A rapid method for detecting specific amplified PCR fragments in microtiter plates.

A simple method is presented to circumvent laborious and time consuming electrophoretic separations of specific PCR amplification products. Specific target DNA is amplified using nucleotides labelled with DIG-dUTP or biotin-dCTP. The labelled PCR products are separated from unincorporated nucleotides or oligonucleotides by using a positively charged DEAE cellulose matrix. Amplification products are visualized directly in the matrix using immunoenzymatic methods or streptavidin-conjugated enzymes. The detection process can be carried out within 2 h, allows the processing of large sample sizes and can potentially be automated.     

A fermentation assay to evaluate the effectiveness of antimicrobial agents on gut microflora

The measurement of gas produced as a fermentation end product in vitro was correlated with absorbance as a measure of bacterial growth and was used as a rapid screening procedure to test the antimicrobial activity of certain essential oil and tannin secondary plant metabolites on gastrointestinal microorganisms from chickens. The assay was optimised using Clostridium perfringens and Lactobacillus fermentum, and tested in antimicrobial assays against C. perfringens; the minimum inhibitory concentration for each essential oil and condensed tannin was determined. The effect of penicillin-G on C. perfringens, in both growth and fermentation assays, was similar, and for all secondary metabolites tested, concentrations that inhibited fermentation were also bacteriocidal. The assay was also used to demonstrate the effect of dietary composition and enzyme supplementation on fermentation of mixed gut microflora in vitro; results are compared with in vivo results for the same dietary treatments. The data demonstrate that the effects of bioactive secondary plant products and feed composition on individual organisms or mixed gut microflora can be tested by analysis of fermentative activity in vitro, and that this provides a rapid assay for testing potential poultry feed additives before in vivo trials.     

A test to evaluate the physical impact on technical performance in soccer

The aim of the study was to develop and examine a test for evaluation of the physical and technical capacity of soccer players. Fourteen youth elite (YE) and seven sub-elite (SE) players performed a physical and technical test (PT-test) consisting of 10 long kicks interspersed with intense intermittent exercise. In addition, a control test (CON-test) without intense exercise was performed. In both cases, the test result was evaluated by the precision of the 10 kicks. The players also performed the Yo-Yo intermittent recovery test level 2 (Yo-Yo IR2). For the SE-players, blood samples were obtained and heart rate was measured before, during, and after the PT-test. A muscle biopsy was collected before and after the PT-test. Coefficient of variation for the PT- and CON-test was 11.7% and 16.0%, respectively. The YE-players performed better (P < 0.05) than the SE-players in both the PT-test (16.3 +/- 0.8 (+/-SE) vs. 13.2 +/- 1.3 points) and CON-test (24.4 +/- 0.7 vs. 20.5 +/- 1.6 points) with no difference in the relative PT-test result (PT-test/CON-test: 0.63 +/- 0.03 vs. 0.64 +/- 0.03). Summed performance of the first 5 repetitions was higher (P < 0.05) than for the last 5 repetitions (8.4 +/- 0.6 vs. 6.9 +/- 0.5; n = 20). The YE-players performed better (P < 0.05) than the SE-players during Yo-Yo IR2 (1023 +/- SE vs. 893 +/- SE m). The mean heart rate during the PT-test was 173 +/- 4 b.p.m. (90 +/- 2% of HRmax). Blood lactate, glucose, and ammonia reached 5.6 +/- 0.7, 6.2 +/- 0.6 mmol L(-1), and 76 +/- 11 umol L(-1) at the end of the test, respectively. After the test muscle CP, glycogen and lactate was 52.9 +/- 6.6, 354 +/- 39, and 25.3 +/- 5.9 mmol kg(-1) d.w., respectively. In summary, the PT-test can be used to evaluate a soccer player's technical skills under conditions similar to intense periods of a soccer game.     

A substrate phage enzyme-linked immunosorbent assay to profile panels of proteases.

It is estimated that proteases comprise nearly 2% of the human genome. Given that the primary structure of all known proteases will soon be available, an important challenge is to define the structure-activity relationships that govern substrate hydrolysis. Ideally this would be accomplished on a genome-wide scale. To this end, we have developed a one-pot phage selection system that yields the substrate recognition profile of multiple proteases from a single round of selection. The system meets five key criteria: (i) multiple proteases can be analyzed simultaneously, (ii) prior knowledge of substrate preference is not required, (iii) information regarding substrate preferences on both side of the scissile bond is obtained, (iv) the system yields selective substrates that distinguish closely related proteases, and (v) semiquantitative information on substrate hydrolysis is obtained, allowing for the assignment of initial rank-order preferences. As an illustration, a phage selection with a mixture of thrombin and factor Xa (serine proteases) along with matrix-metalloproteinase-9 and atrolysin C (metalloproteinases) was performed. Peptide substrates were identified that (i) have high k(cat)/K(m) ratios, (ii) are selective for individual proteases, and (iii) match the sequences of known physiological substrates.     

Comparative field study to evaluate the performance of three different traps for collecting sand flies in northeastern Italy

Three standard methods for collecting sand flies (sticky trap, CDC light trap, and CO₂ trap) were compared in a field study conducted from June to October, 2012, at a site located in the center of a newly established autochthonous focus of canine leishmaniasis in northeastern Italy. Six traps (two sticky traps, two CDC light traps, and two CO₂ traps) were activated at the same time for a single night every two weeks during the season of sand fly activity. A total of 5,667 sand flies were collected and 2,213 identified, of which 82.1% were Phlebotomus perniciosus, 17.4% P. neglectus, 0.3% Sergentomya minuta, and 0.2% P. mascitti. The performances of all traps were influenced by their position inside the site, increasing with proximity to the animal shelters. CO₂ traps were more attractive for females of P. perniciosus and P. neglectus. CDC light traps showed an intermediate efficiency and were more attractive for P. neglectus, compared to other two traps. Results suggest that in northern Italy the CO₂ trap is a suitable sampling method for sand fly monitoring programs that include transmitted pathogen surveillance.     

Open-label,multi-center,non-randomized,single-arm study to evaluate the safety and efficacy of dendritic cell immunotherapy in patients with refractory solid malignancies,on supportive care

Background aimsA phase II clinical trial of an autologous dendritic cell (DC) formulation for the management of refractory solid malignant tumors was conducted across six sites in India with an objective to study safety and efficacy.MethodsA total of 51 patients with refractory cancer (either sex) with life expectancy ≥3 months, Eastern Cooperative Oncology Group score ≤2, available tumor tissue and adequate organ and bone marrow function were recruited. Monocytes obtained by leukapheresis, differentiated into DCs by cytokines and primed with autologous tumor lysate (fresh tissue biopsy or paraffin block). On the 8th day, mature DCs were analyzed for expression of CD40, CD80, CD83, CD86, DC205 and DC209. The treatment regime consisted of six doses (intravenous) over 14 weeks with 2 post-treatment follow-up visits, 6 weeks apart. Safety was assessed at all visits and responses were evaluated on days 58, 100 and 184 or at end of the study.ResultsA total of 38 patients were evaluated for safety and efficacy. One adverse event classified as possibly related was an episode of rigors or chills with mild pyrexia during one infusion. Objective response rate by Response Evaluation Criteria In Solid Tumors was 28.9% (11/38) and immune-related response criteria was 42.1% (16/38); 90% confidence interval for objective response rate was (17.2, 43.3) and (28.5, 56.7) by Response Evaluation Criteria In Solid Tumors and immune-related response criteria, respectively. The median time to treatment progression was >9 weeks. Median overall survival was 397 days. An increase in the expression of interferon-γ was not significant.ConclusionsTherapy was safe. The responses, time to treatment progression and survival are encouraging for patients with aggressive refractory disease.     

Use of phage display for detecting single-nucleotide differences in genes]

Experimental strategy has been developed for selection of mismatched DNA binding phages from library of E. coli f1 filamentous phages carrying random peptide inserts on the surface of bacteriophage particles. The strategy is based on the use of phage display technique, DNA heteroduplexes (with single nucleotide variations), and paramagnetic beads. DNA heteroduplexes have been obtained from biotin-labeled PCR product. During the first stage the phage particles were incubated with DNA heteroduplexes possessing mismatched nucleotides. The next step after elimination of free phages and separation of bound phages from DNA heteroduplexes was subtraction of phages binding with DNA heteroduplexes (without mismatched nucleotides). Phages selected by this method were capable of discriminating DNA heteroduplexes with single nucleotide variations from DNA homoduplexes. Phages immobilized on solid base retain their activity and specificity, and therefore can be used for developing a new screening automated method for detecting point mutations and gene polymorphism.     

Exonuclease cycling assay: an amplified assay for the detection of specific DNA sequences.

An assay is described in which an oligonucleotide probe is specifically digested by lambda exonuclease only when it is annealed to its complementary sequence. In this assay, a cycling effect occurs whereby a small amount of target sequence acts as a specific co-factor in the enzymatic degradation of a larger number of molecules of an oligonucleotide probe. This amplification principle is demonstrated and the effect of the oligonucleotide probe sequence investigated. The necessary steps needed to convert this effect into a useful diagnostic tool are discussed.     

Sensitivity of the phage titer increase test in detecting Listeria

The optimum conditions for performing the phage titer increase test aimed at the detection of Listeria in water and soil have been established.     

Use of psoralen for detecting the conjugated damage to 2 chains in irradiated phage PM2 DNA

Using psoralen for the photochemical cross-linking of DNA chains the authors have demonstrated the formation of conjugated lesions, of both opposite and non-opposite types, in X-irradiated superhelical DNA of PM2 phage. It is suggested that these lesions result from hitting a DNA molecule by a spur.     

A highly specific PCR assay for detecting the fish ectoparasite Amyloodinium ocellatum

Amyloodiniosis, caused by the dinoflagellate ectoparasite Amyloodinium ocellatum, is one of the most serious diseases affecting marine fish in warm and temperate waters. Current diagnostic methods rely entirely on the microscopic identification of parasites on the skin or gills of infested fish. However, subclinical infestations usually go undetected, while no method of detecting the free-swimming, infective (dinospore) stage has been devised. Targeting the parasite's ribosomal DNA region, we have developed a sensitive and specific PCR assay that can detect as little as a single cell from any of the 3 stages of the parasite's life cycle (trophont, tomont, dinospore). This assay performs equally well in a simple artificial seawater medium and in natural seawater containing a plankton community assemblage. The assay is also not inhibited by gill tissue. Sequence analysis of the internal transcribed spacer region of 5 A. ocellatum isolates, obtained from fish in the Red Sea (Israel), eastern Mediterranean Sea (Israel), Adriatic Sea (Italy), Gulf of Mexico (Florida), and from an unknown origin, revealed insignificant variation, indicating that all isolates were the same species. However, 3 of these isolates propagated in cell culture varied in behavior and morphology, and these differences were consistent during at least 2 yr in culture. Thus, our findings do not eliminate the possibility that different strains are in fact 'subspecies' or lower taxa, which may also differ in pathogenic and immunogenic characteristics, environmental tolerance, and other features.