease resistance was identified.

Abstract

Microbe-associated molecular patterns (MAMPs) are highly conserved molecules commonly found in microbes which can be recognized by plant pattern recognition receptors. Recognition triggers a suite of responses including production of reactive oxygen species (ROS) and nitric oxide (NO) and expression changes of defense-related genes. In this study, we used two well-studied MAMPs (flg22 and chitooctaose) to challenge different maize lines to determine whether there was variation in the level of responses to these MAMPs, to dissect the genetic basis underlying that variation and to understand the relationship between MAMP response and quantitative disease resistance (QDR). Naturally occurring quantitative variation in ROS, NO production, and defense genes expression levels triggered by MAMPs was observed. A major quantitative traits locus (QTL) associated with variation in the ROS production response to both flg22 and chitooctaose was identified on chromosome 2 in a recombinant inbred line (RIL) population derived from the maize inbred lines B73 and CML228. Minor QTL associated with variation in the flg22 ROS response was identified on chromosomes 1 and 4. Comparison of these results with data previously obtained for variation in QDR and the defense response in the same RIL population did not provide any evidence for a common genetic basis controlling variation in these traits.