Functional Genomics Characterisation of the Genetic Pathways that Control Kinetochore Function

Kinetochores serve both a structural role linking chromosomes to the mitotic spindle and a regulatory role, controlling the timing of mitosis via the spindle assembly checkpoint. Defects in kinetochore function can lead to aneuploidy, a hallmark of cancer cells. To understand how cells regulate the segregation of their chromosomes during cell division it is important to understand which genes regulate kinetochores function. I have examined the distribution of the outer kinetochore complex DAM1/DASH complex in the budding yeast Saccharomyces cerevisiae using fluorescence imaging. I modified the endogenous DAD4 and SPC42 alleles to introduce the gene encoding yellow fluorescent protein (YFP) and red fluorescent protein (RFP) respectively. I have used the synthetic genetic array technology to transfer these two alleles into all of the ~6000 non-essential gene deletions and essential temperature sensitive mutants in yeast. Fluorescence microscopy of these mutant strains has allowed me to examine quantitative and qualitative kinetochore phenotypes. First, I assessed the levels of YFP fluorescence intensity at the kinetochore as a surrogate for protein concentration to determine changes in DAM1/DASH complex homeostasis. Then, I annotated abnormal distributions of YFP fluorescence by visualisation. I have identified a number of mutants that alter Dad4 homeostasis. For example, ~3% of the non-essential deletions and ~27% of essential conditional alleles produce either an increase or a decrease in Dad4 intensity. I have also identified mutants that cause a mislocalisation of Dad4, some of which represent mitotic spindle defects. Finally, I have investigated the mechanism underlying the phenotype caused by a kinase from the cell wall integrity pathway, Pkc1, and its connection to the kinetochore via the microtubule and actin cytoskeleton. This study highlights the importance of chromatin remodeling complexes, RNA processing enzymes and a number of protein kinases in maintaining kinetochore homeostasis

Antioxidant effects of vitamins in type 2 diabetes: A meta-analysis of randomized controlled trials

© 2018 The Author(s). Background: Vitamins are essential micronutrients with antioxidant potential that may provide a complementary treatment for patients with chronic diseases. Our aim was to assess the effect of vitamin supplementation on the antioxidant status and glycemic index of type 2 diabetes mellitus patients. Methods: We performed a systematic review with meta-analyses. Electronic searches were conducted in PubMed, Scopus, and Web of Science (December 2017). Randomized controlled trials evaluating the effect of any vitamin or vitamin complex supplementation on antioxidant status as primary outcome were included. The outcomes considered were: reduction of malondialdehyde (MDA); augmentation of glutathione peroxidase (GPx); changes in total antioxidant capacity (TAC), enhance in superoxide dismutase enzyme - SOD, and thiobarbituric acid reactive substances (TBARS). Outcomes of glycemic control were also evaluated. Pairwise meta-analyses were performed using software Review Manager 5.3. Results: Thirty trials fulfilled the inclusion criteria, but only 12 could be included in the meta-analyses of antioxidant outcomes. The most commonly studied vitamins were B, C, D and E. Vitamin E was related to significant reduction of blood glucose as well as glycated hemoglobin compared to placebo, while both vitamins C and E were mainly associated with reducing MDA and TBARS and elevating GPx, SOD and TAC, compared to placebo. However, outcome reports in this field are still inconsistent (e.g. because of a lack of standard measures). Conclusions: Supplementation of vitamin E may be a valuable strategy for controlling diabetes complications and enhancing antioxidant capacity. The effects of other micronutrients should be further investigated in larger and well-designed trials to properly place these complementary therapies in clinical practice

Clostridium perfringens epsilon toxin increases the small intestinal permeability in mice and rats

Epsilon toxin is a potent neurotoxin produced by Clostridium perfringens types B and D, an anaerobic bacterium that causes enterotoxaemia in ruminants. In the affected animal, it causes oedema of the lungs and brain by damaging the endothelial cells, inducing physiological and morphological changes. Although it is believed to compromise the intestinal barrier, thus entering the gut vasculature, little is known about the mechanism underlying this process. This study characterizes the effects of epsilon toxin on fluid transport and bioelectrical parameters in the small intestine of mice and rats. The enteropooling and the intestinal loop tests, together with the single-pass perfusion assay and in vitro and ex vivo analysis in Ussing's chamber, were all used in combination with histological and ultrastructural analysis of mice and rat small intestine, challenged with or without C. perfringens epsilon toxin. Luminal epsilon toxin induced a time and concentration dependent intestinal fluid accumulation and fall of the transepithelial resistance. Although no evident histological changes were observed, opening of the mucosa tight junction in combination with apoptotic changes in the lamina propria were seen with transmission electron microscopy. These results indicate that C. perfringens epsilon toxin alters the intestinal permeability, predominantly by opening the mucosa tight junction, increasing its permeability to macromolecules, and inducing further degenerative changes in the lamina propria of the bowel. © 2009 Goldstein et al

Dioxin Toxicity In Vivo Results from an Increase in the Dioxin-Independent Transcriptional Activity of the Aryl Hydrocarbon Receptor

The Aryl hydrocarbon receptor (Ahr) is the nuclear receptor mediating the toxicity of dioxins -widespread and persistent pollutants whose toxic effects include tumor promotion, teratogenesis, wasting syndrome and chloracne. Elimination of Ahr in mice eliminates dioxin toxicity but also produces adverse effects, some seemingly unrelated to dioxin. Thus the relationship between the toxic and dioxin-independent functions of Ahr is not clear, which hampers understanding and treatment of dioxin toxicity. Here we develop a Drosophila model to show that dioxin actually increases the in vivo dioxin-independent activity of Ahr. This hyperactivation resembles the effects caused by an increase in the amount of its dimerisation partner Ahr nuclear translocator (Arnt) and entails an increased transcriptional potency of Ahr, in addition to the previously described effect on nuclear translocation. Thus the two apparently different functions of Ahr, dioxin-mediated and dioxin-independent, are in fact two different levels (hyperactivated and basal, respectively) of a single function

TOMOBFLOW: feature-preserving noise filtering for electron tomography

<p>Abstract</p> <p>Background</p> <p>Noise filtering techniques are needed in electron tomography to allow proper interpretation of datasets. The standard linear filtering techniques are characterized by a tradeoff between the amount of reduced noise and the blurring of the features of interest. On the other hand, sophisticated anisotropic nonlinear filtering techniques allow noise reduction with good preservation of structures. However, these techniques are computationally intensive and are difficult to be tuned to the problem at hand.</p> <p>Results</p> <p>TOMOBFLOW is a program for noise filtering with capabilities of preservation of biologically relevant information. It is an efficient implementation of the Beltrami flow, a nonlinear filtering method that locally tunes the strength of the smoothing according to an edge indicator based on geometry properties. The fact that this method does not have free parameters hard to be tuned makes TOMOBFLOW a user-friendly filtering program equipped with the power of diffusion-based filtering methods. Furthermore, TOMOBFLOW is provided with abilities to deal with different types and formats of images in order to make it useful for electron tomography in particular and bioimaging in general.</p> <p>Conclusion</p> <p>TOMOBFLOW allows efficient noise filtering of bioimaging datasets with preservation of the features of interest, thereby yielding data better suited for post-processing, visualization and interpretation. It is available at the web site <url>http://www.ual.es/%7ejjfdez/SW/tomobflow.html</url>.</p

Network 'small-world-ness': a quantitative method for determining canonical network equivalence

Background: Many technological, biological, social, and information networks fall into the broad class of 'small-world' networks: they have tightly interconnected clusters of nodes, and a shortest mean path length that is similar to a matched random graph (same number of nodes and edges). This semi-quantitative definition leads to a categorical distinction ('small/not-small') rather than a quantitative, continuous grading of networks, and can lead to uncertainty about a network's small-world status. Moreover, systems described by small-world networks are often studied using an equivalent canonical network model-the Watts-Strogatz (WS) model. However, the process of establishing an equivalent WS model is imprecise and there is a pressing need to discover ways in which this equivalence may be quantified. Methodology/Principal Findings: We defined a precise measure of 'small-world-ness' S based on the trade off between high local clustering and short path length. A network is now deemed a 'small-world' if S. 1-an assertion which may be tested statistically. We then examined the behavior of S on a large data-set of real-world systems. We found that all these systems were linked by a linear relationship between their S values and the network size n. Moreover, we show a method for assigning a unique Watts-Strogatz (WS) model to any real-world network, and show analytically that the WS models associated with our sample of networks also show linearity between S and n. Linearity between S and n is not, however, inevitable, and neither is S maximal for an arbitrary network of given size. Linearity may, however, be explained by a common limiting growth process. Conclusions/Significance: We have shown how the notion of a small-world network may be quantified. Several key properties of the metric are described and the use of WS canonical models is placed on a more secure footing

Building Babies - Chapter 16

In contrast to birds, male mammals rarely help to raise the offspring. Of all mammals, only among rodents, carnivores, and primates, males are sometimes intensively engaged in providing infant care (Kleiman and Malcolm 1981). Male caretaking of infants has long been recognized in nonhuman primates (Itani 1959). Given that infant care behavior can have a positive effect on the infant’s development, growth, well-being, or survival, why are male mammals not more frequently involved in “building babies”? We begin the chapter defining a few relevant terms and introducing the theory and hypotheses that have historically addressed the evolution of paternal care. We then review empirical findings on male care among primate taxa, before focusing, in the final section, on our own work on paternal care in South American owl monkeys (Aotus spp.). We conclude the chapter with some suggestions for future studies.Deutsche Forschungsgemeinschaft (HU 1746/2-1) Wenner-Gren Foundation, the L.S.B. Leakey Foundation, the National Geographic Society, the National Science Foundation (BCS-0621020), the University of Pennsylvania Research Foundation, the Zoological Society of San Dieg

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB

Newborn Screening for Homocystinuria Revealed a High Frequency of MAT I/III Deficiency in Iberian Peninsula

Acessível em: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4375120/Homocystinuria due to cystathionine β-synthase deficiency or "classical homocystinuria" is a rare autosomal recessive condition resulting in altered sulfur metabolism with elevated methionine and homocysteine in plasma and homocystine in urine. This condition is characterized by a high clinical heterogeneity, which contributes to late clinical diagnosis, usually only made after irreversible damage has occurred. Treatment is effective if started before clinical symptoms. The analysis of methionine levels by tandem mass spectrometry (MS/MS) allows the newborn screening for homocystinuria, but false-positive results can be frequently obtained and lead to the unwanted identification of methionine adenosyl transferase (MAT I/III) deficiency. This latter condition is biochemically characterized by isolated persistent hypermethioninemia, accompanied in some individuals with slightly elevated levels of homocysteine in plasma. A dominant form of MAT I/III deficiency, associated with mutation p.R264H, seems to be very frequent in the Iberian Peninsula and usually has a clinically benign course. Both these metabolic disorders are screened in Galicia and Portugal since the introduction of the MS/MS technology, in 2000 and 2004, respectively, resulting in the identification of three patients with classical homocystinuria and 44 patients with MAT I/III deficiency. All but one heterozygous parent of MAT I/III patients, identified with the p.R264H mutation, are healthy adults around the age of 30/40. The implementation of a second-tier test for homocysteine in dried blood spots would considerably reduce the number of MAT I/III-deficient patients identified and improve the specificity and positive predictive value for classical homocystinuria screening