Selection of rhizobia for prairie legumes used in restoration and reconstruction programs in Minnesota

The inoculation of indigenous legumes used in revegetation and wetland reconstruction activities in Minnesota presents problems not found in more traditional seedings. Few studies have sought inoculant-quality rhizobia for the legumes involved or examined their interaction in an environment that involves a number of different legumes seeded simultaneously under initially adverse climatic conditions. In this note, we identify effective strains for Astragalus canadensis, Chamaecrista fasciculata, Dalea purpurea, Dalea candida, Desmodium canadense, and Lespedeza capitata. We are currently using these strains to examine the contribution of inoculated legumes to prairie productivity in Minnesota.     

Test and calibration processes for microcalorimeters, with special reference to heat conduction instruments used with aqueous systems

Calorimeters are normally calibrated by use of electrical heaters, but the calibration values derived may not always be representative for the chemical or biological process investigated. It is therefore important to have available chemical test processes by which the electrical calibration values can be controlled. In some cases, electrical calibration should be replaced by chemical calibrations. With particular reference to needs in work with thermophile heat conduction microcalorimeters, a number of test and calibration processes are presented. Some calibration problems for various types of reaction vessel are discussed.     

Model-based workflow for scale-up of process strategies developed in miniaturized bioreactor systems

Miniaturized bioreactor (MBR) systems are routinely used in the development of mammalian cell culture processes. However, scale-up of process strategies obtained in MBR- to larger scale is challenging due to mainly non-holistic scale-up approaches. In this study, a model-based workflow is introduced to quantify differences in the process dynamics between bioreactor scales and thus enable a more knowledge-driven scale-up. The workflow is applied to two case studies with antibody-producing Chinese hamster ovary cell lines. With the workflow, model parameter distributions are estimated first under consideration of experimental variability for different scales. Second, the obtained individual model parameter distributions are tested for statistical differences. In case of significant differences, model parametric distributions are transferred between the scales. In case study I, a fed-batch process in a microtiter plate (4 ml working volume) and lab-scale bioreactor (3750 ml working volume) was mathematically modeled and evaluated. No significant differences were identified for model parameter distributions reflecting process dynamics. Therefore, the microtiter plate can be applied as scale-down tool for the lab-scale bioreactor. In case study II, a fed-batch process in a 24-Deep-Well-Plate (2 ml working volume) and shake flask (40 ml working volume) with two feed media was investigated. Model parameter distributions showed significant differences. Thus, process strategies were mathematically transferred, and model predictions were simulated for a new shake flask culture setup and confirmed in validation experiments. Overall, the workflow enables a knowledge-driven evaluation of scale-up for a more efficient bioprocess design and optimization.     

Classification systems for discrete variables used in forensic anthropology

In recent years a number of papers have been presented on the usefulness of non-metric traits of the cranial and infracranial skeleton in order that one, some few, or a subsample of crania may be allocated to a pair, family, or larger group. These papers have explored (1) which traits should be used, (2) the theoretical implications of assignment, and (3) the methodology for making these assignments. This paper addresses itself to the theoretical implications of assignment and the methodology for making these assignments. Classification techniques based on the Bayes' theorem, weight of evidence procedures, linear discriminant functions, tally method and the Rubison procedure, were utilized in the first level of analysis. The result suggests that methodologies used must contain an accommodation for correlation between bilateral traits and that pairwise classification procedures are often more applicable than multiple classification procedures considering a large number of groups. The accuracy of various methods starts at slightly better than 50%, while the better methods produce results above the 90% level. Results further show that when acceptable assignments are made the theoretical implications of these assignments do not necessarily suggest the use of a particular methodology, and for ease of analysis the simplest methodology should be used.     

Safety and regulation of yeasts used for biocontrol or biopreservation in the food or feed chain

Yeasts have been important components of spontaneous fermentations in food and beverage processing for millennia. More recently, the potential of utilising antagonistic yeasts, e.g. Pichia anomala and Candida spp., for post-harvest biological control of spoilage fungi during storage of plant-derived produce (‘biopreservation’) has been clearly demonstrated. Although some yeast species are among the safest microorganisms known, several have been reported in opportunistic infections in humans, including P. anomala and bakers’ yeast, Saccharomyces cerevisiae. More research is needed about the dominant pathogenicity and virulence factors in opportunistic yeasts, and whether increased utilisation of biopreservative yeasts in general could contribute to an increased prevalence of yeast infections. The regulatory situation for yeasts used in post-harvest biocontrol is complex and the few products that have reached the market are mainly registered as biological pesticides. The qualified presumption of safety (QPS) approach to safety assessments of microorganisms intentionally added to food or feed, recently launched by the European Food Safety Authority, can lead to more efficient evaluations of new products containing microbial species with a sufficient body of knowledge or long-term experience on their safety. P. anomala is one of several yeast species that have been given QPS status, although the status is restricted to use of this yeast for enzyme and metabolite production purposes. With regard to authorisation of new biopreservative yeasts, we recommend that the possibility to regulate microorganisms for food biopreservation as food additives be considered.     

Recombinant DNA approaches for the development of metabolic systems used in in vitro toxicology.

In the past few years there has been considerable progress in the development of mammalian cell systems for use in genetic toxicology by the stable transfer of genes/cDNAs coding for drug metabolizing enzymes directly into the target cell. Alternative approaches have also been developed in which mammalian cells are transiently transfected with cDNAs coding for drug-metabolizing enzymes and S9 preparations expressing a single metabolizing enzyme isolated and used for metabolic activation. Progress in these areas is reviewed here and the relative merits of the different approaches are discussed. Work to date has focused primarily on the cytochrome P450 family of enzymes, although other enzyme systems involved in xenobiotic metabolism have been used. The central theme of this review is the transfer of genetic information to improve the metabolic capability of cell systems used in genetic toxicology. However, a basic philosophy of the review is that genetic manipulation of cultured mammalian cells has the potential for developing systems to be used to better understand chemically induced toxicological effects.     

Numerical simulation of cooling rates in vitrification systems used for oocyte cryopreservation

Oocyte cryopreservation is of key importance in the preservation and propagation of germplasm. Interest in oocyte cryopreservation has increased in recent years due to the application of assisted reproductive technologies in farm animals such as in vitro fertilization, nuclear transfer and the need for the establishment of ova/gene banks worldwide. However, the cryopreservation of the female gamete has been met with limited success mainly due to its small surface-area:volume ratio.In the past decade, several vitrification devices such as open pulled straws (OPS), fine and ultra fine pipette tips, nylon loops and polyethylene films have been introduced in order to manipulate minimal volumes and achieve high cooling rates. However, experimental comparison of cooling rates presents difficulties mainly because of the reduced size of these systems. To circumvent this limitation, a numerical simulation of cooling rates of various vitrification systems immersed in liquid nitrogen was conducted solving the non-stationary heat transfer partial differential equation using finite element method.Results indicate the nylon loop (Cryoloop®) is the most efficient heat transfer system analyzed, with a predicted cooling rate of 180,000 °C/min for an external heat transfer coefficient h = 1000 W/m² K when cooling from 20 to −130 °C; in contrast, the open pulled straw method (OPS) showed the lowest performance with a cooling rate of 5521 °C/min considering the same value of external heat transfer coefficient. Predicted cooling rates of Miniflex® and Cryotop® (polyethylene film system) were 6164 and 37,500 °C/min, respectively, for the same heat transfer coefficient.     

Polish pioneers in research on vertebrate circulatory systems at the Jagiellonian University of Krakow (Poland)

At the end of the XIX century and during the first half of the XX century, Henryk Hoyer and Zygmunt Grodzinski, two eminent Polish researchers, were carring out research on the lymphatic circulatory and blood circulatory systems. Later, these investigations were continued by their students and followers until 1966. Embryos from all groups of vertebrates were used for investigation and on that basis, models of the lymphatic and blood circulatory systems were proposed.     

Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media.

In this study, the quantitative fluctuation of cytoplasmic lipid droplets (LD) and cryotolerance were investigated in bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes developed in different culture systems using serum-free or serum-containing media. The serum-free cultures were grown using IVMD101 medium in conjunction with bovine cumulus/granulosa cell (BCGC) cocultures or IVD101 medium without BCGC cocultures, and the serum-containing cultures were grown in the presence of BCGC cocultures using HPM199 medium supplemented with 5% calf serum (HPM199 + CS). Large numbers of sudanophilic LD were present in the cytoplasm of bovine embryos from 2-cell to hatched blastocyst stages, and the number and size differed between the embryos cultured in serum-free and serum-supplemented media. In the embryos cultured in HPM199 + CS, large (2-6 microm in diameter) sudanophilic LD increased significantly from the morula to the blastocyst stages. Throughout the embryonic development, the embryos developed in serum-free cultures with and without BCGC cocultures had numerous sudanophilic LD, but most of these droplets were small (<2 microm in diameter) and large LD were less numerous than those embryos cultured in HPM199 + CS. Giant LD (>6 microm in diameter) were frequently observed in morulae and blastocysts (including early blastocysts) developed in HPM199 + CS. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of trophoblast and embryonic (inner cell mass) cells of blastocysts cultured in HPM199 + CS. These large LD were identified as osmophilic LD, an indication that these lipid inclusions contained a significant proportion of unsaturated lipids. Many elongated mitochondria were found in embryos developed in IVMD101 and IVD101 at the morula and early blastocyst stages, whereas many of the mitochondria in the morulae developed in HPM199 + CS were of an immature form such as spherical or ovoid shape. The survival and hatching rates of embryos (morulae, early blastocysts, and blastocysts) produced in serum-free media (both IVMD101 and IVD101) after post-thaw culture were superior to those of embryos produced in serum-containing medium. These results showed that bovine embryos cultured in serum-containing medium abnormally accumulated cytoplasmic lipids into their cytoplasm and the excess accumulation of cytoplasmic LD in embryos may affect the cryotolerance of embryos.