Practical implementation of the planar and spatial rotator in a complex tissue: the brain.

In neuroscience, application of widely used stereological local volume estimators, including the planar rotator, is challenged by the combination of a complex tissue organisation and an estimator requirement of either isotropic or vertical sections, i.e. randomly oriented tissue. The spatial rotator is applicable with any tissue orientation but is sensitive to projection artefacts. The challenge is thus to select the most appropriate method for individual analyses. In this study, agreement between estimates of mean cell volume acquired with the vertical planar and the spatial rotator is assessed for two brain regions with different types of cytoarchitecture (motor cortex and hippocampal cornu ammonis 1). The possibility of using the planar rotator in tissues cut in an arbitrary direction is explored and requirements for a theoretically unbiased result as well as histological considerations are provided. LAY DESCRIPTION: Cells may change volume both during disease and with advancing age. Assessment of the volume of individual cells can therefore serve as a useful indicator of general tissue state. Most available methods to estimate cell volume in tissue sections, however, require that the tissue analysed has random orientation. Particularly for complex tissues such as the brain this is a challenge as identification, delineation and subdivision of many brain areas rely heavily on the use of anatomical atlases where illustrations depict the tissue in a few well-known orientations. In this study, the practical application of two different methods for estimating mean cell volumes in tissues cut in a preferred orientation is evaluated. Requirements for the feasibility of cell volume estimation without random tissue orientation as well as histological considerations are provided.

Stereological estimation of particle shape and orientation from volume tensors.

In the present paper, we describe new robust methods of estimating cell shape and orientation in 3D from sections. The descriptors of 3D cell shape and orientation are based on volume tensors which are used to construct an ellipsoid, the Miles ellipsoid, approximating the average cell shape and orientation in 3D. The estimators of volume tensors are based on observations in several optical planes through sampled cells. This type of geometric sampling design is known as the optical rotator. The statistical behaviour of the estimator of the Miles ellipsoid is studied under a flexible model for 3D cell shape and orientation. In a simulation study, the lengths of the axes of the Miles ellipsoid can be estimated with coefficients of variation of about 2% if 100 cells are sampled. Finally, we illustrate the use of the developed methods in an example, involving neurons in the medial prefrontal cortex of rat.

On semiautomatic estimation of surface area.

In this paper, we propose a semiautomatic procedure for estimation of particle surface area. It uses automatic segmentation of the boundaries of the particle sections and applies different estimators depending on whether the segmentation was judged by a supervising expert to be satisfactory. If the segmentation is correct the estimate is computed automatically, otherwise the expert performs the necessary measurements manually. In case of convex particles we suggest to base the semiautomatic estimation on the so-called flower estimator, a new local stereological estimator of particle surface area. For convex particles, the estimator is equal to four times the area of the support set (flower set) of the particle transect. We study the statistical properties of the flower estimator and compare its performance to that of two discretizations of the flower estimator, namely the pivotal estimator and the surfactor. For ellipsoidal particles, it is shown that the flower estimator is equal to the pivotal estimator based on support function measurements along four perpendicular rays. This result makes the pivotal estimator a powerful approximation to the flower estimator. In a simulation study of prolate and oblate ellipsoidal particles, the surfactor also performs well for particles which are not extremely elongated. In particular, the surfactor is not very much affected by the singularity in the surfactor formula or by possible inaccuracies in the necessary angle measurements. We also assess the performance of the semiautomatic procedure in a study of somatostatin positive inhibitory interneurons from mice hippocampi. Only 35% of the cells needed to be analysed manually and an important decrease in workload was obtained by using the semiautomatic approach.

The spatial rotator.

This paper presents a new local volume estimator, the spatial rotator, which is based on measurements on a virtual 3D probe, using computer assisted microscopy. The basic design of the probe builds upon the rotator principle which requires only a few manual intersection markings, thus making the spatial rotator fast to use. Since a 3D probe is involved, it is expected that the spatial rotator will be more efficient than the the nucleator and the planar rotator, which are based on measurements in a single plane. An extensive simulation study shows that the spatial rotator may be more efficient than the traditional local volume estimators. Furthermore, the spatial rotator can be seen as a further development of the Cavalieri estimator, which does not require randomization of sectioning or viewing direction. The tissue may thus be sectioned in any arbitrary direction, making it easy to identify the specific tissue region under study. In order to use the spatial rotator in practice, however, it is necessary to be able to identify intersection points between cell boundaries and test rays in a series of parallel focal planes, also at the peripheral parts of the cell boundaries. In cases where over- and underprojection phenomena are not negligible, they should therefore be corrected for if the spatial rotator is to be applied. If such a correction is not possible, it is needed to avoid these phenomena by using microscopy with increased resolution in the focal plane.

The semi-automatic nucleator.

The nucleator is a well-established manual stereological method of estimating mean cell volume from observations on random cell transects through reference points of the cells. In this paper, we present an automated version of the nucleator that uses automatic segmentation of the boundaries of the cell transects. An expert supervises the process. If the segmentation is judged to be satisfactory, an estimate of the cell volume is calculated automatically on the basis of the whole cell transect. In the remaining cases, the expert intervenes and uses the classical nucleator. The resulting estimator is called the semi-automatic nucleator. In this paper, we study the statistical properties of the semi-automatic nucleator. Formulae for the bias and mean square error are derived. The semi-automatic nucleator may have a small bias but will still in most cases be more efficient than the classical nucleator. Procedures for estimating bias and mean square error from a pilot study are provided. The application of the semi-automatic nucleator is illustrated in a study of somatostatin positive inhibitory interneurons which were genetically labelled with green fluorescent protein (GFP). The cells were sampled with an optical disector. The centre of mass in a central cell transect was used as reference point. It is found in this study that the number of cells needed for obtaining, for instance, a 5% precision of the estimate of mean cell volume is 150 and 189 for the semi-automatic and the classical nucleator, respectively. Taking into account that the time spent analysing one cell is shorter for the semi-automatic nucleator than for the classical nucleator, the semi-automatic nucleator is superior to the classical nucleator.

High-level expression of active human cystatin C in Escherichia coli.

Expression of the human cysteine proteinase inhibitor, cystatin C (CysC) in the cytoplasm of Escherichia coli was studied using a cDNA fragment encoding the cysteine proteinase inhibitor controlled by the phage lambda pR/cI857 system. The yield of CysC was low, probably due to proteolytic degradation. By fusing the cysC cDNA to a DNA fragment encoding the signal peptide of the E. coli outer membrane protein A, it was possible to produce a substantial amount of CysC in the periplasm. The processing of the signal peptide was shown to be quantitative and to result in CysC with the correct N-terminal amino acid. Yields higher than 1000 micrograms CysC/ml can be obtained by initiating the product formation at a moderate temperature (40 degrees C) late in an optimized fermentation process. A method that gives selective extraction of the periplasmic proteins and at the same time stabilizes CysC has been used.

Renal transplantation and HLA-A,B matching. Theoretical considerations concerning pool size.

The rate of improvement in HLA matching quality with increasing pool size is predicted. The expected frequency of HLA-A,B-compatible matches increases rapidly up to approximately 47% for pool size 1,000 and from then on the increase is moderate, whereas for HLA-A,B-identical matches the increase in frequency is more constant, the expected frequencies being approximately 34 and 52% for pool sizes 1,000 and 2,500, respectively. The expected frequency of HLA-A,B fullhouse-identical matches is 33% at a pool size of 2,500. The waiting time for the first available HLA-identical kidney is estimated. For a random recipient there is a 25% chance of obtaining an HLA-identical match among the first 500 kidney donors. For two cooperating pools, it is calculated at which relative transplantation activity the kidney import to export is balanced and also the decrease in waiting time for an identical match when cooperating is estimated. The results of a kidney exchange simulation are presented and they agree well with the theoretical considerations.

Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle.

Muscle contraction causes an increase in activity of 5'-AMP-activated protein kinase (AMPK). This study was designed to determine whether chronic chemical activation of AMPK will increase mitochondrial enzymes, GLUT-4, and hexokinase in different types of skeletal muscle of resting rats. In acute studies, rats were subcutaneously injected with either 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mg/g body wt) in 0.9% NaCl or with 0.9% NaCl alone and were then anesthetized for collection and freezing of tissues. AMPK activity increased in the superficial, white region of the quadriceps and in soleus muscles but not in the deep, red region of the quadriceps muscle. Acetyl-CoA carboxylase (ACC) activity, a target for AMPK, decreased in all three muscle types in response to AICAR injection but was lowest in the white quadriceps. In rats given daily, 1 mg/g body wt, subcutaneous injections of AICAR for 4 wk, activities of citrate synthase, succinate dehydrogenase, and malate dehydrogenase were increased in white quadriceps and soleus but not in red quadriceps. Cytochrome c and delta-aminolevulinic acid synthase levels were increased in white, but not red, quadriceps. Carnitine palmitoyl-transferase and hydroxy-acyl-CoA dehydrogenase were not significantly increased. Hexokinase was markedly increased in all three muscles, and GLUT-4 was increased in red and white quadriceps. These results suggest that chronic AMPK activation may mediate the effects of muscle contraction on some, but not all, biochemical adaptations of muscle to endurance exercise training.

Rena transplantation and HLA-A, B matching. Theoretical considerations concerning pool size.

The rate of improvement in HLA-matching quality with increasing pool size is predicted. The frequency of HLA-A, B compatible matches increases rapidly up to approximately 47% for pool size 1000 and from then on the increase is more moderate while for HLA-A, B fullhouse-identical matches the increase in frequency is slower and more constant, the expected frequency being approximately 33% at a pool size of 2500. The import/export balance between two cooperating pools is estimated for various relative pool sizes and transplantation activities.

The influence of 13 clinical and immunological factors on renal graft survival: a contingency table analysis.

A statistical method which accounts for the heterogeneity of clinical materials is presented and applied to a material of necrokidney transplantations. It is concluded that the recipient sex and the period in which the transplantation was performed are the most important factors (best prognosis for female recipients, best programs for transplantations in the first periods) and that the HLA-match grade has a significant influence on graft survival for male recipients and transplantations with short cold ischemia time. The present analysis has furthermore demonstrate the heterogeneity of the material as judged by the associations between factors and the overestimation of the influence of some of the factors by direct comparisons.

Fusarium graminearum A 3/5 as a novel host for heterologous protein production.

We describe a novel fungal expression system which utilizes the Quorn myco-protein fungus Fusarium graminearum A 3/5. A transformation system was developed for F. graminearum and was used to introduce the coding and regulatory regions of a trypsin gene from Fusarium oxysporum. The protein was efficiently expressed, processed and secreted by the recombinant host strain. In addition, the promoter and terminator of the F. oxysporum trypsin gene have been successfully utilized to drive the expression of a cellulase gene from Scytalidium thermophilum and a lipase gene from Thermomyces lanuginosus in F. graminearum.

A note on the stereological implications of irregular spacing of sections.

Stereological methods for serial sections traditionally assume that the sections are exactly equally spaced. In reality, the spacing and thickness of sections can be quite irregular. This may affect the validity and accuracy of stereological techniques, especially the Cavalieri estimator of volume. We present a new formula for the accuracy of the Cavalieri estimator that includes the effect of random variability in section spacing. A modest amount of variability in section spacing can cause a substantial increase in estimator variance.

On error prediction in circular systematic sampling.

An extended covariogram model is discussed for estimating the precision of circular systematic sampling. The extension is motivated by recent developments in shape analysis of featureless planar objects. Preliminary simulation results indicate that it is important to consider the extended covariogram model.

Shape discrimination by total curvature, with a view to cancer diagnostics.

This paper investigates the use of total curvature for shape discrimination of objects via profiles of their planar sections (not assumed to be star shaped). Methods of estimating total curvature from observation of a finite number of points on the boundary of the object are investigated, including a simple discrete approximation method and various interpolation methods. Total curvature is capable of revealing shape differences on a local scale, as demonstrated by the analysis of two data sets of malignant and normal or benign tumour cell nuclear profiles.

Is 'schizophrenia, schizo-affective type' a useful diagnosis? Ten years experience from Danish psychiatric hospitals.

During the period 1 April 1970 to 31 March 1980, 1,039 admissions (592 males and 447 females) with the main diagnosis schizophrenia, schizo-affective type, were registered in Danish psychiatric hospitals. Based on the Danish Central Psychiatric Register, 114 patients first admitted to psychiatric hospitals in the period 1970 to 1978 with the main diagnosis schizophrenia, schizo-affective type in at least one admission are described. The results show a continued increase in the use of the diagnosis, but the variation in the use both in different psychiatric hospitals and in patients' admission histories is striking. The question, whether the diagnosis is useful in this way, is discussed, and a proposal for a different international classification of schizo-affective psychoses is given.

The efficiency of systematic sampling in stereology and its prediction.

The superior efficiency of systematic sampling at all levels in stereological studies is emphasized and various commonly used ways of implementing it are briefly described. Summarizing recent theoretical and experimental studies a set of very simple estimators of efficiency are presented and illustrated with a variety of biological examples. In particular, a nomogram for predicting the necessary number of points when performing point counting is provided. The very efficient and simple unbiased estimator of the volume of an arbitrary object based on Cavalieri's principle is dealt with in some detail. The efficiency of the systematic fractionating of an object is also illustrated.

Directional analysis of digitized planar sets by configuration counts.

The oriented rose of normal directions of a random set Z can be used to quantify its anisotropy. A method for estimating this quantity from a digitization of Z in a sampling window, i.e. its pixel image, is presented. The image is analysed locally by considering pixel squares of size n x n. This allows us to count the number of different types of n x n configurations in the pixel image. We show that it suffices to restrict attention to the so-called informative configurations. The number of informative configurations increases only polynomially in n. An algorithm to find these informative configurations is presented. Furthermore, estimators of the oriented rose based on counts of observed informative configurations are derived. The procedure is illustrated by a simulated example and an analysis of a microscopic image of steel.

Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate, and salts.

The constitutive cytoplasmic expression in E. coli of human growth hormone (hGH) with different N-terminal extensions (3 or 4 amino acids) has been studied. These hGH precursors were used for in vitro cleavage to obtain the mature, authentic hormone. Small changes in the amino acid extensions of the hGH precursors led to three-fold differences in specific expression rates. The specific expression rate of the hGH precursors was inversely proportional to the ratios of the specific growth rates of plasmid containing and plasmid free cells (micro(+)/micro(-)) and also to the genetic stability. To ensure a satisfactory genetic stability in production fermentors, an hGH precursor with a moderate expression efficiency was chosen.The medium composition and growth conditions were studied, resulting in the choice of a glucose fed batch fermentation process using a complex medium. In this process a yield of 2000 mg/L of met-ala-glu-hGH (MAE-hGH) was obtained. The fermentation process comprised a glucose-limited growth phase followed by a second phase with increased glucose feed and exhaustion of phosphate from the medium. The second phase is characterized by an MAE-hGH production, whereas further biomass formation is blocked. High concentrations of glucose led to reduced specific expression of MAE-hGH--the specific and total yield in batch glucose fermentations is only about 30% of the yield in optimized fed batch fermentations. The physiological background for this was investigated. Chemostat experiments showed that the glucose concentration and the metabolic condition of the cells--i.e. with or without formation of acetate--was not critical per se in order to obtain a high specific yield of MAE-hGH. Therefore it is unlikely that formation of MAE-hGH is catabolite repressed by glucose. Furthermore it was shown that the specific production rate of MAE-hGH was independent of the specific growth rate and it was further demonstrated that the decrease in expression efficiency in glucose batch fermentation was a result of an inhibitory effect of acetic acid. In batch fermentations this inhibitory effect was enhanced by a salt effect caused by increased consumption of acid and base used to control pH. The identity of the acid and the base used are not important in this context. From studies of the expression of other proteins in E. coli. with constitutive as well as inducible promoters we conclude that glucose fed batch processes are often superior to batch processes in the production of heterologous proteins E. coli.

A note on stereological estimation of the volume-weighted second moment of particle volume.

It is shown that for a variety of biological particle shapes, the volume-weighted second moment of particle volume can be estimated stereologically using only the areas of particle transects, which can be estimated manually by point-counting.