Development of a New Approach to Aid in Visual Identification of Murine iPS Colonies Using a Fuzzy Logic Decision Support System

The a priori identification of induced pluripotent stem cells remains a challenge. Being able to quickly identify the most embryonic stem cell-similar induced pluripotent stem cells when validating results could help to reduce costs and save time. In this context, tools based on non-classic logic can be useful in creating aid-systems based on visual criteria. True colonies when viewed at 100x magnification have been found to have the following 3 characteristics: a high degree of border delineation, a more uniform texture, and the absence of a cracked texture. These visual criteria were used for fuzzy logic modeling. We investigated the possibility of predicting the presence of alkaline phosphatase activity, typical of true induced pluripotent stem cell colonies, after 25 individuals, with varying degrees of experience in working with murine iPS cells, categorized the images of 136 colonies based on visual criteria. Intriguingly, the performance evaluation by area under the ROC curve (16 individuals with satisfactory performance), Spearman correlation (all statistically significant), and Cohen''s Kappa agreement analysis (all statistically significant) demonstrates that the discriminatory capacity of different evaluators are similar, even those who have never cultivated cells. Thus, we report on a new system to facilitate visual identification of murine- induced pluripotent stem cell colonies that can be useful for staff training and opens the possibility of exploring visual characteristics of induced pluripotent stem cell colonies with their functional peculiarities. The fuzzy model has been integrated as a web-based tool named “2see-iPS” which is freely accessed at http://genetica.incor.usp.br/2seeips/.     

Thiocyanate concentration in saliva of cystic fibrosis patients

Thiocyanates (SCN-) are ubiquitous in nature. There are indispensable part of host defense system that act as a substrate for lactoperoxidase (LPO). In our study we present initial data on SCN- concentration in saliva of CF patients in comparison to healthy non-smokers and healthy smokers. 5 ml of saliva was collected from each subject to a sterile tube and thiocyanate concentration was measured in each sample. The results of the measurements are presented on Fig. 1. Mean concentration of SCN- in saliva of CF patients was 0.031 +/- 0.0052 g/l, in healthy non-smokers 0.039 +/- 0.0048 g/l and in healthy smokers 0.048 +/- 0.0161 g/l. The differences between each group were statistically significant. Studies on larger group of patients and probably on different material (BALF or induced sputum) should present interesting data complementing the in vitro studies.     

A model investigation of the impact of ventilation-perfusion mismatch on oxygenation during apnea in preterm infants

Ventilation-perfusion (V/Q) mismatch is a prominent feature of preterm infants and adults with lung disease. V/Q mismatch is known to cause arterial hypoxemia under steady-state conditions, and has been proposed as the cause of rapid arterial oxygen desaturation during apnea. However, there is little evidence to support a role for V/Q mismatch in the dynamic changes in arterial oxygenation that occur during apnea. Using a mathematical model, we quantified the effect of V/Q mismatch on the rate of desaturation during apnea to ascertain whether it could lead to rates of up to 10% s^-1 as observed in preterm infants. We used a lung-body model for the preterm infant that incorporated 50 parallel alveolar-capillary units that were ventilated and perfused with the severity of V/Q mismatch (σ) defined conventionally according to σ=S.D. of the distribution of V/Q ratios. Average desaturation rate 10 s from apnea onset was strongly elevated with worsening V/Q mismatch as a result of an earlier desaturation of low V/Q units compared with high V/Q units. However, V/Q mismatch had little impact after apnea onset, with peak desaturation rate only substantially increased if mismatching caused a lowered resting arterial O₂ saturation. In conclusion, V/Q mismatch causes a more immediate onset of desaturation during apnea, and therefore places preterm infants and adults with lung disease at risk of hypoxemic dips. However, V/Q mismatch does not accelerate desaturation rate beyond apnea onset and cannot, therefore, explain the rapid desaturation observed during recurrent apnea in preterm infants.     

Leptin increases maternal investment

The primary goal of virtually all organisms is to produce genetic offspring, thereby passing on their genes to future generations. Offspring production, however, is limited by available resources within an environment. Moreover, distributing sufficient energy among competing physiological systems is challenging and can result in trade-offs between self-maintenance and offspring investment when resources are limited. In the current study, we tested the hypothesis that the adipose hormone leptin is involved in mediating energetic trade-offs between competing physiological systems. Specifically, we tested the effects of elevated maternal leptin on investment into offspring production versus self maintenance (immune function), in the Siberian hamster (Phodopus sungorus). The current study provides the first evidence that leptin serves as a signal to mothers of available energy resulting in epigenetic effects. Therefore, elevated leptin allows females to retain more embryos to parturition, and rear more offspring to weaning via reduced maternal infanticide. Innate immune response was suppressed seemingly as a result of these enlarged litters, suggesting that the observed fitness increase is not without costs to the mother. Collectively, these findings suggest that leptin plays a critical role in allowing mothers to determine how much energy to invest in the production and care of young versus self-maintenance.     

Influence of the nature of the sterol on the behavior of palmitic acid/sterol mixtures and their derived liposomes

The phase behavior of mixtures formed with palmitic acid (PA) and one of the following sterols (dihydrocholesterol, ergosterol, 7-dehydrocholesterol, stigmasterol and stigmastanol), in a PA/sterol molar ratio of 3/7, has been characterized by IR and ²H NMR spectroscopy at different pH. Our study shows that it is possible to form liquid-ordered (lo) lamellar phases with these binary non-phospholipid mixtures. The characterization of alkyl chain dynamics of PA in these systems revealed the large ordering effect of the sterols. It was possible to extrude these systems, using standard extrusion techniques, to form large unilamellar vesicles (LUVs), except in the case of ergosterol-containing mixture. The resulting LUVs displayed a very limited passive permeability consistent with the high sterol concentration. In addition, the stability of these PA/sterol self-assembled bilayers was also found to be pH-sensitive, therefore, potentially useful as nanovectors. By examining different sterols, we could establish some correlations between the structure of these bilayers and their permeability properties. The structure of the side chain at C17 of the sterol appears to play a prime role in the mixing properties with fatty acid.     

The relationship between different forms of human alpha-mannosidase.

The tissue distribution and some properties of human alpha-mannosidase (alpha-D-mannoside mannohydrolase EC 3.2.1.24) have been studied. The acidic forms of the enzyme were fairly stable, whereas the neutral forms easily lost enzymic activity. The acidic forms were sensitive to neuraminidase but the neutral forms were unaffected. The experiments indicate that the acidic components are closely related to each other, differing only in sialic acid content and possibly conformation. The neutral forms of the enzyme are probably quite different from the acidic forms both in structure and cellular function.     

A MACROMUTATION IN TRIPSACUM DACTYLOIDES (POACEAE): CONSEQUENCES FOR SEED SIZE,GERMINATION, AND SEEDLING ESTABLISHMENT

A recessive allele of a gene in Tripsacum dactyloides L. (eastern gamagrass) changes staminate florets to pistillate or hermaphrodite, and restores fertility to suppressed florets. There were ten to 25 times more seeds in the mutant pistillate form, and these were 0.32 to 0.59 times smaller than seeds from the normal form. Seeds from pistillate plants had significantly lower germination rates (22% vs. 50%), and seedlings grew 20% slower than those of normal plants in a greenhouse experiment. Pistillate seedling survival rates were lower in both high- (18.8% vs. 62.6%) and low- (52.8% vs. 72.6%) competition environments in a field experiment, and surviving seedlings were smaller. The maternal parent of volunteer seedlings next to a plantation of normal and pistillate plants was determined by dissecting the attached fruitcases of 1,313 seedlings. Pistillate plants in the plantation produced 90% of all seeds falling on the site but only 29% of the volunteer seedlings. The pistillate macromutation is not likely to spread in the wild due to morphological constraints on seed size and packaging.     

Central Nervous System Pathology Progresses Independently of KC and CXCR2 in Globoid-Cell Leukodystrophy

Globoid-cell Leukodystrophy (GLD; Krabbe’s disease) is a rapidly progressing inherited demyelinating disease caused by a deficiency of the lysosomal enzyme Galactosylceramidase (GALC). Deficiency of GALC leads to altered catabolism of galactosylceramide and the cytotoxic lipid, galactosylsphingosine (psychosine). This leads to a rapidly progressive fatal disease with spasticity, cognitive disability and seizures. The murine model of GLD (Twitcher; GALC−/−) lacks the same enzyme and has similar clinical features. The deficiency of GALC leads to oligodendrocyte death, profound neuroinflammation, and the influx of activated macrophages into the CNS. We showed previously that keratinocyte chemoattractant factor (KC) is highly elevated in the CNS of untreated Twitcher mice and significantly decreases after receiving a relatively effective therapy (bone marrow transplantation combined with gene therapy). The action of KC is mediated through the CXCR2 receptor and is a potent chemoattractant for macrophages and microglia. KC is also involved in oligodendrocyte migration and proliferation. Based on the commonalities between the disease presentation and the functions of KC, we hypothesized that KC and/or CXCR2 contribute to the pathogenesis of GLD. Interestingly, the course of the disease is not significantly altered in KC- or CXCR2-deficient Twitcher mice. There is also no alteration in inflammation or demyelination patterns in these mice. Furthermore, transplantation of CXCR2-deficient bone marrow does not alter the progression of the disease as it does in other models of demyelination. This study highlights the role of multiple redundant cytokines and growth factors in the pathogenesis of GLD.     

Selective Media for the Detection of Revertants in Cultures of Glycogen-deficient Mutants

Glycogen-deficient yeast mutants grew more slowly than wild-type cells on certain nonfermentable carbon substrates. Revertant colonies could be detected on these lines.