Molecular signatures of proliferation and quiescence in hematopoietic stem cells

Stem cells resident in adult tissues are principally quiescent, yet harbor enormous capacity for proliferation to achieve self renewal and to replenish their tissue constituents. Although a single hematopoietic stem cell (HSC) can generate sufficient primitive progeny to repopulate many recipients, little is known about the molecular mechanisms that maintain their potency or regulate their self renewal. Here we have examined the gene expression changes that occur over a time course when HSCs are induced to proliferate and return to quiescence in vivo. These data were compared to data representing differences between naturally proliferating fetal HSCs and their quiescent adult counterparts. Bioinformatic strategies were used to group time-ordered gene expression profiles generated from microarrays into signatures of quiescent and dividing stem cells. A novel method for calculating statistically significant enrichments in Gene Ontology groupings for our gene lists revealed elemental subgroups within the signatures that underlie HSC behavior, and allowed us to build a molecular model of the HSC activation cycle. Initially, quiescent HSCs evince a state of readiness. The proliferative signal induces a preparative state, which is followed by active proliferation divisible into early and late phases. Re-induction of quiescence involves changes in migratory molecule expression, prior to reestablishment of homeostasis. We also identified two genes that increase in both gene and protein expression during activation, and potentially represent new markers for proliferating stem cells. These data will be of use in attempts to recapitulate the HSC self renewal process for therapeutic expansion of stem cells, and our model may correlate with acquisition of self renewal characteristics by cancer stem cells.     

Organ slices for the evaluation of human drug toxicity

Human organ slices, an in vitro model representing the multicellular and functional features of in vivo tissue, is a promising model for characterizing mechanisms of drug-induced organ injury and for identifying biomarkers of organ injury. Target organ injury is a significant clinical issue. In vitro models, which compare human and animal tissue to improve the extrapolation of animal in vivo studies for predicting human outcome, will contribute to improving drug candidate selection and to defining species susceptibilities in drug discovery and development programs. A critical aspect to the performance and outcome of human organ slice studies is the use of high quality tissue, and the use of culture conditions that support optimum organ slice survivability, in order to accurately reproduce mechanisms of organ injury in vitro. The attribute of organ slices possessing various cell types and interactions contributes to the overall biotransformation, inflammatory response and assessment of injury. Regional differences and changes in morphology can be readily evaluated by histology and special stains, similar to tissue obtained from in vivo studies. The liver is the major organ of which slice studies have been performed, however the utility of extra-hepatic derived slices, as well as co-cultures is increasing. Recent application of integrating gene expression, with human organ slice function and morphology demonstrate the increased potential of this model for defining the molecular and biochemical pathways leading to drug-induced tissue changes. By gaining a more detailed understanding of the mechanisms of drug-induced organ injury, and by correlating clinical measurements with drug-induced effects in the in vitro models, the vision of human in vitro models to identify more sensitive and discriminating markers of organ damage is attainable.     

Musashi Protein-directed Translational Activation of Target mRNAs Is Mediated by the Poly(A) Polymerase,Germ Line Development Defective-2

The mRNA-binding protein, Musashi, has been shown to regulate translation of select mRNAs and to control cellular identity in both stem cells and cancer cells. Within the mammalian cells, Musashi has traditionally been characterized as a repressor of translation. However, we have demonstrated that Musashi is an activator of translation in progesterone-stimulated oocytes of the frog Xenopus laevis, and recent evidence has revealed Musashi''s capability to function as an activator of translation in mammalian systems. The molecular mechanism by which Musashi directs activation of target mRNAs has not been elucidated. Here, we report a specific association of Musashi with the noncanonical poly(A) polymerase germ line development defective-2 (GLD2) and map the association domain to 31 amino acids within the C-terminal domain of Musashi. We show that loss of GLD2 interaction through deletion of the binding domain or treatment with antisense oligonucleotides compromises Musashi function. Additionally, we demonstrate that overexpression of both Musashi and GLD2 significantly enhances Musashi function. Finally, we report a similar co-association also occurs between murine Musashi and GLD2 orthologs, suggesting that coupling of Musashi to the polyadenylation apparatus is a conserved mechanism to promote target mRNA translation.     

Allosteric drugs: the interaction of antitumor compound MKT-077 with human Hsp70 chaperones

Hsp70 (heat shock protein 70 kDa) chaperones are key to cellular protein homeostasis. However, they also have the ability to inhibit tumor apoptosis and contribute to aberrant accumulation of hyperphosphorylated tau in neuronal cells affected by tauopathies, including Alzheimer's disease. Hence, Hsp70 chaperones are increasingly becoming identified as targets for therapeutic intervention in these widely abundant diseases. Hsp70 proteins are allosteric machines and offer, besides classical active-site targets, also opportunities to target the mechanism of allostery. In this work, it is demonstrated that the action of the potent anticancer compound MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride) occurs through a differential interaction with Hsp70 allosteric states. MKT-077 is therefore an “allosteric drug.” Using NMR spectroscopy, we identify the compound's binding site on human HSPA8 (Hsc70). The binding pose is obtained from NMR-restrained docking calculations, subsequently scored by molecular-dynamics-based energy and solvation computations. Suggestions for the improvement of the compound's properties are made on the basis of the binding location and pose.     

Which States Matter? An Application of an Intelligent Discretization Method to Solve a Continuous POMDP in Conservation Biology

When managing populations of threatened species, conservation managers seek to make the best conservation decisions to avoid extinction. Making the best decision is difficult because the true population size and the effects of management are uncertain. Managers must allocate limited resources between actively protecting the species and monitoring. Resources spent on monitoring reduce expenditure on management that could be used to directly improve species persistence. However monitoring may prevent sub-optimal management actions being taken as a result of observation error. Partially observable Markov decision processes (POMDPs) can optimize management for populations with partial detectability, but the solution methods can only be applied when there are few discrete states. We use the Continuous U-Tree (CU-Tree) algorithm to discretely represent a continuous state space by using only the states that are necessary to maintain an optimal management policy. We exploit the compact discretization created by CU-Tree to solve a POMDP on the original continuous state space. We apply our method to a population of sea otters and explore the trade-off between allocating resources to management and monitoring. We show that accurately discovering the population size is less important than management for the long term survival of our otter population.     

Maximising return on conservation investment in the conterminous USA

Efficient conservation planning requires knowledge about conservation targets, threats to those targets, costs of conservation and the marginal return to additional conservation efforts. Systematic conservation planning typically only takes a small piece of this complex puzzle into account. Here, we use a return‐on‐investment (ROI) approach to prioritise lands for conservation at the county level in the conterminous USA. Our approach accounts for species richness, county area, the proportion of species' ranges already protected, the threat of land conversion and land costs. Areas selected by a complementarity‐based greedy heuristic using our full ROI approach provided greater averted species losses per dollar spent compared with areas selected by heuristics accounting for richness alone or richness and cost, and avoided acquiring lands not threatened with conversion. In contrast to traditional prioritisation approaches, our results highlight conservation bargains, opportunities to avert the threat of development and places where conservation efforts are currently lacking.     

Effects of Taurine Supplementation on Hepatic Markers of Inflammation and Lipid Metabolism in Mothers and Offspring in the Setting of Maternal Obesity

Maternal obesity is associated with obesity and metabolic disorders in offspring. However, intervention strategies to reverse or ameliorate the effects of maternal obesity on offspring health are limited. Following maternal undernutrition, taurine supplementation can improve outcomes in offspring, possibly via effects on glucose homeostasis and insulin secretion. The effects of taurine in mediating inflammatory processes as a protective mechanism has not been investigated. Further, the efficacy of taurine supplementation in the setting of maternal obesity is not known. Using a model of maternal obesity, we examined the effects of maternal taurine supplementation on outcomes related to inflammation and lipid metabolism in mothers and neonates. Time-mated Wistar rats were randomised to either: 1) control : control diet during pregnancy and lactation (CON); 2) CON supplemented with 1.5% taurine in drinking water (CT); 3) maternal obesogenic diet (high fat, high fructose) during pregnancy and lactation (MO); or 4) MO supplemented with taurine (MOT). Maternal and neonatal weights, plasma cytokines and hepatic gene expression were analysed. A MO diet resulted in maternal hyperinsulinemia and hyperleptinemia and increased plasma glucose, glutamate and TNF-α concentrations. Taurine normalised maternal plasma TNF-α and glutamate concentrations in MOT animals. Both MO and MOT mothers displayed evidence of fatty liver accompanied by alterations in key markers of hepatic lipid metabolism. MO neonates displayed a pro-inflammatory hepatic profile which was partially rescued in MOT offspring. Conversely, a pro-inflammatory phenotype was observed in MOT mothers suggesting a possible maternal trade-off to protect the neonate. Despite protective effects of taurine in MOT offspring, neonatal mortality was increased in CT neonates, indicating possible adverse effects of taurine in the setting of normal pregnancy. These data suggest that maternal taurine supplementation may ameliorate the adverse effects observed in offspring following a maternal obesogenic diet but these effects are dependent upon prior maternal nutritional background.     

Low utilization of circulating glucose after food withdrawal in Snell dwarf mice

Glucose metabolism is altered in long-lived people and mice. Although it is clear that there is an association between altered glucose metabolism and longevity, it is not known whether this link is causal or not. Our current hypothesis is that decreased fasting glucose utilization may increase longevity by reducing oxygen radical production, a potential cause of aging. We observed that whole body fasting glucose utilization was lower in the Snell dwarf, a long-lived mutant mouse. Whole body fasting glucose utilization may be reduced by a decrease in the production of circulating glucose. Our isotope labeling analysis indicated both gluconeogenesis and glycogenolysis were suppressed in Snell dwarfs. Elevated circulating adiponectin may contribute to the reduction of glucose production in Snell dwarfs. Adiponectin lowered the appearance of glucose in the media over hepatoma cells by suppressing gluconeogenesis and glycogenolysis. The suppression of glucose production by adiponectin in vitro depended on AMP-activated protein kinase, a cell mediator of fatty acid oxidation. Elevated fatty acid oxidation was indicated in Snell dwarfs by increased utilization of circulating oleic acid, reduced intracellular triglyceride content, and increased phosphorylation of acetyl-CoA carboxylase. Finally, protein carbonyl content, a marker of oxygen radical damage, was decreased in Snell dwarfs. The correlation between high glucose utilization and elevated oxygen radical production was also observed in vitro by altering the concentrations of glucose and fatty acids in the media or pharmacologic inhibition of glucose and fatty acid oxidation with 4-hydroxycyanocinnamic acid and etomoxir, respectively.