Distance and direction, but not light cues, support response reversal learning.

Across three experiments, we examined the cuing properties of metric (distance and direction) and nonmetric (lighting) cues in different tasks. In Experiment 1, rats were trained on a response problem in a T-maze, followed by four reversals. Rats that experienced a change in maze orientation (Direction group) or a change in the length of the start arm (Distance group) across reversals showed facilitation of reversal learning relative to a group that experienced changes in room lighting across reversals. In Experiment 2, rats learned a discrimination task more readily when distance or direction cues were used than when light cues were used as the discriminative stimuli. In Experiment 3, performance on a go/no-go task was equivalent using both direction and lighting cues. The successful use of both metric and nonmetric cues in the go/no-go task indicates that rats are sensitive to both types of cues and that the usefulness of different cues is dependent on the nature of the task.

Monoallelic ABCC8 mutations are a common cause of diazoxide-unresponsive diffuse form of congenital hyperinsulinism.

ABCC8 encodes a subunit of the ß-cell potassium channel (KATP ) whose loss of function is responsible for congenital hyperinsulinism (CHI). Patients with two recessive mutations of ABCC8 typically have severe diffuse forms of CHI unresponsive to diazoxide. Some dominant ABCC8 mutations are responsible for a subset of diffuse diazoxide-unresponsive forms of CHI. We report the analysis of 21 different ABCC8 mutations identified in 25 probands with diazoxide-unresponsive diffuse CHI and carrying a single mutation in ABCC8. Nine missense ABCC8 mutations were subjected to in vitro expression studies testing traffic efficiency and responses of mutant channels to activation by MgADP and diazoxide. Eight of the 9 missense mutations exhibited normal trafficking. Seven of the 8 mutants reaching the plasma membrane had dramatically reduced response to MgADP or to diazoxide (<10% of wild-type response). In our cohort, dominant KATP mutations account for 22% of the children with diffuse unresponsive-diazoxide CHI. Their clinical phenotype being indistinguishable from that of children with focal CHI and diffuse CHI forms due to two recessive KATP mutations, we show that functional testing is essential to make the most reliable diagnosis and offer appropriate genetic counseling.

Investigating the performance and cost-effectiveness of the simple ultrasound-based rules compared to the risk of malignancy index in the diagnosis of ovarian cancer (SUBSONiC-study): protocol of a prospective multicenter cohort study in the Netherlands.

BACKGROUND: Estimating the risk of malignancy is essential in the management of adnexal masses. An accurate differential diagnosis between benign and malignant masses will reduce morbidity and costs due to unnecessary operations, and will improve referral to a gynecologic oncologist for specialized cancer care, which improves outcome and overall survival. The Risk of Malignancy Index is currently the most commonly used method in clinical practice, but has a relatively low diagnostic accuracy (sensitivity 75-80% and specificity 85-90%). Recent reports show that other methods, such as simple ultrasound-based rules, subjective assessment and (Diffusion Weighted) Magnetic Resonance Imaging might be superior to the RMI in the pre-operative differentiation of adnexal masses. METHODS/DESIGN: A prospective multicenter cohort study will be performed in the south of The Netherlands. A total of 270 women diagnosed with at least one pelvic mass that is suspected to be of ovarian origin who will undergo surgery, will be enrolled. We will apply the Risk of Malignancy Index with a cut-off value of 200 and a two-step triage test consisting of simple ultrasound-based rules supplemented -if necessary- with either subjective assessment by an expert sonographer or Magnetic Resonance Imaging with diffusion weighted sequences, to characterize the adnexal masses. The histological diagnosis will be the reference standard. Diagnostic performances will be expressed as sensitivity, specificity, positive and negative predictive values and likelihood ratios. DISCUSSION: We hypothesize that this two-step triage test, including the simple ultrasound-based rules, will have better diagnostic accuracy than the Risk of Malignancy Index and therefore will improve the management of women with adnexal masses. Furthermore, we expect this two-step test to be more cost-effective. If the hypothesis is confirmed, the results of this study could have major effects on current guidelines and implementation of the triage test in daily clinical practice could be a possibility. TRIAL REGISTRATION: ClinicalTrials.gov: registration number NCT02218502.

Genetic analysis of ageing: role of oxidative damage and environmental stresses.

Evolutionary theory predicts substantial interspecific and intraspecific differences in the proximal mechanisms of ageing. Our goal here is to seek evidence for common ('public') mechanisms among diverse organisms amenable to genetic analysis. Oxidative damage is a candidate for such a public mechanism of ageing. Long-lived strains are relatively resistant to different environmental stresses. The extent to which these stresses produce oxidative damage remains to be established.

The Werner syndrome protein is a DNA helicase.

Werner syndrome (WS) is an uncommon autosomal recessive disorder characterized by premature aging. The clinical manifestations of WS, including atherosclerosis and osteoporosis, appear early in adulthood, and death in the fourth to sixth decade commonly ensues from myocardial infarction or cancer. In accord with the aging phenotype, cells from WS patients have a reduced replicative life span in culture. Genomic instability is observed at the cytogenetic level in the form of chromosome breaks and translocations and at the molecular level by multiple large deletions. The Werner syndrome gene (WRN) has recently been cloned. The predicted product is a 1,432-amino-acid protein whose central domain is homologous to members of the RecQ family of DNA helicases. Such homology does not necessarily mean that WRN encodes an active helicase. For example, the Saccharomyces cerevisiae RAD26 gene protein and the human transcription-repair coupling factor CSB (Cockayne syndrome 8) are highly homologous to known helicases, yet neither encodes an active helicase. Moreover, the Bloom's syndrome gene (BLM), discovered before WRN, is also homologous to the RecQ family of DNA helicases, though we still await demonstration that it encodes an active helicase. Here we report that the WS protein does indeed catalyze DNA unwinding.

Increased vulnerability of hippocampal neurons to excitotoxic necrosis in presenilin-1 mutant knock-in mice.

Excitotoxicity, a form of neuronal injury in which excessive activation of glutamate receptors results in cellular calcium overload, has been implicated in the pathogenesis of Alzheimer disease (AD), although direct evidence is lacking. Mutations in the presenilin-1 (PS1) gene on chromosome 14 are causally linked to many cases of early-onset inherited AD (refs. 5,6). We generated PS1 mutant mice (PS1M146VKI) that express the PS1 M146V targeted allele at normal physiological levels. Although PS1M146VKI mice have no overt mutant phenotype, they are hypersensitive to seizure-induced synaptic degeneration and necrotic neuronal death in the hippocampus. Cultured hippocampal neurons from PS1M146VKI mice have increased vulnerability to death induced by glutamate, which is correlated with perturbed calcium homeostasis, increased oxidative stress and mitochondrial dysfunction. Agents that suppress calcium influx or release and antioxidants protect neurons against the excitotoxic action of the PS1 mutation. These findings establish a direct link between a genetic defect that causes AD and excitotoxic neuronal degeneration, and indicate new avenues for therapeutic intervention in AD patients.

Protocol for translabial 3D-ultrasonography for diagnosing levator defects (TRUDIL): a multicentre cohort study for estimating the diagnostic accuracy of translabial 3D-ultrasonography of the pelvic floor as compared to MR imaging.

BACKGROUND: Pelvic organ prolapse (POP) is a condition affecting more than half of the women above age 40. The estimated lifetime risk of needing surgical management for POP is 11%. In patients undergoing POP surgery of the anterior vaginal wall, the re-operation rate is 30%. The recurrence risk is especially high in women with a levator ani defect. Such defect is present if there is a partially or completely detachment of the levator ani from the inferior ramus of the symphysis. Detecting levator ani defects is relevant for counseling, and probably also for treatment. Levator ani defects can be imaged with MRI and also with Translabial 3D ultrasonography of the pelvic floor. The primary aim of this study is to assess the diagnostic accuracy of translabial 3D ultrasonography for diagnosing levator defects in women with POP with Magnetic Resonance Imaging as the reference standard. Secondary goals of this study include quantification of the inter-observer agreement about levator ani defects and determining the association between levator defects and recurrent POP after anterior repair. In addition, the cost-effectiveness of adding translabial ultrasonography to the diagnostic work-up in patients with POP will be estimated in a decision analytic model. METHODS/DESIGN: A multicentre cohort study will be performed in nine Dutch hospitals. 140 consecutive women with a POPQ stage 2 or more anterior vaginal wall prolapse, who are indicated for anterior colporapphy will be included. Patients undergoing additional prolapse procedures will also be included. Prior to surgery, patients will undergo MR imaging and translabial 3D ultrasound examination of the pelvic floor. Patients will be asked to complete validated disease specific quality of life questionnaires before surgery and at six and twelve months after surgery. Pelvic examination will be performed at the same time points. Assuming a sensitivity and specificity of 90% of 3D ultrasound for diagnosing levator defects in a population of 120 women with POP, with a prior probability of levator ani defects of 40%, we will be able to estimate predictive values with good accuracy (i.e. confidence limits of at most 10% below or above the point estimates of positive and negative predictive values).Anticipating 3% unclassifiable diagnostic images because of technical reasons, and a further safety margin of 10% we plan to recruit 140 patients. TRIAL REGISTRATION: Nederlands trial register NTR2220.

Reinitiation of DNA synthesis in senescent human fibroblasts upon fusion with cells of unlimited growth potential.

Postreplicative, "senescent" human fibroblasts were fused to HeLa or to SV-40 transformed human fibroblasts with Sendai virus. DNA synthesis was reinitiated in senescent nuclei in a high proportion of the heterodikaryons. The [3H]thymidine labeling index of senescent fibroblast nuclei in heteropolykaryons was a function of the ratio of HeLa to senescent nuclei.

Parasexual cycle in cultivated human somatic cells.

Somatic segregation for three different autosomes was demonstrated in two strains of human diploid fibroblasts derived from subjects known to be heterozygous for chromosomal variants. Recombinant diploid cells appeared within cultures of tetraploid clones isolted from mass cultures. Tetraploid cells regularly occur in mass cultures and within clones of diploid cells. Such a parasexual cycle (2n-->4n-->2n), with recombination of entire linkage groups, could from the basis of a beginning formal genetic analysis in man.

Positional cloning of the Werner's syndrome gene.

Werner's syndrome (WS) is an inherited disease with clinical symptoms resembling premature aging. Early susceptibility to a number of major age-related diseases is a key feature of this disorder. The gene responsible for WS (known as WRN) was identified by positional cloning. The predicted protein is 1432 amino acids in length and shows significant similarity to DNA helicases. Four mutations in WS patients were identified. Two of the mutations are splice-junction mutations, with the predicted result being the exclusion of exons from the final messenger RNA. One of the these mutations, which results in a frameshift and a predicted truncated protein, was found in the homozygous state in 60 percent of Japanese WS patients examined. The other two mutations are nonsense mutations. The identification of a mutated putative helicase as the gene product of the WS gene suggests that defective DNA metabolism is involved in the complex process of aging in WS patients.

Deficient neurogenesis in forebrain-specific presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces.

To examine the in vivo function of presenilin-1 (PS1), we selectively deleted the PS1 gene in excitatory neurons of the adult mouse forebrain. These conditional knockout mice were viable and grew normally, but they exhibited a pronounced deficiency in enrichment-induced neurogenesis in the dentate gyrus. This reduction in neurogenesis did not result in appreciable learning deficits, indicating that addition of new neurons is not required for memory formation. However, our postlearning enrichment experiments lead us to postulate that adult dentate neurogenesis may play a role in the periodic clearance of outdated hippocampal memory traces after cortical memory consolidation, thereby ensuring that the hippocampus is continuously available to process new memories. A chronic, abnormal clearance process in the hippocampus may conceivably lead to memory disorders in the mammalian brain.

Induction of adenine salvage in mouse cell lines deficient in adenine phosphoribosyltransferase.

Adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) pseudorevertant cell lines were isolated under selective conditions requiring adenine salvage for survival; yet they were found to be deficient in measurable APRT activity and resistant to the purine analog 2'6'-diaminopurine (DAP) (M.S. Turker, J. A. Tischfield, P. Rabinovitch, P.J. Stambrook, J.J. Trill, A.C. Smith, C.E. Ogburn, and G.M. Martin, manuscript in preparation). Adenine salvage was examined in two APRT pseudorevertant cell lines, their two APRT homozygous deficient parental cell lines, and a genotypic APRT revertant cell line (i.e., one with measurable APRT activity and DAP sensitivity). Adenine accumulation was observed in both revertant phenotypes and was demonstrated by high-performance liquid chromatography to be linked with adenine metabolism. The ability to salvage adenine declined substantially in the pseudorevertant cell lines when they were removed from selective media containing inhibitors of de novo 5'-AMP synthesis (alanosine and azaserine); for one pseudorevertant cell line this decline was accelerated by the addition of DAP to the medium. The readdition of alanosine or azaserine to the growth medium of the pseudorevertant lines induced adenine salvage to its previous levels. An APRT-like cross-reacting material was found in the pseudorevertant cell lines, although its relationship to adenine salvage is unknown. A low level of constitutive adenine salvage was found in the parental APRT-deficient lines, and it was also possible to induce adenine salvage in these cell lines. These findings suggest a novel regulatory mechanism for adenine salvage.

The Werner syndrome protein is involved in RNA polymerase II transcription.

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)-dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40-60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II-dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid-protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.

Mitochondrial-Targeted Catalase: Extended Longevity and the Roles in Various Disease Models.

The free-radical theory of aging was proposed more than 50 years ago. As one of the most popular mechanisms explaining the aging process, it has been extensively studied in several model organisms. However, the results remain controversial. The mitochondrial version of free-radical theory of aging proposes that mitochondria are both the primary sources of reactive oxygen species (ROS) and the primary targets of ROS-induced damage. One critical ROS is hydrogen peroxide, which is naturally degraded by catalase in peroxisomes or glutathione peroxidase within mitochondria. Our laboratory developed mice-overexpressing catalase targeted to mitochondria (mCAT), peroxisomes (pCAT), or the nucleus (nCAT) in order to investigate the role of hydrogen peroxide in different subcellular compartments in aging and age-related diseases. The mCAT mice have demonstrated the largest effects on life span and healthspan extension. This chapter will discuss the mCAT phenotype and review studies using mCAT to investigate the roles of mitochondrial oxidative stresses in various disease models, including metabolic syndrome and atherosclerosis, cardiac aging, heart failure, skeletal muscle pathology, sensory defect, neurodegenerative diseases, and cancer. As ROS has been increasingly recognized as essential signaling molecules that may be beneficial in hormesis, stress response and immunity, the potential pleiotropic, or adverse effects of mCAT are also discussed. Finally, the development of small-molecule mitochondrial-targeted therapeutic approaches is reviewed.

Machine learning-based models to predict modes of toxic action of phenols to Tetrahymena pyriformis.

The phenols are structurally heterogeneous pollutants and they present a variety of modes of toxic action (MOA), including polar narcotics, weak acid respiratory uncouplers, pro-electrophiles, and soft electrophiles. Because it is often difficult to determine correctly the mechanism of action of a compound, quantitative structure-activity relationship (QSAR) methods, which have proved their interest in toxicity prediction, can be used. In this work, several QSAR models for the prediction of MOA of 221 phenols to the ciliated protozoan Tetrahymena pyriformis, using Chemistry Development Kit descriptors, are reported. Four machine learning techniques (ML), k-nearest neighbours, support vector machine, classification trees, and artificial neural networks, have been used to develop several models with higher accuracies and predictive capabilities for distinguishing between four MOAs. They showed global accuracy values between 95.9% and 97.7% and area under Receiver Operator Curve values between 0.978 and 0.998; additionally, false alarm rate values were below 8.2% for training set. In order to validate our models, cross-validation (10-folds-out) and external test-set were performed with good behaviour in all cases. These models, obtained with ML techniques, were compared with others previously reported by other researchers, and the improvement was significant.

Flow cytometry and scrape-loading/dye transfer as a rapid quantitative measure of intercellular communication in vitro.

We describe two flow cytometric assays performed on populations of cells which have been stained with various fluorescent tracer molecules by the scrape-loading technique. One assay uses a simple one-color analysis on a flow cytometer by quantitating the fluorescence intensity of scrape-loaded lucifer yellow CH (LY) in individual cells. The other assay utilizes a two-color analysis on a cell sorter whereby cells which are initially loaded (donors) are identified by their uptake of both rhodamine isothiocyanate-dextran and LY, whereas the recipients of dye transfer are identified as having LY only. Agents which have been shown to inhibit intercellular communication in other assays exhibit similar blocking activity in LY transfer and this is readily quantitated by flow cytometry. The two-color analysis has the added advantage of being able to identify both donors and recipients in a highly quantitative manner.

Preparation of cell-size unilamellar liposomes with high captured volume and defined size distribution.

Cell-size unilamellar vesicles were made by removing solvents from microscopic chloroform spherules containing smaller water droplets within. The average diameter of the vesicles in a typical preparation was 9.2 mum, comparable to that of human erythrocytes (7 mum). The standard deviation of the size distribution was 3.0 mum. The unilamellarity and bilayer unit membrane of vesicles were demonstrated by transmission electron microscopy. Materials so far successfully incorporated into vesicles include glucose, sucrose, Arsenazo III and Ponceau S dyes, thymidine triphosphate, methotrexate, agarose, collagen, ferritin, polyadenylic aid, DNA, and whole bacteria. The captured volume per milligram of lipids (up to 144 microliter/mg) was almost an order of magnitude greater than the highest value reported in the literature to date (up to 15.6 microliter/mg) (Szoka, F.C. and Papahadjopoulos, D. (1978) Proc. Natl. Acad, Sci. U.S.A. 75, 4194-4198).

Preparation of multivesicular liposomes.

A novel type of liposome, named here multivesicular liposomes, was prepared by evaporation of organic solvents from chloroform-ether spherules suspended in water. Within each spherule were numerous water droplets that contained solutes to be trapped in liposomes upon solvent evaporation. Liposome preparations of different average diameters were made, varying from 29 +/- 10 microns to 5.6 +/- 1.7 microns. The liposomes were morphologically characterized by light microscopy and transmission electron microscopy. Materials successfully trapped within the liposomes ranged in molecular size from glucose to nucleic acids. Extremely high percentages of encapsulation (up to 89%) were achieved.

Cellular Werner phenotypes in mice expressing a putative dominant-negative human WRN gene.

Mutations at the Werner helicase locus (WRN) are responsible for the Werner syndrome (WS). WS patients prematurely develop an aged appearance and various age-related disorders. We have generated transgenic mice expressing human WRN with a putative dominant-negative mutation (K577M-WRN). Primary tail fibroblast cultures from K577M-WRN mice showed three characteristics of WS cells: hypersensitivity to 4-nitroquinoline-1-oxide (4NQO), reduced replicative potential, and reduced expression of the endogenous WRN protein. These data suggest that K577M-WRN mice may provide a novel mouse model for the WS.

QuBiLs-MAS method in early drug discovery and rational drug identification of antifungal agents.

The QuBiLs-MAS approach is used for the in silico modelling of the antifungal activity of organic molecules. To this effect, non-stochastic (NS) and simple-stochastic (SS) atom-based quadratic indices are used to codify chemical information for a comprehensive dataset of 2478 compounds having a great structural variability, with 1087 of them being antifungal agents, covering the broadest antifungal mechanisms of action known so far. The NS and SS index-based antifungal activity classification models obtained using linear discriminant analysis (LDA) yield correct classification percentages of 90.73% and 92.47%, respectively, for the training set. Additionally, these models are able to correctly classify 92.16% and 87.56% of 706 compounds in an external test set. A comparison of the statistical parameters of the QuBiLs-MAS LDA-based models with those for models reported in the literature reveals comparable to superior performance, although the latter were built over much smaller and less diverse datasets, representing fewer mechanisms of action. It may therefore be inferred that the QuBiLs-MAS method constitutes a valuable tool useful in the design and/or selection of new and broad spectrum agents against life-threatening fungal infections.