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In this study a well-characterized pathological mutation at nucleotide position 3243 of human mitochondrial DNA was introduced into human [[rho].sup.0] teratocarcinoma (NT2) cells. In cloned and mixed populations of NT2 cells heteroplasmic for the mutation, mitotic segregation toward increasing levels of mutant mitochondrial DNA always occurred. Rapid segregation was frequently followed by complete loss of mitochondrial DNA. These findings support the idea that pathological mitochondrial DNA mutations are particularly deleterious in specific cell types, which can explain some of the tissue-specific aspects of mitochondrial DNA diseases. Moreover, these findings suggest that mitochondrial DNA depletion may be an important and widespread feature of mitochondrial DNA disease.

The mitochondrial genotype of heteroplasmic human cell lines containing the pathological np 3243 mtDNA mutation, plus or minus its suppressor at np 12300, has been followed over long periods in culture. Cell lines containing various different proportions of mutant mtDNA remained generally at a consistent, average heteroplasmy value over at least 30 wk of culture in nonselective media and exhibited minimal mitotic segregation, with a segregation number comparable with mtDNA copy number ([is greater than or equal to] 1000). Growth in selective medium of cells at 99% np 3243 mutant mtDNA did, however, allow the isolation of clones with lower levels of the mutation, against a background of massive cell death. As a rare event, cell lines exhibited a sudden and dramatic diversification of heteroplasmy levels, accompanied by a shift in the average heteroplasmy level over a short period ([is less than] 8 wk), indicating selection. One such episode was associated with a gain of chromosome 9. Analysis of respiratory phenotype and mitochondrial genotype of cell clones from such cultures revealed that stable heteroplasmy values were generally reestablished within a few weeks, in a reproducible but clone-specific fashion. This occurred independently of any straightforward phenotypic selection at the individual cell-clone level. Our findings are consistent with several alternate views of mtDNA organization in mammalian cells. One model that is supported by our data is that mtDNA is found in nucleoids containing many copies of the genome, which can themselves be heteroplasmic, and which are faithfully replicated. We interpret diversification and shifts of heteroplasmy level as resulting from a reorganization of such nucleoids, under nuclear genetic control. Abrupt remodeling of nucleoids in vivo would have major implications for understanding the developmental consequences of heteroplasmy, including mitochondrial disease phenotype and progression.

Ca2+ signaling plays major role in cardiac contractility. Alterations in Ca2+ signaling can be seen in arrhythmogenesis associated with cardiac disorders and heart failures. By analyzing the Ca2+ cycling of cardiomyocytes with the help of Ca2+ imaging, basic cardiac functionality, cardiac disorders and drug responses can be studied more thoroughly.We have generated spontaneously beating cardiomyocytes from induced pluripotent stem cell lines derived from patients with different cardiac disorders. Ca2+ cycling studies have revealed substantial defects and abnormalities in calcium signaling of these cardiomyocytes, presumably reflecting the cardiac phenotype observed in the patients. Ca2+ signaling abnormalities can be seen as variable frequency and amplitude and they can be categorized by their form. The analysis of these abnormalities is extremely important to study and understand different cardiac diseases and drug responses. So far this analysis has been done manually by researcher without any generally accepted analysis criteria's. However this way of analysis is subjective and slow and repeatability of analysis may be poor. To overcome these issues, we have developed Ca2+ data analysis software based on interactive visualization.The Ca2+ data analysis software allows to explore and distinguish different Ca2+ signal patterns from recordings and categorize the abnormalities objectively. It will speed up the analysis of cardiomyocyte Ca2+ signals and provide a specific analysis criteria's to analyze Ca2+ abnormalities in cardiomyocytes. This analysis software can be exploited to study basic disease pathology, to screen drugs, and to optimize drug therapy in a patient-specific manner. [ABSTRACT FROM PUBLISHER]

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have revolutionized cardiovascular research. Abnormalities in Ca 2+ transients have been evident in many cardiac disease models. We have shown earlier that, by exploiting computational machine learning methods, normal Ca 2+ transients corresponding to healthy CMs can be distinguished from diseased CMs with abnormal transients. Here our aim was to study whether it is possible to separate different genetic cardiac diseases (CPVT, LQT, HCM) on the basis of Ca 2+ transients using machine learning methods. Classification accuracies of up to 87% were obtained for these three diseases, indicating that Ca 2+ transients are disease-specific. By including healthy controls in the classifications, the best classification accuracy obtained was still high: approximately 79%. In conclusion, we demonstrate as the proof of principle that the computational machine learning methodology appears to be a powerful means to accurately categorize iPSC-CMs and could provide effective methods for diagnostic purposes in the future.

The focus of this research is on automated identification of the quality of human induced pluripotent stem cell (iPSC) colony images. iPS cell technology is a contemporary method by which the patient's cells are reprogrammed back to stem cells and are differentiated to any cell type wanted. iPS cell technology will be used in future to patient specific drug screening, disease modeling, and tissue repairing, for instance. However, there are technical challenges before iPS cell technology can be used in practice and one of them is quality control of growing iPSC colonies which is currently done manually but is unfeasible solution in large-scale cultures. The monitoring problem returns to image analysis and classification problem. In this paper, we tackle this problem using machine learning methods such as multiclass Support Vector Machines and several baseline methods together with Scaled Invariant Feature Transformation based features. We perform over 80 test arrangements and do a thorough parameter value search. The best accuracy (62.4%) for classification was obtained by using a k-NN classifier showing improved accuracy compared to earlier studies.

The purpose of this paper is to examine how well the human induced pluripotent stem cell (hiPSC) colony images can be classified using error-correcting output codes (ECOC). Our image dataset includes hiPSC colony images from three classes (bad, semigood, and good) which makes our classification task a multiclass problem. ECOC is a general framework to model multiclass classification problems. We focus on four different coding designs of ECOC and apply to each one of them k -Nearest Neighbor ( k -NN) searching, naïve Bayes, classification tree, and discriminant analysis variants classifiers. We use Scaled Invariant Feature Transformation (SIFT) based features in classification. The best accuracy (62.4%) is obtained with ternary complete ECOC coding design and k -NN classifier (standardized Euclidean distance measure and inverse weighting). The best result is comparable with our earlier research. The quality identification of hiPSC colony images is an essential problem to be solved before hiPSCs can be used in practice in large-scale. ECOC methods examined are promising techniques for solving this challenging problem.

Comprehensive functioning of Ca2+ cycling is crucial for excitation-contraction coupling of cardiomyocytes (CMs). Abnormal Ca2+ cycling is linked to arrhythmogenesis, which is associated with cardiac disorders and heart failure. Accordingly, we have generated spontaneously beating CMs from induced pluripotent stem cells (iPSC) derived from patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), which is an inherited and severe cardiac disease. Ca2+ cycling studies have revealed substantial abnormalities in these CMs. Ca2+ transient analysis performed manually lacks accepted analysis criteria, and has both low throughput and high variability. To overcome these issues, we have developed a software tool, AnomalyExplorer based on interactive visualization, to assist in the classification of Ca2+ transient patterns detected in CMs. Here, we demonstrate the usability and capability of the software, and we also compare the analysis efficiency to manual analysis. We show that AnomalyExplorer is suitable for detecting normal and abnormal Ca2+ transients; furthermore, this method provides more defined and consistent information regarding the Ca2+ abnormality patterns and cell line specific differences when compared to manual analysis. This tool will facilitate and speed up the analysis of CM Ca2+ transients, making it both more accurate and user-independent. AnomalyExplorer can be exploited in Ca2+ cycling analysis to study basic disease pathology and the effects of different drugs.

Virtual reality methods and equipment can be used to create stimulations for several psychophysiological measurements. Such stimulations can be flexibly modified and their versatility is wide; in principle even such could be prepared that are not possible in the real physical world like virtual flying in the space. For the generation of virtual reality stimulations we constructed a three-dimensional (3D) graphics engine to measure body balance variations in response to emotional stimulation. The stimulation was implemented as a virtual reality scene, where the subject watched a street view with a human figure approaching the subject at a constant speed. The functioning of the system was tested by measuring body balance variations while the approaching figure's facial expression was angry, neutral, or happy. The results showed that our young, healthy subjects were slightly, but still statistically significantly affected by the stimulation of the approaching figure. Thus, the whole system offers a tool for controlled experimental studies for analyzing body balance, for example, during stimulating approach-withdrawal behavior. There are several opportunities to further develop new visual psychophysiological stimulations and test forms.

We constructed a connectionist model with multilayer perceptron networks for the simulation of word production errors in the Finnish language. Such errors occur as slips of the tongue in healthy subjects and even more often in aphasic patients suffering from brain damage. Word production errors are of theoretical interest as they open an avenue to inner workings of the language production system. Here we present our coding schemes for semantic and phoneme information of words, investigate the general properties of the model, and compare the results of the model against empirical naming error data from ten aphasic patients. The model was reasonable in simulating word production errors in this heterogeneous group of aphasic patients.

Functional genomics methods are used to investigate the huge amount of information contained in genomes. Numerous experimental methods rely on the use of oligo- or polynucleotides. Nucleotide strand hybridization forms the underlying principle for these methods. For all these techniques, the probes should be unique for analyzed genes. In addition to being unique for the studied genes, the probes should fulfill a large number of criteria to be usable and valid. The criteria include for example, avoidance of self-annealing, suitable melting temperature and nucleotide composition. We developed a method for searching unique and valid oligonucleotides or probes for genes so that there is not even a similar (approximate) occurrence in any other location of the whole genome. By using probe size 25, we analyzed 17 complete genomes representing a wide range of both prokaryotic and eukaryotic organisms. More than 92% of all the genes in the investigated genomes contained valid oligonucleotides. Extensive statistical tests were performed to characterize the properties of unique and valid oligonucleotides. Unique and valid oligonucleotides were relatively evenly distributed in genes except for the beginning and end, which were somewhat overrepresented. The flanking regions in eukaryotes were clearly underrepresented among suitable oligonucleotides. In addition to distributions within genes, the effects on codon and amino acid usage were also studied.

Mutations in NOD2 have been shown to associate with increased susceptibility to Crohn's disease. A recent Polish study linked the truncating NOD2 3020insC variant with an increased risk of colorectal cancer (CRC) at an older age (>50 years) of disease onset, with an odds ratio of 2.23. We studied the possible contribution of the 3020insC variant to CRC risk in a series of 1,042 Finnish population-based patients from which 926 samples were successfully analyzed and in 348 anonymous cancer-free controls. The frequency of the 3020insC mutation was 3.7% in both CRC patients (34 of 926, including 1 homozygote) and cancer-free controls (13 of 348; odds ratio, 0.98; confidence interval, 0.51-1.88). Contrary to the Polish study, there was no significant difference in the mutation rates between CRC patients > 50 years of age (25 of 576; 4.3%) and controls in the present series. We studied respective tumor tissue DNAs of all patients displaying heterozygous 3020insC changes for loss of heterozygosity. Loss of heterozygosity at NOD2 was observed in only 1 of the 33 CRC samples. Our results suggest that NOD2 3020insC alone does not contribute to CRC risk. If this variant predisposes to CRC, additional factors not present in the Finnish population need to be involved.

Hereditary paraganglioma syndrome has recently been shown to be caused by germline heterozygous mutations in three (SDHB, SDHC, and SDHD) of the four genes that encode mitochondrial succinate dehydrogenase. Extraparaganglial component neoplasias have never been previously documented. In a population-based registry of symptomatic presentations of phaeochromocytoma/paraganglioma comprising 352 registrants, among whom 16 unrelated registrants were SDHB mutation positive, one family with germline SDHB mutation c.847-50delTCTC had two members with renal cell carcinoma (RCC), of solid histology, at ages 24 and 26 years. Both also had paraganglioma. A registry of early-onset RCCs revealed a family comprising a son with clear-cell RCC and his mother with a cardiac tumor, both with the germline SDHB R27X mutation. The cardiac tumor proved to be a paraganglioma. All RCCs showed loss of the remaining wild-type allele. Our observations suggest that germline SDHB mutations can predispose to early-onset kidney cancers in addition to paragangliomas and carry implications for medical surveillance.

People relying much on vision in the control of posture are known to have an elevated risk of falling. Dependence on visual control is an important parameter in the diagnosis of balance disorders. We have previously shown that virtual reality (VR) methods can be used to produce visual stimuli that affect balance, but suitable stimuli need to be found. In this study, the effect of six different VR stimuli on the balance of 22 healthy test subjects was evaluated using force platform posturography. We report in more detail and expand the results published earlier. According to the tests two of the stimuli have a significant destabilizing effect on balance. In addition a significant displacement effect on the subject's center of pressure (COP) was found. Thus it is shown that the design of VR stimuli to cause different effects on the control of balance is possible.

The concentration and histological distribution of hyaluronan, a tumor promoting extracellular matrix polysaccharide, and the activity of hyaluronidase, a potential source of angiogenic hyaluronan oligosaccharides, were analyzed in malignant epithelial (n = 24), borderline (n = 8), benign epithelial (n = 20), functional cyst (n = 21), and normal (n = 5) tissue samples of human ovary. Hyaluronan concentration increased specifically in cancers (P = 0.001), particularly in grade 3 tumors (>49-fold) and in metastases (>89-fold). Hyaluronan staining in the tissues correlated with hyaluronan concentration (P = 0.002). Hyaluronidase activity slightly decreased from semimalignant through low grade to high grade tumors (P = 0.041). Therefore, hyaluronan accumulation, but not hyaluronidase activation, is associated with the aggressiveness of ovarian epithelial cancer.

Purpose: Cyclooxygenase (Cox) is the key enzyme in conversion of arachidonic acid to prostanoids. Two Cox genes have been cloned, and expression of Cox-2 mRNA and protein has been shown to be elevated in several human malignancies and in animal models of carcinogenesis. The purpose of this study was to investigate Cox-2 protein expression in human gastric dysplasias and adenocarcinomas., Experimental Design: Performance of several Cox-2 antibodies was evaluated, after which Cox-2 protein expression was studied in 67 gastric cancer specimens and in eight definitive dysplasias by using immunohistochemistry., Results: Cox-2 positivity was detected in 58% (25/43) of the intestinal-type (well-differentiated) tumors and 6% (1/18) of diffuse-type (poorly differentiated) tumors. Consistent with these data, we detected higher expression of Cox-2 mRNA, protein, and enzymatic activity in well-differentiated gastric cancer cell lines (MKN-28 and MKN-74) when compared with poorly differentiated cell lines (HSC-39 and KATO III). Cox-2 immunoreactivity was localized to the carcinoma cells, but the stroma of the tumors was negative. However, strong Cox-2 positivity was consistently detected in stromal cells at sites of erosions and ulcerations. Furthermore, four of nine (44%) definitive dysplasias of the stomach that showed no evidence of invasion were positive for Cox-2., Conclusions: Cox-2 is expressed by the neoplastic cells in the intestinal-type gastric adenocarcinoma and by precarcinogenic (dysplastic) lesions leading to this disease.

A replication error (RER) phenotype has been documented both in sporadic colorectal tumors and in tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC). In the current study 8 of 49 (16%) sporadic colorectal cancers (CRCs) and 25 of 29 (86%) CRCs from HNPCC patients were found to be RER+. All 9 (100%) CRCs from HNPCC patients with germline mutations of the mismatch repair gene MSH2 were found to be RER+, while 16 of 20 CRCs from HNPCC kindreds unlinked or not studied for linkage to MSH2 were RER+. Corresponding analysis in colorectal adenomas revealed that only 1 of 33 (3%) sporadic tumors but 8 of 14 (57%) HNPCC tumors were RER+. Moreover, RER was found in all 6 extracolonic cancers (endometrium, 2; kidney, 1; stomach, 1; duodenum, 1; and ovary, 1) derived from members of HNPCC families. These data suggest the involvement of mismatch repair deficiency in the premalignant stage of tumorigenesis in HNPCC cases, and suggest that mismatch repair genes (MSH2 or others) are defective in the germline of nearly all these patients.