Subjects and Objects: Metaphysics, Biology, Consciousness, and Cognition

Over the past half-century, the Freeman laboratory has accumulated a large volume of data and a correspondingly extensive interpretive framework centered around an alternative perspective on brain function, that of dynamical systems. The purpose of this paper is first briefly to summarise this work, and bring it into dialogue with other perspectives. The contents of consciousness are seen as an inevitably sparse sample of events in the perception–action cycle. The paper proceeds to an attempt to elucidate the contents of this sparse sample. A critical concept is that of selfhood, and how it presents itself phenomenologically. It is argued that our experience of selfhood is an artifact of the necessity of preservation of subject/object relations. As this part of metaphysics has been badly misunderstood, we scrutinise it at some length in quantum mechanics, biology, and cognition. It is argued that Pattee's approach, while valuable, tends to premature closure on these issues. The paper then proceeds to outline a view on how selfhood is best considered wrt the immune response. This is interrelated with phenomenology, and some data are adduced to support this central hypothesis. Finally, consequences for the social sciences in general are hinted at.     

Similarity indices,sample size and diversity

Summary The effect of sample size and species diversity on a variety of similarity indices is explored. Real values of a similarity index must be evaluated relative to the expected maximum value of that index, which is the value obtained for samples randomly drawn from the same universe, with the diversity and sample sizes of the real samples. It is shown that these expected maxima differ from the theoretical maxima, the values obtained for two identical samples, and that the relationship between expected and theoretical maxima depends on sample size and on species diversity in all cases, without exception. In all cases but one (the Morisita index) the expected maxima depend strongly to fairly strongly on sample size and diversity. For some of the more useful indices empirical equations are given to calculate the expected maximum value of the indices to which the observed values can be related at any combination of sample sizes. It is recommended that the Morisita index be used whenever possible to avoid the complex dealings with effects of sample size and diversity; however, when previous logarithmic transformation of the data is required, which often may be the case, the Morisita-Horn or the Renkonen indices are recommended.     

Multiplex PCR and a chromogenic DNA macroarray for the detection of Listeria monocytogens, Staphylococcus aureus, Streptococcus agalactiae, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio parahaemolyticus, Salmonella spp. and Pseudomonas fluorescens in milk and meat samples

Food products, such as milk and meat products including cheese, milk powder, fermented milk, sausage, etc. are susceptible to the contamination by pathogenic and deteriorative bacteria. These bacteria include Listeria monocytogens, Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7, Salmonella spp., Vibrio parahaemolyticus, Streptococcus agalactiae and Pseudomonas fluorescens, etc. Traditional methods for the detection of these microorganisms are laborious and time consuming. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and PCR primers for the detection of aforementioned microorganisms. By using two sets of multiplex PCR, followed by a chromogenic macroarray system, these organisms in milk or other food products could be simultaneously detected. When the system was used for the inspection of milk or meat homogenate containing 10(0) target cells per milliliter or gram of the sample, all these bacterial species could be identified after an 8h pre-enrichment step. The system consisting of a multiplex PCR step followed by macroarray allowed us to detect multiple target bacterial species simultaneously without the use of agarose gel electrophoresis. Compared to the commonly used multiplex PCR method, this approach has the additional advantage of detecting more bacterial strains because some bacterial strains generate PCR products with the same molecular sizes which can be differentiated by macroarray but not by electrophoresis.     

Sampling effort, regression method, and the shape and slope of size–abundance relations

1. Despite a substantial body of work there remains much disagreement about the form of the relationship between organism abundance and body size. In an attempt at resolving these disagreements the shape and slope of samples from simulated and real abundance–mass distributions were assessed by ordinary least squares regression (OLS) and the reduced major axis method (RMA).
2. It is suggested that the data gathered by ecologists to assess these relationships are usually truncated in respect of density. Under these conditions RMA gives slope estimates which are consistently closer to the true slopes than OLS regression.
3. The triangular relationships reported by some workers are found over smaller mass and abundance ranges than linear relations. Scatter in slope estimates is much greater and positive slopes more common at small sample sizes and sample ranges. These results support the notion that inadequate and truncated sampling is responsible for much of the disagreement reported in the literature.
4. The results strongly support the notion that density declines with increasing body mass in a broad, linear band with a slope around −1. However there is some evidence to suggest that this overall relation results from a series of component relations with slopes which differ from the overall slope.     

A thermoelectric, heat-flux-controlled cooling stage for measuring freezing parameters of small plants

A new cooling system was developed which permits the accurate recording of the freezing temperatures of small samples which contain less than 1 mg of water. The cooling system consists of a thermoelectric cooling stage with automatically controlled heat exchange between sample and coolant and of a sample-temperature recording unit. The heat exchange controller keeps the latent heat dissipation small during freezing, thus it is also possible to record an accurate freezing plateau in small samples like buds, leaves, conifer needles, short apices, and roots as well as small tissue samples like leaf disks, isolated bark, cambium or wood.Construction and performance of the system are discussed. Results of cooling experiments are presented to illustrate the system's performance as well as some of the problems encountered with test material of varying sizes and water contents. Based upon this experience we may recommend the use of this system for various cryobiological investigations.     

Fluorometric assay for urea in urine, plasma, and tubular fluid

The variation in the sensitivity of proteins to some commonly used protein assay procedures was estimated by a calculation of the ideal titer per unit weight of protein for a sample of 350 proteins. On the basis of such calculations, the variation expected of nitrogen-based assay procedures is expected to be small, and such procedures are considered to give a more consistent quantification of a variety of proteins than other commonly used assay procedure.     

Growth and secular trend in school-children from Cento, Ferrara, Italy

Growth parameters were surveyed in a sample of 296 Italian children, 6-9 years old, from Cento (Ferrara, Emilia-Romagna). The comparison with children from the same town measured in 1974-75 show changes in some parameters, suggesting an ongoing secular trend. To better understand the observed weight increase and the sex difference, we also evaluated body composition and motricity. The analysis of the present sample is a preliminary part of a longitudinal study dealing with modifications of body composition and motor capacity induced by growth. In our sample the children are growing according to the Italian reference standard. The females present weight, height and Body Mass Index (BMI) values comparable to the 50th centile, while the males present higher values of weight, skinfold thicknesses and BMI. Sex differences in the motor performance were noted. A methodological comparison of obesity assessments based on BMI and percentage of body fat (%F) shows similar conclusions but somewhat different results.     

Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry

Sphingolipids are a highly diverse category of compounds that serve not only as components of biologic structures but also as regulators of numerous cell functions. Because so many of the sphingolipids in a biological system are bioactive and are often closely related structurally and metabolically (for example, complex sphingolipids<-->ceramide<-->sphingosine<-->sphingosine 1-phosphate), to understand the role(s) of sphingolipids in a given context one must conduct a "sphingolipidomic" analysis-i.e., a structure-specific and quantitative measurement of all of these compounds, or at least all members of a critical subset. Liquid chromatography tandem mass spectrometry (LC MS/MS) is currently the only technology with the requisite structural specificity, sensitivity, quantitative precision, and relatively high-throughput capabilities for such analyses in small samples ( approximately 10(6) cells). This review describes a series of protocols that have been developed for the relatively rapid analysis of all of the molecular species from 3-ketosphinganines through sphingomyelins and some glycosphingolipids (including all the compounds that are presently regarded as sphingolipid "second messengers") using normal- and reverse-phase LC to separate isometric and isobaric species (such as glucosylceramides and galactosylceramides) in combination with triple quadrupole (for MS/MS) and hybrid quadrupole-ion trap (for MS3) mass spectrometry. Also discussed are some of the issues remaining to be resolved in the analysis of the full sphingolipidome.     

Estimating the number,frequency, and dominance of S-alleles in a natural population of Arabidopsis lyrata(Brassicaceae) with sporophytic control of self-incompatibility

In homomorphic plant self-incompatibility (SI) systems, large numbers of alleles may be maintained at a single Mendelian locus. Most estimators of the number of alleles present in natural populations are designed for gametophytic self-incompatibility systems (GSI) in which the recognition phenotype of the pollen is determined by its own haploid genotype. In sporophytic systems (SSI), the recognition phenotype of the pollen is determined by the diploid genotype of its parent, and dominance differs among alleles. We describe research aimed at estimates of S-allele numbers in a natural population of Arabidopsis lyrata (Brassicaceae), whose SSI system has recently been described. Using a combination of pollination studies and PCR-based identification of alleles at a locus equivalent to the Brassica SRK gene, we identified and sequenced 11 putative alleles in a sample of 20 individuals from different maternal seed sets. The pollination results indicate that we have not amplified all alleles that must be present. Extensive partial incompatibility, nonreciprocal compatibility differences, and evidence of weakened expression of SI in some genotypes, prevent us from determining the exact number of missing alleles based only on cross-pollination data. Although we show that none of the theoretical models currently proposed is completely appropriate for estimating the number of alleles in this system, we estimate that there are between 13 and 16 different S-alleles in our sample, probably between 16 and 25 alleles in the population, and discuss the relative frequency of alleles in relation to dominance.     

ATP, ADP, and magnesium mixed solutions: in vitro 31P NMR characterization and in vivo application to lens

The difference between the 31P NMR resonance position of the beta-peak of ADP and the gamma-peak of ATP at a fixed monovalent ion concentration level and pH is shown in vitro to depend only on free magnesium concentration. This difference can vary by more than 1 ppm depending on the pH of the solution and free magnesium concentration. Using 31P NMR and ion-selective electrodes we have constructed experimental curves to show how calibration of experimental results can be achieved. Theoretical calculations can be used to predict the functional dependence of the chemical shift difference on the above parameters. However, using the reported ion dissociation constants the fit was not exact and the discrepancy increased at higher pH values. To demonstrate that this technique can yield valid in vivo results and to analyze a previously unreported system where free magnesium levels vary, sample spectra from lenses and enucleated eyes are given. The data show a disagreement between the present methods and the more conventional ATP method. The reason for this difference is not known although some possible reasons are suggested.     

ABO segregation distortion in Visakhapatnam, India.

Distortions in mother-infant, mother-child and father-child segregation for the ABO system as well as in the sex of offspring are described in a sample from a maternity service and from families of Visakhapatnam, India. Some distortions follow the expected fetomaternal incompatibility depression, others the expected feto-maternal induction of tolerance, and some remain unexplained. A differential action of selective factors on male and female fetuses, infants and children was also found, but no hypothesis could be postulated to explain it. The mother-infant matrix was found to be different from the mother-child matrix probably due to the inclusion of the reproductive time only in the mother-infant matrix. Unexpectedly, father-child segregation distortions were also found.     

Extremely acidic, pendulous cave wall biofilms from the Frasassi cave system, Italy

The sulfide-rich Frasassi cave system hosts an aphotic, subsurface microbial ecosystem including extremely acidic (pH 0-1), viscous biofilms (snottites) hanging from the cave walls. We investigated the diversity and population structure of snottites from three locations in the cave system using full cycle rRNA methods and culturing. The snottites were composed primarily of bacteria related to Acidithiobacillus species. Other populations present in the snottites included Thermoplasmata group archaea, bacteria related to Sulfobacillus, Acidimicrobium, and the proposed bacterial lineage TM6, protists, and filamentous fungi. Based on fluorescence in situ hybridization population counts, Acidithiobacillus are key members of the snottite communities, accompanied in some cases by smaller numbers of archaea related to Ferroplasma and other Thermoplasmata. Diversity estimates show that the Frasassi snottites are among the lowest-diversity natural microbial communities known, with one to six prokaryotic phylotypes observed depending on the sample. This study represents the first in-depth molecular survey of cave snottite microbial diversity and population structure, and contributes to understanding of rapid limestone dissolution and cave formation by microbially mediated sulfuric acid speleogenesis.     

Separation, Identification, and Characterization of Microorganisms by Capillary Electrophoresis

The use of capillary electrophoresis (CE) for the analysis, identification, and characterization of microorganisms has been gaining in popularity. The advantages of CE, such as small sample requirements, minimal sample preparation, rapid and simultaneous analysis, ease of quantitation and identification, and viability assessment, make it an attractive technique for the analysis of microbial analytes. As this instrumental method has evolved, higher peak efficiencies have been achieved by optimizing CE conditions, such as pH, ionic strength, and polymer additive concentration. Experimental improvements have allowed better quantitation and more accurate results. Many practical applications of this technique have been investigated. Viability and identification of microbes can be accomplished in a single analysis. This is useful for evaluation of microbial analytes in consumer products. Diagnosis of microbe-based diseases is now possible, in some cases, without the need for culture methods. Microbe-molecule, virus-antibody, or bacteria-antibiotic interactions can be monitored using CE, allowing for the screening of possible drug candidates. Fermentation can be monitored using this system. This instrumental approach can be adapted to many different applications, including assessing the viability of sperm cells. Progress has been made in the development of microelectrophoresis instrumentation. These advances will eventually allow the development of small, dedicated devices for the rapid, repetitive analyses of specific microbial samples. Although these methods may never fully replace traditional approaches, they are proving to be a valuable addition to the collection of techniques used to analyze, quantitate, and characterize microbes. This review outlines the recent developments in this rapidly growing field.     

Bio-banking in microbiology: from sample collection to epidemiology, diagnosis and research

Millions of biological samples, including cells of human, animal or bacterial origin, viruses, serum/plasma or DNA/RNA, are stored every year throughout the world for diagnostics and research. The purpose of this review is to summarize the resources necessary to set up a bio-banking facility, the challenges and pitfalls of sample collection, and the most important techniques for separation and storage of samples. Biological samples can be stored for up to 30 years, but specific protocols are required to reduce the damage induced by preservation techniques. Software dedicated to biological banks facilitate sample registration and identification, the cataloguing of sample properties (type of sample/specimen, associated diseases and/or therapeutic protocols, environmental information, etc.), sample tracking, quality assurance and specimen availability. Bio-bank facilities must adopt good laboratory practices and a stringent quality control system and, when required, comply with ethical issues.     

Corridors for migration between large subdivided populations, and the structured coalescent

We study the ancestral genetic process for samples from two large, subdivided populations that are connected by migration to, from, and within a small set of subpopulations, or demes. We consider convergence to an ancestral limit process as the numbers of demes in the two large, subdivided populations tend to infinity. We show that the ancestral limit process for a sample includes a recent instantaneous adjustment to the sample size and structure followed by a more ancient process that is identical to the usual structured coalescent, but with different scaled parameters. This justifies the application of a modified structured coalescent to some hierarchically structured populations.     

A new transparent handy sampler for collecting water from lakes,rivers and seas

A handy sampler for collecting water from lakes, rivers and seas is described. The sampler consists of an exchangeable sample tube and a new sample-holding system. The system comprises an upper disk, a lower disk, a pair of arms with a guide pin and a semicircular arm guide with a groove. The water sample collected into the acryl tube is held without flow by a tight tube-closing system. Contamination of the sample by chemical and biological materials from the sample tube is minimized by exchanging the tube with a clean one at each sampling station. This new sampler is light, simple and useful for sampling of water from surface to bottom.     

Limit of blank, limit of detection and limit of quantitation

* Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) are terms used to describe the smallest concentration of a measurand that can be reliably measured by an analytical procedure. * LoB is the highest apparent analyte concentration expected to be found when replicates of a blank sample containing no analyte are tested. LoB = mean(blank) + 1.645(SD(blank)). * LoD is the lowest analyte concentration likely to be reliably distinguished from the LoB and at which detection is feasible. LoD is determined by utilising both the measured LoB and test replicates of a sample known to contain a low concentration of analyte. * LoD = LoB + 1.645(SD (low concentration sample)). * LoQ is the lowest concentration at which the analyte can not only be reliably detected but at which some predefined goals for bias and imprecision are met. The LoQ may be equivalent to the LoD or it could be at a much higher concentration.     

Simple, defensible sample sizes based on cost efficiency

Summary .   The conventional approach of choosing sample size to provide 80% or greater power ignores the cost implications of different sample size choices. Costs, however, are often impossible for investigators and funders to ignore in actual practice. Here, we propose and justify a new approach for choosing sample size based on cost efficiency, the ratio of a study's projected scientific and/or practical value to its total cost. By showing that a study's projected value exhibits diminishing marginal returns as a function of increasing sample size for a wide variety of definitions of study value, we are able to develop two simple choices that can be defended as more cost efficient than any larger sample size. The first is to choose the sample size that minimizes the average cost per subject. The second is to choose sample size to minimize total cost divided by the square root of sample size. This latter method is theoretically more justifiable for innovative studies, but also performs reasonably well and has some justification in other cases. For example, if projected study value is assumed to be proportional to power at a specific alternative and total cost is a linear function of sample size, then this approach is guaranteed either to produce more than 90% power or to be more cost efficient than any sample size that does. These methods are easy to implement, based on reliable inputs, and well justified, so they should be regarded as acceptable alternatives to current conventional approaches.     

Corridors for migration between large subdivided populations,and the structured coalescent

We study the ancestral genetic process for samples from two large, subdivided populations that are connected by migration to, from, and within a small set of subpopulations, or demes. We consider convergence to an ancestral limit process as the numbers of demes in the two large, subdivided populations tend to infinity. We show that the ancestral limit process for a sample includes a recent instantaneous adjustment to the sample size and structure followed by a more ancient process that is identical to the usual structured coalescent, but with different scaled parameters. This justifies the application of a modified structured coalescent to some hierarchically structured populations.     

Separation, identification, and characterization of microorganisms by capillary electrophoresis.

The use of capillary electrophoresis (CE) for the analysis, identification, and characterization of microorganisms has been gaining in popularity. The advantages of CE, such as small sample requirements, minimal sample preparation, rapid and simultaneous analysis, ease of quantitation and identification, and viability assessment, make it an attractive technique for the analysis of microbial analytes. As this instrumental method has evolved, higher peak efficiencies have been achieved by optimizing CE conditions, such as pH, ionic strength, and polymer additive concentration. Experimental improvements have allowed better quantitation and more accurate results. Many practical applications of this technique have been investigated. Viability and identification of microbes can be accomplished in a single analysis. This is useful for evaluation of microbial analytes in consumer products. Diagnosis of microbe-based diseases is now possible, in some cases, without the need for culture methods. Microbe-molecule, virus-antibody, or bacteria-antibiotic interactions can be monitored using CE, allowing for the screening of possible drug candidates. Fermentation can be monitored using this system. This instrumental approach can be adapted to many different applications, including assessing the viability of sperm cells. Progress has been made in the development of microelectrophoresis instrumentation. These advances will eventually allow the development of small, dedicated devices for the rapid, repetitive analyses of specific microbial samples. Although these methods may never fully replace traditional approaches, they are proving to be a valuable addition to the collection of techniques used to analyze, quantitate, and characterize microbes. This review outlines the recent developments in this rapidly growing field.