The affinity of [3H]quinuclidinyl benzilate and some other radioligands for glass microfiber and cellulose filters in some common liquid scintillation solvents

The optimum conditions for measuring radioactivity in the filtration assay of muscarinic cholinoceptors with tritiated quinuclidinyl benzilate are to use Whatman GF/B filters and to add a simple toluene scintillant to them while they are still damp. Practically all the radioactive material is then slowly extracted into the scintillant and high counting efficiencies are obtained after 24 h. Dried filters, or dry filters in control experiments in the absence of receptor, adsorb much of the radioactivity with a 30% reduction in counting efficiency. Other scintillants were able to extract the radioactive material from dry filters, but were generally not preferable to toluene. The GF/B filters performed better than other glass microfiber and cellulose filters in terms of retention of receptor-bound ligand, rapid filtration rates, and low filter blanks. Toluene is unsuitable as a scintillant with GF/B filters for some other radioligands examined.     

The effect of filtration method on the efficiency of environmental DNA capture and quantification via metabarcoding

Environmental DNA (eDNA) is a promising tool for rapid and noninvasive biodiversity monitoring. eDNA density is low in environmental samples, and a capture method, such as filtration, is often required to concentrate eDNA for downstream analyses. In this study, six treatments, with differing filter types and pore sizes for eDNA capture, were compared for their efficiency and accuracy to assess fish community structure with known fish abundance and biomass via eDNA metabarcoding. Our results showed that different filters (with the exception of 20‐μm large‐pore filters) were broadly consistent in their DNA capture ability. The 0.45‐μm filters performed the best in terms of total DNA yield, probability of species detection, repeatability within pond and consistency between ponds. However performance of 0.45‐μm filters was only marginally better than for 0.8‐μm filters, while filtration time was significantly longer. Given this trade‐off, the 0.8‐μm filter is the optimal pore size of membrane filter for turbid, eutrophic and high fish density ponds analysed here. The 0.45‐μm Sterivex enclosed filters performed reasonably well and are suitable in situations where on‐site filtration is required. Finally, prefilters are applied only if absolutely essential for reducing the filtration time or increasing the throughput volume of the capture filters. In summary, we found encouraging similarity in the results obtained from different filtration methods, but the optimal pore size of filter or filter type might strongly depend on the water type under study.     

Microfluorometric filter determination of DNA in sucrose density gradients

A simple filter method for the fluorometric estimation of DNA in alkaline and neutral sucrose gradient fractions with DABA·2HCl is discussed. Alpha-450 membrane filters of regenerated cellulose (Gelman Instrument Co., Ann Arbor, Michigan) were used. In the proposed method washing of the DNA precipitate as well as the reaction with DABA·2HCl were performed directly on the filters, thus avoiding repeated washing and centrifugations of DNA precipitates applied hitherto in analogous fluorometric techniques. A good coincidence of the results concerning localization of DNA sedimentation profiles determined by radioisotopic and fluorometric methods was obtained. The method is very convenient for DNA estimation in alkaline and neutral sucrose density gradient fractions obtained by ultracentrifugation of DNA of nonproliferating cells where DNA labeling is very difficult, and in the case of human lymphocytes even impossible without stimulation for blastic transformation. Its other advantages are a considerably simplified procedure and a higher precision with respect to other fluorometric methods for determination of DNA.     

Croatian bibliometric analysis, 2000-2007

To develop search filters and retrieve information estimating the Croatian scientific output (SO) focusing on Public Health (PH) and Preventive Medicine (PM) in MEDLINE. A PubMed search of the MEDLINE database was performed to retrieve articles added to this database between 2000 and 2007. Search filters inspired by previous strategies were applied involving 'geographical', 'place of publication', 'subject' and 'language of publication' aspects. An evaluation of the geographical filter performance was done and sensitivity and specificity were calculated. There were obtained publications in several languages, originated in Croatia, published in Croatia and/or abroad. The Croatian SO in the field of PH-PM was obtained for the same period of time by combining search filters. The evaluation of the filter performance showed sensitivity 95.56% and specificity 100%. The filters constructed permitted the retrieval of the Croatian eight years research output. Increased tendency was observed in the global SO evolution and in the PH-PM area as well. The main languages of publication were English and Croatian. This study is a contribution to research in the field of scientific documentation and further analysis is recommended in constructing and developing search filters to retrieve and focus on specific information.     

The effects of alignment error and alignment filtering on the sitewise detection of positive selection

When detecting positive selection in proteins, the prevalence of errors resulting from misalignment and the ability of alignment filters to mitigate such errors are not well understood, but filters are commonly applied to try to avoid false positive results. Focusing on the sitewise detection of positive selection across a wide range of divergence levels and indel rates, we performed simulation experiments to quantify the false positives and false negatives introduced by alignment error and the ability of alignment filters to improve performance. We found that some aligners led to many false positives, whereas others resulted in very few. False negatives were a problem for all aligners, increasing with sequence divergence. Of the aligners tested, PRANK's codon-based alignments consistently performed the best and ClustalW performed the worst. Of the filters tested, GUIDANCE performed the best and Gblocks performed the worst. Although some filters showed good ability to reduce the error rates from ClustalW and MAFFT alignments, none were found to substantially improve the performance of PRANK alignments under most conditions. Our results revealed distinct trends in error rates and power levels for aligners and filters within a biologically plausible parameter space. With the best aligner, a low false positive rate was maintained even with extremely divergent indel-prone sequences. Controls using the true alignment and an optimal filtering method suggested that performance improvements could be gained by improving aligners or filters to reduce the prevalence of false negatives, especially at higher divergence levels and indel rates.     

Cell-mediated immunity to Friend virus-induced leukemia. I. Modification of 125IUdR release cytotoxicity assay for use with suspension target cells.

Cell-mediated cytotoxic reactivity of C57BL/6 mice against syngeneic FBL-3 cells, a Friend virus-induced leukemia, was measured by the 125IUdR release assay with tumor target cells in suspension. The tests could be performed either with Linbro tissue culture plates(16-mm wells) or with Microtest II plates (6-mm wells). The former could only be harvested manually; the latter could be harvested mechanically by an automatic harvesting apparatus which permitted larger scale tests. With either plate, it was found that careful preparation of the target cells and of the attacker cells has important effects on the results obtained. When performed under optimal test conditions, the 125IUdR assay can be used as a very simple, objective, and reproducible assay for testing cell-mediated cytotoxicity.     

Virus safety of plasma products using 20 nm instead of 15 nm filtration as virus removing step

During the manufacture of human plasma derivatives, a series of complementary measures are undertaken to prevent transmission of blood-borne viruses. Virus filtration using 15 nm (Planova15N) filters has successfully been implemented in manufacturing processes for various plasma derivatives primarily because virus filtration is a technique, mild for proteins, that can effectively remove even small non-lipid-enveloped viruses, such as HAV and parvovirus B19. However, the use of 15 nm filters has limitations with regard to protein capacity of the filters and the process flow, resulting in an expensive manufacturing step. Therefore, studies were performed to test whether the use of 20 nm (Planova20N) filters, having different characteristics compared to 15 nm filters, can be an alternative for the use of 15 nm filters.It is shown that 20 nm filtration can be an alternative for 15 nm filtration. However, the virus removal capacity of the 20 nm filters depends on the plasma product that is filtered. Therefore, an optimisation study must be performed with regard to process parameters such as pressure, pH and protein concentration for each plasma product. In this study, using optimised conditions, the virus removal capacity of 20 nm filters appears to be comparable or even better when compared to that of 15 nm filters.     

Connectivity matrix method for analyses of biological networks and its application to atom-level analysis of a model network of carbohydrate metabolism

An approach for analysis of biological networks is proposed. In this approach, named the connectivity matrix (CM) method, all the connectivities of interest are expressed in a matrix. Then, a variety of analyses are performed on GNU Octave or Matlab. Each node in the network is expressed as a row vector or numeral that carries information defining or characterising the node itself. Information about connectivity itself is also expressed as a row vector or numeral. Thus, connection of node n1 to node n2 through edge e is expressed as [n1, n2, e], a row vector formed by the combination of three row vectors or numerals, where n1, n2 and e indicate two different nodes and one connectivity, respectively. All the connectivities in any given network are expressed as a matrix, CM, each row of which corresponds to one connectivity. Using this CM method, intermetabolite atom-level connectivity is investigated in a model metabolic network composed of the reactions for glycolysis, oxidative decarboxylation of pyruvate, citric acid cycle, pentose phosphate pathway and gluconeogenesis.     

Enzyme-Linked Immunosorbent Assay of Substance P: A Study in the Eye

A solid phase enzyme-linked immunosorbent assay for quantitation of substance P is presented. The assay measures the capacity of soluble substance P to compete with the solid phase antigen for a limited quantity of specific substance P antibody. The solid-phase antigen consists of a synthetic substance P.poly-D-glutamic acid conjugate coated to polystyrene micro-ELISA plate wells. Soluble substance P and antibodies to substance P are first preincubated together and then added to the wells containing solid-phase antigen. Subsequently the wells are incubated with anti-antibodies conjugated to alkaline phosphatase. The wells are finally incubated with p-nitrophenyl phosphate an the absorbance is read in a spectrophotometer 16--24 hr after the start of the assay. The threshold for detection of substance P was 5--10 pg per well (0.25 ml). Substance P was extracted from rabbit eyes and the values obtained with the present method are compared with previously reported values based on radioimmunoassay.     

Filtering Decanting Water in Fly-Ash Lagoons Using Geotextiles — An Experimental Study

Disposal of fly-ash at thermal power stations is carried out in the form of ash slurry by mixing the ash with water. The ash slurry is stored in an ash pond. The water in the slurry is gradually drained out, through decanting wells, earthen embankments, and over spillways, leaving the ash to be deposited in the pond. It is observed that the design of decanting wells presently used allows a considerable amount of fly-ash to flow out with the water, leading to the contamination of soil and water courses. Further, it is observed that the failure of earthen embankments of ash ponds, which results in major damage to the environment, is mainly due to ineffective functioning of filters. This article presents the details of an experimental investigation carried out to study the role of geotextiles in improving the performance of decanting wells and different drainage systems in earthen embankments. Two series of experiments were carried out. In the first series a model-decanting well was tested with two different Geotextiles linings. In the second series, a number of model earthen embankments were tested with and without geotextiles. The economics of providing geotextiles in earthen embankments was studied. The results indicated that the use of Geotextiles improve the performance of decanting wells by reducing the amount of ash flowing into the decanting well and drainage system. Further, the use of Geotextiles has shown an improvement in the performance of earthen embankment used for ash ponds. The use of Geotextiles in earthen embankment is shown to be economical compared to the conventional drainage system.     

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.     

A Procedure for the Measurement of Hormone Receptors Using a Cell Harvesting Machine

Abstract

A procedure has been developed that will rapidly measure the binding of hormones and growth factors to suspensions of intact cells. Radiolabeled ligand is bound to cells in 96-well cluster plates, and the cells are then collected and washed on glass fiber filters using a cell harvester. Up to twelve wells can be processed simultaneously in less than a minute. This rapid washing and harvesting method is convenient to use with a large number of samples, and the amount of bound ligand is saturable and proportional to cell number. This procedure should be applicable to suspension cultures, loosely adhering monolayer cultures, and other cellular preparations that will bind to porous filters. Using this procedure, we have shown that HeLa S₃ cells grown in suspension cultures saturably bind to epidermal growth factor (EGF). The quantity of EGF bound to HeLa S₃ cells can be increased by adding glucocorticoids to the culture medium.     

Direction-specific recruitment of rotator cuff muscles during bench press and row

Recent studies indicate that rotator cuff (RC) muscles are recruited in a reciprocal, direction-specific pattern during shoulder flexion and extension exercises. The main purpose of this study was to determine if similar reciprocal RC recruitment occurs during bench press (flexion-like) and row (extension-like) exercises. In addition, shoulder muscle activity was comprehensively compared between bench press and flexion; row and extension; and bench press and row exercises. Electromyographic (EMG) activity was recorded from 9 shoulder muscles sites in 15 normal volunteers. All exercises were performed at 20, 50 and 70% of subjects’ maximal load. EMG data were normalized to standard maximal voluntary contractions. Infraspinatus activity was significantly higher than subscapularis during bench press, with the converse pattern during the row exercise. Significant differences in activity levels were found in pectoralis major, deltoid and trapezius between the bench press and flexion exercises and in lower trapezius between the row and extension exercises. During bench press and row exercises, the recruitment pattern in each active muscle did not vary with load. During bench press and row exercises, RC muscles contract in a reciprocal direction-specific manner in their role as shoulder joint dynamic stabilizers to counterbalance antero-posterior translation forces.     

Simple anthropometric and physical performance tests to predict maximal box-lifting ability

Box-lifting ability is an important characteristic of military personnel. The purpose of this paper was to determine the usefulness of the upright row free weight exercise and simple anthropometric tests to predict maximal box-lifting performance that simulates the loading of military supply vehicles. Two groups of adults performed maximal box lifts to 1.4 m (study 1) and 1.7 m (study 2), respectively. All subjects were also tested for upright row 1 repetition maximum (1RM) strength, body mass, height, and body composition. In study 1, a remarkably good prediction of maximal box-lift performance to 1.4 m (42 +/- 12 kg) was obtained from a regression equation including the variables body mass, body composition, and upright row 1RM. Approximately 95% of the variation in 1.4-m box-lifting performance could be accounted for. In contrast, in study 2, only 80% of the variation in 1.7-m box-lifting performance (51 +/- 15 kg) could be accounted for by the best predictor equation. Upright row 1RM strength appears to be a useful tool in the prediction of box-lifting ability to approximately chest height for most adults, probably due to a close match between the muscle groups and contraction modes required during both tasks. Military or other organizations could use the data reported here to substitute simple anthropometry and a 1RM test of strength and for the direct assessment of 1.4-m box-lifting performance.     

Efficacy of filter types for detecting Campylobacter jejuni and Campylobacter coli in environmental water samples by polymerase chain reaction.

A previously developed polymerase chain reaction (PCR) amplification of a target region in the flaA Campylobacter flagellin gene was evaluated and adapted for use with environmental water samples. The ability to detect Campylobacter jejuni or Campylobacter coli in seeded water samples was tested with various filters after concentration and freeze-thaw lysis of the bacterial cells. A nonradioactive probe for the amplified flagellin gene fragment detected as little as 1 to 10 fg of genomic DNA and as few as 10 to 100 viable C. jejuni cells per 100 ml of water filtered onto Fluoropore (Millipore Corp.) filters. No amplification was obtained with cellulose acetate filters, most likely because of binding of the DNA to the filter. Concentration and lysis of target cells on Fluoropore and Durapore (Millipore Corp.) filters allowed PCR to be performed in the same reaction tube without removing the filters. This methodology was then adapted for use with environmental water samples. The water supply to a broiler chicken production farm was suspected as the source of C. jejuni known to be endemic in grow-out flocks at the farm, despite the inability to culture the organisms by standard methods. The filtration-PCR method detected Campylobacter DNA in more than half of the farm water samples examined. Amplified campylobacter DNA was not detected in small volumes of regional surface water samples collected on a single occasion in February. The filtration-PCR amplification method provided a basis for detection of C. jejuni and C. coli in environmental waters with a high degree of specificity and sensitivity.     

Variables Influencing Extraction of Nucleic Acids from Microbial Plankton (Viruses,Bacteria, and Protists) Collected on Nanoporous Aluminum Oxide Filters

Anodic aluminum oxide (AAO) filters have high porosity and can be manufactured with a pore size that is small enough to quantitatively capture viruses. These properties make the filters potentially useful for harvesting total microbial communities from water samples for molecular analyses, but their performance for nucleic acid extraction has not been systematically or quantitatively evaluated. In this study, we characterized the flux of water through commercially produced nanoporous (0.02 μm) AAO filters (Anotop; Whatman) and used isolates (a virus, a bacterium, and a protist) and natural seawater samples to test variables that we expected would influence the efficiency with which nucleic acids are recovered from the filters. Extraction chemistry had a significant effect on DNA yield, and back flushing the filters during extraction was found to improve yields of high-molecular-weight DNA. Using the back-flush protocol, the mass of DNA recovered from microorganisms collected on AAO filters was ≥100% of that extracted from pellets of cells and viruses and 94% ± 9% of that obtained by direct extraction of a liquid bacterial culture. The latter is a minimum estimate of the relative recovery of microbial DNA, since liquid cultures include dissolved nucleic acids that are retained inefficiently by the filter. In conclusion, we demonstrate that nucleic acids can be extracted from microorganisms on AAO filters with an efficiency similar to that achievable by direct extraction of microbes in suspension or in pellets. These filters are therefore a convenient means by which to harvest total microbial communities from multiple aqueous samples in parallel for subsequent molecular analyses.     

Development and application of new positively charged filters for recovery of bacteriophages from water

Electronegative and electropositive filters were compared for the recovery of indigenous bacteriophages from water samples, using the VIRADEL technique. Fiber glass and diatomaceous earth filters displayed low adsorption and recovery, but an important increase of the adsorption percentage was observed when the filters were treated with cationic polymers (about 99% adsorption). A new methodology of virus elution was developed in this study, consisting of the slow passage of the eluent through the filter, thus increasing the contact time between eluent and virus adsorbed on the filters. The use of this technique allows a maximum recovery of 71.2% compared with 46.7% phage recovery obtained by the standard elution procedure. High percentages (over 83%) of phage adsorption were obtained with different filters from 1-liter aliquots of the samples, except for Virosorb 1-MDS filters (between 1.6 and 32% phage adsorption). Phage recovery by using the slow passing of the eluent depended on the filter type, with recovery ranging between 1.6% for Virosorb 1-MDS filters treated with polyethyleneimine and 103.2% for diatomaceous earth filters treated with 0.1% Nalco.     

Development and application of new positively charged filters for recovery of bacteriophages from water.

Electronegative and electropositive filters were compared for the recovery of indigenous bacteriophages from water samples, using the VIRADEL technique. Fiber glass and diatomaceous earth filters displayed low adsorption and recovery, but an important increase of the adsorption percentage was observed when the filters were treated with cationic polymers (about 99% adsorption). A new methodology of virus elution was developed in this study, consisting of the slow passage of the eluent through the filter, thus increasing the contact time between eluent and virus adsorbed on the filters. The use of this technique allows a maximum recovery of 71.2% compared with 46.7% phage recovery obtained by the standard elution procedure. High percentages (over 83%) of phage adsorption were obtained with different filters from 1-liter aliquots of the samples, except for Virosorb 1-MDS filters (between 1.6 and 32% phage adsorption). Phage recovery by using the slow passing of the eluent depended on the filter type, with recovery ranging between 1.6% for Virosorb 1-MDS filters treated with polyethyleneimine and 103.2% for diatomaceous earth filters treated with 0.1% Nalco.     

Changes in the transparency of dentin: computerized densitometric analysis

Computerized densitometry was utilized to carry out a quantitative and distributive study of dentin transparency in dental roots from different age individuals. By means of original software S.A.M. (Shape Analytical Morphometry) a densitometric reading of transilluminated whole dental root was performed and so obtained densitometric matrixes were worked for grey level classification and correspondent areas evaluation, false colour restitution, pseudotridimensional and row/column barr graphs, isodensity outlines and density inclination maps. Results are discussed for both age evaluation in forensic application and for altered dentin recognition in pathological conditions.     

Inheritance of kernel row number, a multicategorical threshold trait of maize ears

Information about the inheritance of threshold traits is scarce, especially in plants. We examined the genetic control of kernel row number in maize (Zea mays). Knowledge of this inheritance is especially important because it is a primary component of grain yield. This trait has a discontinuous distribution. Characters like these are conceptualized as threshold traits. Crosses were made between the inbred line Geneze 3 (G3) with many kernel rows and the inbreds Argentino IV (A4) and Dente de Cravo (DC), with fewer kernel rows. The F(1) and F(2) generations and the backcrosses BC(11) and BC(21) were obtained for the combinations G3 x A4 and G3 x DC. These populations were evaluated under field conditions, and the kernel row number was determined by direct counting of approximately 14, 140 and 75 ears for the F(1), F(2) and backcrosses, respectively. Genetic control was determined through estimates of generation means and variance analysis and was also performed by Wright's method for threshold traits. It was found that genetic control is predominantly due to additive alleles. The component a, was greater than zero, additive variance was positive and the variance of dominance did not differ from zero. In the F(2) generation, the range of the kernel row number was 10 to 28 in G3 x A4, while in G3 x DC it was 12 to 26. Inheritance of the number of kernel rows, estimated by the two methods, gave similar results. This correspondence is due to adjusting of the data to the normal distribution.