In situ nick translation at the electron microscopic level: a tool for studying the location of DNAse I-sensitive regions within the cell

The in situ nick translation method was adapted to the ultrastructural level, to study the location of DNAse I-sensitive sequences within the cell. Ultra-thin sections of Lowicryl-embedded cells were incubated in a medium containing DNAse I, DNA polymerase I, and all four deoxyribonucleotides, some being biotinylated. The nick-translated sites were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern is closely dependent on the DNAse I concentration in the nick-translation medium. The method reveals with great precision the specific DNAse I-sensitive regions within the nucleus. This technique can be used to discriminate between active and inactive regions of interphase chromatin.     

Advanced technologies for studying circulating tumor cells at the protein level

Metastasis is the main cause of cancer death. As the tumor progresses, cells from the primary tumor site are shed into the bloodstream as circulating tumor cells (CTCs). Eventually, these cells colonize other organs and form distant metastases. It is therefore imperative that we gain a better understanding of the biological characteristics of CTCs for development of novel treatment modalities to minimize metastasis-associated cancer deaths. In recent years, rapid developments in technologies for the study of CTCs have taken place. We now have a variety of tools for the isolation and examination of CTCs which were not available before. This review introduces some commonly used protein markers in CTC investigations and summarizes a few advanced technologies which have been successfully applied for studying CTC biology at the protein level.     

Methodology to determine failure characteristics of planar soft tissues using a dynamic tensile test

Predicting the injury risk in automotive collisions requires accurate knowledge of human tissues, more particularly their mechanical properties under dynamic loadings. The present methodology aims to determine the failure characteristics of planar soft tissues such as skin, hollow organs and large vessel walls. This consists of a dynamic tensile test, which implies high-testing velocities close to those in automotive collisions. To proceed, I-shaped tissue samples are subjected to dynamic tensile tests using a customized tensile device based on the drop test principle. Data acquisition has especially been adapted to heterogeneous and soft biological tissues given that standard measurement systems (considered to be global) have been completed with a non-contact and full-field strain measurement (considered to be local). This local measurement technique, called the Image Correlation Method (ICM) provides an accurate strain analysis by revealing strain concentrations and avoids damaging the tissue. The methodology has first been applied to human forehead skin and can be further expanded to other planar soft tissues. The failure characteristics for the skin in terms of ultimate stress are 3 MPa +/- 1.5 MPa. The ultimate global longitudinal strains are equal to 9.5%+/-1.9% (Green-Lagrange strain), which contrasts with the ultimate local longitudinal strain values of 24.0%+/-5.3% (Green-Lagrange strain). This difference is a consequence of the tissue heterogeneity, clearly illustrated by the heterogeneous distribution of the local strain field. All data will assist in developing the tissue constitutive law that will be implemented in finite element models.     

Chemical components of cells at the microscopic level

Thin slices of potato tuber can be used to demonstrate, at the cellular level, the presence of protein, carbohydrate, and nucleic acids. The presence of fat can readily be demonstrated with avocado     

A simple device for harvesting biological fluids at the microscopic level

A simple aspirating device is described which permits repeated samples of biological materials to be taken near or at the microscopic level. It features an inner coaxial tube which delivers a harvesting fluid into the very tip of the collecting needle, permitting metabolic activity in the sample to be quenched immediately. The device is easily constructed from common laboratory material.     

A computational approach to studying ageing at the individual level

The ageing process is actively regulated throughout an organism''s life, but studying the rate of ageing in individuals is difficult with conventional methods. Consequently, ageing studies typically make biological inference based on population mortality rates, which often do not accurately reflect the probabilities of death at the individual level. To study the relationship between individual and population mortality rates, we integrated in vivo switch experiments with in silico stochastic simulations to elucidate how carefully designed experiments allow key aspects of individual ageing to be deduced from group mortality measurements. As our case study, we used the recent report demonstrating that pheromones of the opposite sex decrease lifespan in Drosophila melanogaster by reversibly increasing population mortality rates. We showed that the population mortality reversal following pheromone removal was almost surely occurring in individuals, albeit more slowly than suggested by population measures. Furthermore, heterogeneity among individuals due to the inherent stochasticity of behavioural interactions skewed population mortality rates in middle-age away from the individual-level trajectories of which they are comprised. This article exemplifies how computational models function as important predictive tools for designing wet-laboratory experiments to use population mortality rates to understand how genetic and environmental manipulations affect ageing in the individual.     

Immunocytochemical staining of Histoplasma capsulatum at the electron microscopic level

Summary Rabbits were immunized with histoplasmin emulsified in Freund's complete adjuvant. Antibody raised in these rabbits was exposed to Histoplasma capsulatum yeast cells, either in tissue culture medium, or after in vitro or in vivo phagocytosis by mouse macrophages. The sites of antibody binding were identified using an immunoperoxidase technique. At least two sites of antibody binding were identified, one to the fungal cell wall and the other to the outer cell membrane. Within 6 h after phagocytosis by macrophages, fungal cell walls appeared roughened, with what appeared to be cell wall antigen released into the phagolysosome, appearing associated with the phagolysosome membrane, and possibly adjacent macrophage cytoplasm. Similar staining of fungal antigen was noted in alveolar macrophages which had ingested Histoplasma capsulatum after a respiratory challenge. This method may be useful in detailing the host/pathogen interactions which occur in human pulmonary histoplasmosis.     

Structure of human chromosomes visualized at the electron microscopic level

A newly developed technique allows cytological (light microscope level) chromosome preparations to be examined at the electron microscopic level. Ultrathin (50 nm) sections of highly condensed Hela cell metaphase chromosomes show the characteristic mitotic chromosome morphology. In addition a fibrous network (presumably chromosome fibers) can be seen within them. Fibers appear to be gathered at foci along each chromatid. Treatment of chromosomes with trypsin in a trypsin/G-banding procedure reduces the amount of staining material at the electron microscopic level and results in more prominent foci. Thicker (100 nm) sections of less condensed chromosomes prepared from human lymphocytes display a banding pattern similar to G-banding, even without pretreatment with proteases.     

Scintillation autoradiography at the light microscopic level: A review

Summary This article reviews the reported attempts to expose autoradiographs, intended for examination by light microscopy, in the presence of scintillation fluid in order to increase efficiency and thus shorten exposure times. The scintillation process is reviewed, together with the use of comparable techniques in the autoradiography of chromatograms and electrophoresis gels. The conflicting reports on the usefulness of scintillation autoradiography in light microscopy are then discussed, and explanations sought for the wide diversity of claims made for the technique. Many of the more optimistic claims can be explained on the basis that the normal autoradiographs used for comparison had unduly low efficiencies caused by inadequate drying of the emulsion and consequent latent image fading. The techniques used are unlikely to have produced any increase in efficiency through the scintillation process. Optimal conditions for obtaining such increases are derived from theoretical considerations.     

One-domain approach for studying multiphase transport phenomena in biofilm growing systems

The one-domain approach (ODA) was used as an alternative to solve fluid–biofilm interfacial behavior in a 2-D model for diffusion–reaction–convection coupled with prediction of irregular growth of biofilms via a cellular automaton strategy. The simulations exhibited errors of <7% compared with the porosity of a previously reported capillary experimental system. Additionally, biofilm surface geometrical aspects were satisfactorily compared with reports of experimental and similar rigorously simulated benchmark systems. The method developed was applied to simulate typical biofilm systems predicting recirculation flow patterns, interface concentration profiles, and clogging of the inlet section of the capillary tube, which are phenomena that affect the efficiency of diverse biotechnological applications, including membrane bioreactors and biofilters. The ODA method applied to the governing equations of momentum and mass transfer combined with a cellular automaton algorithm is a suitable and straightforward approach for modeling solid-state fermentation at different sophistication levels.     

Cause-effect modeling as a general method for describing and studying phenomena in complex hierarchical systems

A strict definition of a cause-effect model of a complex phenomenon is given, and the rules for presenting such models in the form of cause-effect diagrams are formulated. The relationship between the cause-effect modeling and conventional methods of mathematical modeling is analyzed. Examples of the cause-effect models (diagrams) of phenomena of various physical nature are presented, and the application of these models to some specific problems is shown. In particular, the mechanism of renormalizing the rate constants of chemical reactions is considered in terms of dissipative resonance. An example of renormalizing the parameters of climate sensitivity and the relaxation time of the Earth’s climatic system in terms of a two-component (CO₂ + H₂O) greenhouse effect is considered.     

Novel two-phase sampling designs for studying binary outcomes

In biomedical cohort studies for assessing the association between an outcome variable and a set of covariates, usually, some covariates can only be measured on a subgroup of study subjects. An important design question is—which subjects to select into the subgroup to increase statistical efficiency. When the outcome is binary, one may adopt a case-control sampling design or a balanced case-control design where cases and controls are further matched on a small number of complete discrete covariates. While the latter achieves success in estimating odds ratio (OR) parameters for the matching covariates, similar two-phase design options have not been explored for the remaining covariates, especially the incompletely collected ones. This is of great importance in studies where the covariates of interest cannot be completely collected. To this end, assuming that an external model is available to relate the outcome and complete covariates, we propose a novel sampling scheme that oversamples cases and controls with worse goodness-of-fit based on the external model and further matches them on complete covariates similarly to the balanced design. We develop a pseudolikelihood method for estimating OR parameters. Through simulation studies and explorations in a real-cohort study, we find that our design generally leads to reduced asymptotic variances of the OR estimates and the reduction for the matching covariates is comparable to that of the balanced design.     

Novel methods for studying normal and disordered erythropoiesis

Erythropoiesis is a process during which multipotential hematopoietic stem cells proliferate, differentiate and eventually form mature erythrocytes. Interestingly, unlike most cell types, an important feature of erythropoiesis is that following each mitosis the daughter cells are morphologically and functionally different from the parent cell from which they are derived, demonstrating the need to study erythropoiesis in a stage-specific manner. This has been impossible until recently due to lack of methods for isolating erythroid cells at each distinct developmental stage. This review summarizes recent advances in the development of methods for isolating both murine and human erythroid cells and their applications. These methods provide powerful means for studying normal and impaired erythropoiesis associated with hematological disorders.     

Indirect hemagglutination test at the ultrastructural level]

A study on ultra-thin sections was made of the preparations of agglutinate produced during the reaction of the immunoglobulin erythrocytic diagnostic agent with dry corpuscular Rickettsia prowazeki antigen, fluoresceine isothiocyanate labeled, and also SRBC used for the preparation of the diagnostic agent after formalinization, tannin treatment, sensitization with hyperimmune horse serum immunoglobulins and lyophilization, respectively. Formalin and tannin treatment of erythrocytes failed to be reflected on the ultrastructure of their cellular membranes; the treatment with hemosensitin was accompanied by the appearance of spheroid protrusions of the erythrocyte cytoplasmic membrane with the preservation of its three-layer structure. Specific interaction of sensitized erythrocytes with the antigen corpuscles was expressed morphologically in their apposition or connection through a gap of 20--30 nm.