Experimental and numerical investigations on the effect of fracture geometry and fracture aperture distribution on flow and solute transport in natural fractures.

The impact of fracture geometry and aperture distribution on fluid movement and on non-reactive solute transport was investigated experimentally and numerically in single fractures. For this purpose a hydrothermally altered and an unaltered granite drill core with axial fractures were investigated. Using three injection and three extraction locations at top and bottom of the fractured cores, different dipole flow fields were examined. The conservative tracer (Amino-G) breakthrough curves were measured using fluorescence spectroscopy. Based on 3-D digital data obtained by micro-computed tomography 2.5-D numerical models were generated for both fractures by mapping the measured aperture distributions to the 2-D fracture geometries (x-y plane). Fluid flow and tracer transport were simulated using COMSOL Multiphysics®. By means of numerical simulations and tomographic imaging experimentally observed breakthrough curves can be understood and qualitatively reproduced. The experiments and simulations suggest that fluid flow in the altered fracture is governed by the 2-D fracture geometry in the x-y plane, while fluid flow in the unaltered fracture seems to be controlled by the aperture distribution. Moreover, we demonstrate that in our case simplified parallel-plate models fail to describe the experimental findings and that pronounced tailings can be attributed to complex internal heterogeneities. The results presented, implicate the necessity to incorporate complex domain geometries governing fluid flow and mass transport into transport modeling.

LINE-1 induces hTERT and ensures telomere maintenance in tumour cell lines.

A hallmark of cancer cells is an activated telomere maintenance mechanism, which allows prolonged survival of the malignant cells. In more than 80% of tumours, telomeres are elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Cancer cells are also characterized by expression of active LINE-1 elements (L1s, long interspersed nuclear elements-1). L1 elements are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Using L1-knockdown (KD), we show for the first time that L1 is critical for telomere maintenance in telomerase-positive tumour cells. The reduced length of telomeres in the L1-KD-treated cells correlated with an increased rate of telomere dysfunction foci, a reduced expression of shelterin proteins and an increased rate of anaphase bridges. The decreased telomere length was associated with a decreased telomerase activity and decreased telomerase mRNA level; the latter was increased upon L1 overexpression. L1-KD also led to a decrease in mRNA and protein expression of cMyc and KLF-4, two main transcription factors of telomerase and altered mRNA levels of other stem-cell-associated proteins such as CD44 and hMyb, as well as a corresponding reduced growth of spheroids. The KD of KLF-4 or cMyc decreased the level of L1-ORF1 mRNA, suggesting a specific reciprocal regulation with L1. Thus, our findings contribute to the understanding of L1 as a pathogenicity factor in cancer cells. As L1 is only expressed in pathophysiological conditions, L1 now appears to be target in the rational treatment of telomerase-positive cancer.

Synchrotron-based X-ray tomographic microscopy for visualization of three-dimensional collagen matrices.

INTRODUCTION: Three-dimensional collagen matrices (3D-CMs) may be visualized by cumbersome reconstructions of serial sections. We report here on the method of synchrotron-based X-ray tomographic microscopy (SRXTM) to image 3D-CMs in native tissue probes. MATERIAL AND METHODS: SRXTM of 3D-CMs (mucoderm®, mucograft®) was performed at the TOMCAT beamline of the Swiss Light Source (SLS) at the Paul Scherrer Institute (Villigen, Switzerland). RESULTS: SRXTM combines the advantages of high-resolution scanning electron microscopy (SEM) imaging with the low-resolution reconstructions of micro-CT (µCT) imaging. It may be used to non-destructively visualize and analyze structures within the 3D-CMs without the need of serial sectioning and reconstruction. CONCLUSION: High-resolution SRXTM is a useful tool in analyzing the topology and morphometry of structures in 3D-CMs. The outcome justifies the efforts in sophisticated data processing. CLINICAL RELEVANCE: SRXTM may help to understand the clinical characteristics of 3D-CMs in more detail.

Natural micro-scale heterogeneity induced solute and nanoparticle retardation in fractured crystalline rock.

We studied tracer (Tritiated Water (HTO); Tritium replaces one of the stable hydrogen atoms in the H(2)O molecule) and nanoparticle (quantum dots (QD)) transport by means of column migration experiments and comparison to 3D CFD modeling. Concerning the modeling approach, a natural single fracture was scanned using micro computed tomography (µCT) serving as direct input for the model generation. The 3D simulation does not incorporate any chemical processes besides the molecular diffusion coefficient solely reflecting the impact of fracture heterogeneity on mass (solute and nanoparticles) transport. Complex fluid velocity distributions (flow channeling and flowpath heterogeneity) evolve as direct function of fracture geometry. Both experimental and simulated solute and colloidal breakthrough curves show heavy tailing (non-Fickian transport behavior), respectively. Regarding the type of quantum dots and geochemical conditions prevailing (Grimsel ground water chemistry, QD and diorite surface charge, respectively and porosity of the Äspö diorite drill core) experimental breakthrough of the quantum dots always arrives faster than the solute tracer in line with the modeling results. Besides retardation processes like sorption, filtration, straining or matrix diffusion, the results show that natural 3D fracture heterogeneity represents an important additional retardation mechanism for solutes and colloidal phases. This is clearly verified by the numerical simulations, where the 3D real natural fracture geometry and the resulting complex flow velocity distribution is the only possible process causing solute/nanoparticle retardation. Differences between the experimental results and the simulations are discussed with respect to uncertainties in the µCT measurements and experimental and simulation boundary conditions, respectively.

Boron determination in liver tissue by combining quantitative neutron capture radiography (QNCR) and histological analysis for BNCT treatment planning at the TRIGA Mainz.

The typical primary malignancies of the liver are hepatocellular carcinoma and cholangiocarcinoma, whereas colorectal liver metastases are the most frequently occurring secondary tumors. In many cases, only palliative treatment is possible. Boron neutron capture therapy (BNCT) represents a technique that potentially destroys tumor tissue selectively by use of externally induced, locally confined secondary particle irradiation. In 2001 and 2003, BNCT was applied to two patients with colorectal liver metastases in Pavia, Italy. To scrutinize the rationale of BNCT, a clinical pilot study on patients with colorectal liver metastases was carried out at the University of Mainz. The distribution of the (10)B carrier (p-borono-phenylalanine) in the liver and its uptake in cancerous and tumor-free tissue were determined, focusing on a potential correlation between the uptake of p-borono-phenylalanine and the biological characteristics of cancerous tissue. Samples were analyzed using quantitative neutron capture radiography of cryosections combined with histological analysis. Methodological aspects of the combination of these techniques and results from four patients enrolled in the study are presented that indicate that the uptake of p-borono-phenylalanine strongly depends on the metabolic activity of cells.

Insulin receptor binding in pork brain: different affinities of porcine and human insulin.

The insulin receptor binding of human and pork insulin was measured in four different pork brain tissues. In medial hypothalamus, infundibulum of pituitary gland, neurohypophysis, and adenohypophysis insulin receptors were found. Binding of mono-125I-(TyrA14)-insulin proceeded rapidly at 37 degrees C, pH 7.4, and reached a steady state for human and pork insulin at 15 minutes. Biosynthetic human insulin and semisynthetic human insulin dissociated more rapidly than pork insulin. In the four tested brain tissues the specific insulin binding per mg protein differed significantly (2p less than 0.01). The receptor affinity constant of human insulin was significantly (2p less than 0.01) lower in comparison to pork insulin in the four brain tissues studied. This study demonstrates that there are insulin receptors in pork brain tissue, that the amounts of these receptors vary depending on the area and that the brain insulin receptors react differently to pork and human insulin.

Different potencies of biosynthetic human and purified porcine insulin.

The glucose clamp technique was used to compare the biological activity of purified porcine insulin and Biosynthetic Human Insulin (BHI). An intravenous bolus of 0.1 U/kg BW was injected in eight male volunteers, and the glucose was clamped at baseline values (euglycemic clamp). Serum insulin, serum C-peptide and plasma glucose did not differ between porcine and human insulin. The insulin induced glucose consumption differed significantly (p less than 0.007) between purified porcine insulin (50.5 +/- 5.2 [SEM] g/2h) and Biosynthetic Human Insulin (63.5 +/- 4.5 g/2h). Purified porcine insulin induced a hormonal response with significantly (p less than 0.05) elevated concentrations of serum growth hormone (12.1 +/- 0.25 ng/ml) and serum cortisol (161.4 +/- 28.6 ng/ml), which were not observed following Biosynthetic Human Insulin (serum growth hormone: 2.6 +/- 0.2 ng/ml; serum cortisol: 117.3 +/- 14.8 ng/ml). The data confirm earlier results indicating hormonal and metabolic differences between human and porcine insulin.

Treatment with human insulin (recombinant DNA) in diabetic subjects pretreated with pork or beef insulin: first results of a multicenter study.

In two double-blind studies 66 insulin-dependent diabetic subjects pretreated with pork insulin were changed to human insulin (recombinant DNA) or a purified pork insulin preparation (regular and NPH insulin). Sixty-five patients previously pretreated with beef insulin were transferred, in a randomized, double-blind fashion, to human insulin and purified beef insulin of the same preparations (regular and NPH insulin). Patients' metabolic control, as demonstrated by fasting and 1-h postprandial blood glucose, HbA1c, and daily insulin dosage, over 4 mo was unchanged in our four groups compared with the values before changing insulin preparation. No severe hypoglycemic attacks or skin reactions were reported.

Crossover study with human insulin (recombinant DNA) in type I diabetic subjects.

The activity of three combinations of regular and NPH human insulin (recombinant DNA) has been compared to that of an intermediate-acting pork insulin in a crossover study in type I diabetic subjects. After a prephase, the patients injected each insulin subcutaneously for 1 wk in different order. During the 5-wk period, daily glucose profiles were self-monitored, except at the end of each week, when blood was sampled for the determination of glucose, HbA1, glycosylated albumin, C-peptide, glucagon, and routine laboratory parameters. Fasting as well as mean daily glucose and mean amplitude of glucose excursions were similar during the treatment with pork and the various human insulin preparations. There was also no significant difference in C-peptide, glucagon, HbA1 or the routine laboratory parameters with each insulin tested. Glycosylated albumin, however, was significantly lower during the test period with intermediate-acting pork insulin and human insulin (25:75 regular:NPH), when compared with the prephase. We conclude that in type I diabetic subjects human insulin in special galenic preparations shows very similar metabolic activity to pork insulin.

Pharmacodynamics of human insulin (recombinant DNA)--regular, NPH, and mixtures--obtained by the Gerritzen method in healthy volunteers.

The study was concerned with the comparison of the action profile of regular and NPH preparations of human insulin (recombinant DNA) and of PPI (pork purified insulin). In addition, the action profiles of some mixtures (10:90, 15:85, 20:80, 25:75, 30:70) of regular and NPH human insulin were evaluated. The comparisons were based on the Gerritzen test. There was no statistically significant difference in the time-course of the blood glucose levels after administration of NPH human insulin and NPH PPI. A tendency was noted, however, that NPH human insulin has a faster onset of action and that the serum glucose minimum for NPH human insulin lasts longer. The serum glucose curves after the application of regular and NPH human insulin initially lie closer together than after the respective PPI preparations. In the later phase, regular human insulin interferes less with the NPH curves. This means that combinations of regular and NPH human insulin may have a clinically useful action profile. No skin reaction or other adverse reaction was detected after application of human insulin and no antibodies against human insulin and Escherichia coli protein were found.

Different counterregulatory responses to human insulin (recombinant DNA) and purified pork insulin.

The biologic effect of human insulin (recombinant DNA) and purified pork insulin (PPI) was compared during insulin-induced hypoglycemia at two intravenous dosages 0.075 and 0.1 U/kg body wt in healthy volunteers. Serum insulin concentrations and plasma glucose curves were identical. PPI induced a significantly (P less than 0.05) higher output of epinephrine, growth hormone, and cortisol at both doses. Less inhibition (P less than 0.05) of endogenous insulin secretion was observed for human insulin at 0.1 kg body wt. An elevated incidence of sweating during hypoglycemia was related to epinephrine secretion. The results indicate that homologous insulin produces in vivo effects which are different from those produced by heterologous insulin.

Comparative studies on intermediary metabolism and hormonal counterregulation following human insulin (recombinant DNA) and purified pork insulin in man.

Human insulin (recombinant DNA) and purified pork insulin (PPI) were administered intravenously at a dosage of 0.075 U/kg in eight healthy men. Both insulins exerted the same hypoglycemic effect with the same restoration pattern to normal glucose levels at the end of the test. Differences were found with respect to a stronger antilipolytic and antiketogenic effect of human insulin; also the reactive rise of both compounds at the end of the test is less under human insulin in comparison with PPI. In spite of the same glucose nadir, the pattern of hormonal counterregulation is different under human insulin in comparison with PPI. There was less epinephrine and glucagon and practically no prolactin secretion following human insulin. Growth hormone secretion is augmented under human insulin. The clinical significance of these results under long-term treatment with human insulin has to be assessed.

Inhibition of pancreatic glucagon responses to arginine by human insulin (recombinant DNA) and purified porcine insulin in normal and diabetic subjects.

This study investigates and compares human insulin (recombinant DNA) and purified porcine insulin (PPI) in healthy volunteers and in type II diabetic patients, in terms of whether both these insulins were capable of influencing in a different manner pancreatic glucagon, C-peptide, and free fatty acids (FFA) concentrations. The findings reveal that the beta-cell of human pancreas apparently recognizes human insulin more readily than PPI, as assessed by the inhibition of C-peptide, and a similar conclusion follows for the alpha-cell; this conclusion is underscored by the inhibited glucagon values. The delayed increments of glucagon under human insulin following arginine stimulation may be the result of a more rapid insulin absorption from subcutaneous tissue and a greater biologic action of this insulin in comparison with the PPI. Finally, human insulin has additional properties as demonstrated by its stronger antilipolytic effects.

The inhibition of cancer cell stickiness, a model for investigation of platelet aggregation inhibitors in vivo. Effect of the sulfonyl urea derivatives, glibenclamide, gliclazide, and HB180, as well as the carboxylic acid derivative, meglitinide.

Employing the test model which we developed for the investigation of platelet adhesiveness and aggregation in vivo, experiments demonstrated that the sulfonyl urea derivatives, glibenclamide, gliclazide, and HB 180, as well as the carboxylic acid derivative, meglitinide, are able to inhibit, in a dose-dependent relationship, the adherence of i.v. injected Walker-256-carcinosarcoma cells to the vascular endothelium of the rat mesentery, as well as to reduce significantly the rate of instantly occurring terminal tumor cell embolism of the lung. Since venous blood platelet count in surviving animals is inversely proportional to the number of the tumor cells which adhere to the vascular endothelium, one can deduce that tumor cell embolism is an immediate result of a massively occurring disseminated intravascular coagulation (DIC) which may be induced by i.v. injection of thromboplastic active carcinosarcoma cells and leads primarily to a drastic platelet count reduction. All four substances inhibit this platelet count reduction as well as the directly correlated tumor cell embolism mortality rate in a linear dose-dependent fashion. Their action can therefore be explained as being mediated via an inhibition of platelet adhesion and aggregation to the circulating tumor cells. Our proof of platelet aggregation in vivo correlates with the results obtained by Klaff et al. (1979), as far as a normalization of the pathologically increased platelet aggregation tendency in vitro in diabetics following 4-6 weeks of therapy with the sulfonyl urea derivatives glibenclamide and gliclazide.