Methodological considerations for conducting tumor surveys of fishes

The usefulness of tumor surveys in indicating the availability of carcinogenic contaminants in ecosystems hinges largely on decisions made during the planning process. Species used as indicator organisms for monitoring programs are best selected from those known to be susceptible to epizootics of neoplasms. Lesions which can be quickly located and preserved in the field and for which a data base exists to help interpretation include liver neoplasms and external neoplasms. Since tumor frequencies increase with age and may change from season to season, studies should hold these variables constant among locations. During the design of the study investigators should determine the number of fish they need to examine in order to achieve the degree of resolution desired. Finally the field crew must be familiar with fish anatomy and necropsy techniques as well as storage and handling of the chosen preservative.     

A review and evaluation of study design considerations for site-specifically assessing the health of fish populations

Since there are not any well developed procedures for site-specifically evaluating the health of fish populations, most field surveillance programs have been restricted to collecting information on chemistry, toxicity, bioaccumulation, biochemical alterations orin situ benthic community structure. Identification of the mechanism and significance of contaminant effects on fish populations depends on the ability to identify changes and to distinguish changes in survival, food availability or food conversion efficiency, from coincidental changes associated with alterations in habitat or natural variability. Preliminary identification of the characteristics of adult fish can be used to cost-effectively focus financial resources on the alterations which are of relevance to interpretation of impacts and identification of causal factors. Proper interpretation requires that appropriate attention be given to monitoring level and strategy, selection of species and timing of sampling, sample size requirements and choice of reference site.     

Methodological considerations on the study of sexual dimorphism in past human populations

The degree of sexual dimorphism in human populations is influenced by stress, social role and by labour division. However, studies on ethnographic populations provided contradictory results. Unfortunately, most of these studies were based on stature only, which, as we could observe in a survey on pre-protohistoric circum-Medirerranean samples, is a poor indicator of functionally related dimorphism. A number of skeletal measurements were examined: skull, stature, transverse trunk diameters, long bones length, circumference and section, in order to assess their usefulness as indicators of functionally related dimorphism. The best indicators were represented by section and circumference of the long bones of the limbs, followed by cross-shoulder breadth (biclavicular length), stature and limb bone length, facial measurements, cranial measurements and sacral breadth. From the methodological point of view, it was found that:

1. It is better to calculate the index of dimorphism for each trait or set of traits within each sample. Then a weighted average of all the available samples is taken. The index derived from pooling a number of samples does not make biological sense. In pooled samples the distinction of between versus within sample differences is obscured;
2. It is better to combine an index which is based on the difference between averages and one which takes variability into account, because variability can also be an aspect of sexual dimorphism;
3. It is better to apply some allometric correction to the measurements used. For instance, the log transformation produces clearer trends of differential dimorphism among the various traits.

     

Pleiotropic functions of brain proteinases: Methodological considerations and search for caspase substrates

Analysis of the literature and our own data suggest that the so-called “apoptotic” proteinases play important roles in brain function. However, mechanisms of their involvement in normal neuronal plasticity remain obscure. One of the main reasons for this is broad substrate specificity of proteinases; the number of potential substrates of each can reach several thousands. Obviously, a real approach to study functions of “apoptotic” proteinases, caspase-3 in particular, is to identify their intracellular substrates. It is the nature of a substrate that defines the direction of signal transduction or metabolic changes; therefore, identification of molecular partners of particular proteases should be the key study, not just measuring its activity or respective protein or mRNA expression. This approach will allow studying regulatory mechanisms not only for proteinases, but also for other pleiotropic enzymes usually possessing broad substrate specificity.     

Methodological considerations for the human platelet 5-HT2A receptor binding kinetic assay

Analysis of an extensive database of human platelet 5-HT2A receptor binding assays has been conducted in order to identify factors that may affect the assay results. Despite anecdotal reports that storage of frozen platelet pellets may affect 5-HT2A binding affinity and capacity, no quantitative study has been reported in the literature. Analysis of binding data for 373 frozen samples with a storage time up to three years is presented in this paper. It is shown that prolonged storage significantly decreases binding. The loss of binding capacity begins in the first six month of storage. Bmax declines by half after 17 month. The impact of storage time on the binding affinity is much smaller. There is only about 20% increase in the value of affinity K(D) during the half-life of Bmax. Differences in sample storage time may partly explain discrepancies in results between different research groups. Nonspecific binding due to binding to filter material diminishes accuracy and reliability of the binding assays as a result of a decrease in the ratio of specific to nonspecific ratio. A data analysis based on our suggested mathematical model shows that this effect depends on tissue concentration in test tube and becomes pronounced when the concentration is below 0.1 mg protein/ml (at 0.2 nM of ligand). Above 0.1 mg protein/ml, percentage of specific to total binding exceeds 65%, which is an acceptable level for the ratio. The majority of the binding studies reported in the literature employed a tissue concentration more than 0.5 mg/ml, well above the minimal limit sufficient for a reliable assay. However, development of microassays to conserve precious tissue must take the limit into consideration.     

Reactions involving carbamyl phosphate in the presence of precipitated calcium phosphate with formation of pyrophosphate: A model for primitive energy-conservation pathways

The formation of carbamyl phosphate (CAP) in dilute solutions of cyanate (NCO^–) and orthophosphate (Pi) was measured both in the absence and in the presence of a precipitated matrix of calcium phosphate (Pi.Ca). The second-order rate constant and the free energy of CAP synthesis were not modified by the presence of the solid matrix, indicating that synthesis occurs in the homogeneous Pi-containing solution. The elimination reaction of CAP to form NCO^– and Pi followed first-order kinetics and the rate constant was the same whether or not calcium phosphate was present. Elimination was not complete, and the steady level of remaining CAP was that expected from the free energy of synthesis. The formation of pyrophosphate (PPi) was detected in CAP-containing medium only in the presence of calcium, showing a close correlation with the amount of precipitated Pi.Ca. Phosphorolysis of CAP followed a sigmoidal time course, compatible with adsorption of CAP to the solid matrix as a prelude to transphosphorylation. Addition of 5-AMP and of short linear polyphosphates inhibited phosphorolysis of CAP. It is proposed that the presence of a solid phosphate matrix and the relative concentrations of cyano compounds, as well as those of nucleotides and inorganic polyphosphates, could have played a crucial role in the conservation of chemical energy of CAP and in its use in prebiotic phosphorylation reactions.     

Orbital floor reconstruction using calcium phosphate cement paste: an animal study

Orbital floor defects were created in 10 New Zealand white rabbits and were reconstructed using an injectable calcium phosphate paste. These animals were euthanized at 2, 4, 8, and 12 months after implantation and were examined for biocompatibility and osteoconductivity. Grossly, implants were found to be adherent to the floor and covered with fibrous tissues. There was no sign of infection, extrusion, or migration of implant within the orbit and maxilla. The orbital floor was completely restored. Histological examination showed active new bone formation that encroached within the implant and gradually increased in density with time. Maxillary mucosa and glands were likewise reconstituted. Thin fibrovascular tissues were seen on top of and within the surface of the implant, and few to slight inflammatory cells were seen. Microradiography showed direct apposition between the new bone and the implant. These findings compare favorably with previously published reports on the biocompatibility and osteoconductivity of calcium phosphate cement. The authors believe that, together with ease of use and structural integrity, calcium phosphate paste can be useful in orbital floor reconstruction.     

Methodological and empirical considerations when assessing freshwater ecosystem service provision in a developing city context: Making the best of what we have

This study contributes to both the methodological and empirical literature by developing an integrative approach to assessing temporal and spatial change in riparian ecosystem service delivery by drawing on available and diverse data sets. These data sets act as multiple lines of evidence in supporting comparisons between data sets to test the validity of developed methods and the application of such methods. In order to synthesise these data as well as to determine the fluctuations in riparian ecosystem service provision a scoring system was developed. Methodologically, the scoring system proved informative across the majority of ecosystem services categories, showing close to 80% similarity in outcomes when comparing the scoring method to trends in long-term water quality measurements. Other benefits of the scoring system included its design simplicity, cost-effectiveness, and applicability and replicability across various urban settings. Empirically, the data sets used support the findings of the ecosystem services scoring exercise and suggests that fluctuations in ecosystem service delivery through time and across the river reaches are linked to land-use change and other human activities. Findings suggest that as water leaves an urban protected area and travels across transformed and impacted landscapes, the results are poor water quality and diminished ability of rivers to yield ecosystem services the further the river flows into the urban setting. Urbanisation and changes in land-uses in developing city contexts is therefore shown to affect potential ecosystem services benefits, necessitating increasing management interventions.     

Methodological challenges in assessing the environmental status of a marine ecosystem: case study of the Baltic Sea

Assessments of the environmental status of marine ecosystems are increasingly needed to inform management decisions and regulate human pressures to meet the objectives of environmental policies. This paper addresses some generic methodological challenges and related uncertainties involved in marine ecosystem assessment, using the central Baltic Sea as a case study. The objectives of good environmental status of the Baltic Sea are largely focusing on biodiversity, eutrophication and hazardous substances. In this paper, we conduct comparative evaluations of the status of these three segments, by applying different methodological approaches. Our analyses indicate that the assessment results are sensitive to a selection of indicators for ecological quality objectives that are affected by a broad spectrum of human activities and natural processes (biodiversity), less so for objectives that are influenced by a relatively narrow array of drivers (eutrophications, hazardous substances). The choice of indicator aggregation rule appeared to be of essential importance for assessment results for all three segments, whereas the hierarchical structure of indicators had only a minor influence. Trend-based assessment was shown to be a useful supplement to reference-based evaluation, being independent of the problems related to defining reference values and indicator aggregation methodologies. Results of this study will help in setting priorities for future efforts to improve environmental assessments in the Baltic Sea and elsewhere, and to ensure the transparency of the assessment procedure.     

Methodological considerations for determining cleanup limits for uranium in treated and untreated soils

Uranium‐contaminated soils are present at various locations across the U.S. where uranium was processed for nuclear fuels or atomic weapons. Important issues in dealing with such contamination include the assessment of the potential health risks associated with human exposures to the residual uranium and the determination of safe levels of U in soils that have been treated by a given technology. This paper reviews pertinent aspects of the health risks posed by uranium in soils and discusses various methodological considerations that must be dealt with in developing cleanup limits for U in treated soils. Of special concern is the development of remediation limits that are closely tied to a set of monitoring requirements for determining compliance with derived limits. Key issues addressed include characterization of the bioavailability of uranium compounds in food and water, determination of a safe level of uranium in kidney tissue, estimation of the health risks associated with the uranium daughter products radium and radon, assessment of the potential for ground‐water contamination, biogeochemical characterization of soil‐treatment processes, and specification of appropriate monitoring and statistical protocols for analyzing treated and untreated soils.     

A comparison of calcium phosphate coprecipitation and electroporation

Plasmid-based transfection assays provide a rapid means to measure homologous and nonhomologous recombination in mammalian cells. Often it is of interest to examine the stimulation of recombination by DNA damage induced by radiation, genotoxic chemicals, or nucleases. Transfection is frequently performed by using calcium phosphate coprecipitation (CPP), because this method is well suited for handling large sample sets, and it does not require expensive reagents or equipment. Alternative transfection methods include lipofection, microinjection, and electroporation. Since DNA strand breaks are known to stimulate both homologous and nonhomologous recombination, the induction of nonspecific damage during transfection would increase background recombination levels and thereby reduce the sensitivity of assays designed to detect the stimulation of recombination by experimentally induced DNA damage. In this article, we compare the stimulatory effects of nuclease-induced double-strand breaks (DSBs) on homologous and nonhomologous recombination for molecules transfected by CPP and by electroporation. Although electroporation yielded fewer transfectants, both nonhomologous and homologous recombination were stimulated by nuclease-induced DSBs to a greater degree than with CPP. Ionizing radiation is an effective agent for inducing DNA strand breaks, but previous studies using CPP generally showed little or no stimulation of homologous recombination among plasmids damaged with ionizing radiation. By contrast, we found clear dose-dependent enhancement of recombination with irradiated plasmids transfected using electroporation. Thus, electroporation provides a higher signal-to-noise ratio for transfection-based studies of damage-induced recombination, possibly reflecting less nonspecific damage to plasmid DNA during transfection of mammalian cells.     

Bone regeneration: molecular and cellular interactions with calcium phosphate ceramics

Calcium phosphate bioceramics are widely used in orthopedic and dental applications and porous scaffolds made of them are serious candidates in the field of bone tissue engineering. They have superior properties for the stimulation of bone formation and bone bonding, both related to the specific interactions of their surface with the extracellular fluids and cells, ie, ionic exchanges, superficial molecular rearrangement and cellular activity.     

Novel autophagy modulators: Design and synthesis of (+)-epogymnolactam analogues and structure-activity relationship

(+)-Epogymnolactam (1) was discovered as a novel autophagy inducer from a culture of Gymnopus sp. in our laboratory. To determine structure-activity relationships among (+)-epogymnolactam analogues comparing with cerulenin (2), we synthesized 5 analogues including (?)-epogymnolactam (3) having each different functional group, and 3 analogues with different side-chain lengths. Five analogues, 3, 4, 5, 6, and 7 did not significantly increase the ratio of LC3-II to LC3-I as an autophagy marker in NIH3T3 cells. These results suggest that presence and stereochemistry of (2R,3S)-epoxy group and cyclic syn-form (1b) of 1 are important for the activity as autophagy inducer. Hexyl analogue (8) as well as 1 having butyl side-chain dose-dependently increased the ratio of LC3-II to LC3-I, whereas octyl analogue (9) and 2 rather decreased the ratio. Decyl analogue (10) did not give a change in the ratio. Although 8 seemed to be an excellent autophagy inducer, it dose-dependently increased SQSTM1 (p62) as in the case of 2, whereas 1 showed a slight dose-dependent decrease of p62 as an index of autophagic protein degradation. These observations suggest that 8 is an autophagy modulator with different molecular target from 1 or 2.     

A new genetic model for calcium induced autophagy and ER-stress in Drosophila photoreceptor cells

Cytoplasmic Ca²⁺ overload is known to trigger autophagy and ER-stress. Furthermore, ER-stress and autophagy are commonly associated with degenerative pathologies, but their role in disease progression is still a matter of debate, in part, owing to limitations of existing animal model systems. The Drosophila eye is a widely used model system for studying neurodegenerative pathologies. Recently, we characterized the Drosophila protein, Calphotin, as a cytosolic immobile Ca²⁺ buffer, which participates in Ca²⁺ homeostasis in Drosophila photoreceptor cells. Exposure of calphotin hypomorph flies to continuous illumination, which induces Ca²⁺ influx into photoreceptor cells, resulted in severe Ca²⁺-dependent degeneration. Here we show that this degeneration is autophagy and ER-stress related. Our studies thus provide a new model in which genetic manipulations trigger changes in cellular Ca²⁺ distribution. This model constitutes a framework for further investigations into the link between cytosolic Ca²⁺, ER-stress and autophagy in human disorders and diseases.     

Biomimetic calcium phosphate mineralization with multifunctional elastin-like recombinamers

Biomimetic hybrid materials based on a polymeric and an inorganic component such as calcium phosphate are potentially useful for bone repair. The current study reports on a new approach toward biomimetic hybrid materials using a set of recombinamers (recombinant protein materials obtained from a synthetic gene) as crystallization additive for calcium phosphate. The recombinamers contain elements from elastin, an elastic structural protein, and statherin, a salivary protein. Via genetic engineering, the basic elastin sequence was modified with the SN(A)15 domain of statherin, whose interaction with calcium phosphate is well-established. These new materials retain the biocompatibility, "smart" nature, and desired mechanical behavior of the elastin-like recombinamer (ELR) family. Mineralization in simulated body fluid (SBF) in the presence of these recombinamers reveals surprising differences. Two of the polymers inhibit calcium phosphate deposition (although they contain the statherin segment). In contrast, the third polymer, which has a triblock structure, efficiently controls the calcium phosphate formation, yielding spherical hydroxyapatite (HAP) nanoparticles with diameters from 1 to 3 nm after 1 week in SBF at 37 °C. However, at lower temperatures, no precipitation is observed with any of the polymers. The data thus suggest that the molecular design of ELRs containing statherin segments and the selection of an appropriate polymer structure are key parameters to obtain functional materials for the development of intelligent systems for hard tissue engineering and subsequent in vivo applications.