Human in vitro reporter model of neuronal development and early differentiation processes

<p>Abstract</p> <p>Background</p> <p>During developmental and adult neurogenesis, doublecortin is an early neuronal marker expressed when neural stem cells assume a neuronal cell fate. To understand mechanisms involved in early processes of neuronal fate decision, we investigated cell lines for their capacity to induce expression of doublecortin upon neuronal differentiation and develop <it>in vitro </it>reporter models using doublecortin promoter sequences.</p> <p>Results</p> <p>Among various cell lines investigated, the human teratocarcinoma cell line NTERA-2 was found to fulfill our criteria. Following induction of differentiation using retinoic acid treatment, we observed a 16-fold increase in doublecortin mRNA expression, as well as strong induction of doublecortin polypeptide expression. The acquisition of a neuronal precursor phenotype was also substantiated by the establishment of a multipolar neuronal morphology and expression of additional neuronal markers, such as Map2, βIII-tubulin and neuron-specific enolase. Moreover, stable transfection in NTERA-2 cells of reporter constructs encoding fluorescent or luminescent genes under the control of the doublecortin promoter allowed us to directly detect induction of neuronal differentiation in cell culture, such as following retinoic acid treatment or mouse Ngn2 transient overexpression.</p> <p>Conclusion</p> <p>Induction of doublecortin expression in differentiating NTERA-2 cells suggests that these cells accurately recapitulate some of the very early events of neuronal determination. Hence, the use of reporter genes under the control of the doublecortin promoter in NTERA-2 cells will help us to investigate factors involved early in the course of neuronal differentiation processes. Moreover the ease to detect the induction of a neuronal program in this model will permit to perform high throughput screening for compounds acting on the early neuronal differentiation mechanisms.</p

Characterization of human metapneumoviruses isolated from patients in North America.

Human metapneumovirus (HMPV) was recently identified in The Netherlands and was linked to acute respiratory tract illness. In this study, 11 isolates from 10 patients with respiratory disease from Quebec, Canada, were tested by a reverse-transcriptase polymerase chain reaction based on the fusion protein gene. Identified sequences were consistent with HMPV. The patients were 2 months to 87 years of age (median age, 58 years) and presented with acute respiratory tract illness during the winter season. Sequence studies of the nucleocapsid, fusion, and polymerase genes identified 2 main lineages of HMPV and cocirculation of both lineages during the same year. These findings support a previous finding that HMPV is a human respiratory pathogen that merits further study

A cross scale investigation of galena oxidation and controls on mobilization of lead in mine waste rock.

Abstract Galena and Pb-bearing secondary phases are the main sources of Pb in the terrestrial environment. Oxidative dissolution of galena releases aqueous Pb and SO4 to the surficial environment and commonly causes the formation of anglesite (in acidic environments) or cerussite (in alkaline environments). However, conditions prevalent in weathering environments are diverse and different reaction mechanisms reflect this variability at various scales. Here we applied complementary techniques across a range of scales, from nanometers to 10 s of meters, to study the oxidation of galena and accumulation of secondary phases that influence the release and mobilization of Pb within a sulfide-bearing waste-rock pile. Within the neutral-pH pore-water environment, the oxidation of galena releases Pb ions resulting in the formation of secondary Pb-bearing carbonate precipitates. Cerussite is the dominant phase and shannonite is a possible minor phase. Dissolved Cu from the pore water reacts at the surface of galena, forming covellite at the interface. Nanometer scale characterization suggests that secondary covellite is intergrown with secondary Pb-bearing carbonates at the interface. A small amount of the S derived from galena is sequestered with the secondary covellite, but the majority of the S is oxidized to sulfate and released to the pore water

Optimal Fleet Composition via Dynamic Programming and Golden Section Search

In this paper, we consider an optimization problem arising in vehicle fleet management. The problem is to construct a heterogeneous vehicle fleet in such a way that cost is minimized subject to a constraint on the overall fleet size. The cost function incorporates fixed and variable costs associated with the fleet, as well as hiring costs that are incurred when vehicle requirements exceed fleet capacity. We first consider the simple case when there is only one type of vehicle. We show that in this case the cost function is convex, and thus the problem can be solved efficiently using the well-known golden section method. We then devise an algorithm, based on dynamic programming and the golden section method, for solving the general problem in which there are multiple vehicle types. We conclude the paper with some simulation results

c-Myc Regulates Self-Renewal in Bronchoalveolar Stem Cells

BACKGROUND: Bronchoalveolar stem cells (BASCs) located in the bronchoalveolar duct junction are thought to regenerate both bronchiolar and alveolar epithelium during homeostatic turnover and in response to injury. The mechanisms directing self-renewal in BASCs are poorly understood. METHODS: BASCs (Sca-1(+), CD34(+), CD31(-) and, CD45(-)) were isolated from adult mouse lung using FACS, and their capacity for self-renewal and differentiation were demonstrated by immunostaining. A transcription factor network of 53 genes required for pluripotency in embryonic stem cells was assessed in BASCs, Kras-initiated lung tumor tissue, and lung organogenesis by real-time PCR. c-Myc was knocked down in BASCs by infection with c-Myc shRNA lentivirus. Comprehensive miRNA and mRNA profiling for BASCs was performed, and significant miRNAs and mRNAs potentially regulated by c-Myc were identified. We explored a c-Myc regulatory network in BASCs using a number of statistical and computational approaches through two different strategies; 1) c-Myc/Max binding sites within individual gene promoters, and 2) miRNA-regulated target genes. RESULTS: c-Myc expression was upregulated in BASCs and downregulated over the time course of lung organogenesis in vivo. The depletion of c-Myc in BASCs resulted in decreased proliferation and cell death. Multiple mRNAs and miRNAs were dynamically regulated in c-Myc depleted BASCs. Among a total of 250 dynamically regulated genes in c-Myc depleted BASCs, 57 genes were identified as potential targets of miRNAs through miRBase and TargetScan-based computational mapping. A further 88 genes were identified as potential downstream targets through their c-Myc binding motif. CONCLUSION: c-Myc plays a critical role in maintaining the self-renewal capacity of lung bronchoalveolar stem cells through a combination of miRNA and transcription factor regulatory networks

A Systems Biology Approach Identifies Molecular Networks Defining Skeletal Muscle Abnormalities in Chronic Obstructive Pulmonary Disease

Chronic Obstructive Pulmonary Disease (COPD) is an inflammatory process of the lung inducing persistent airflow limitation. Extensive systemic effects, such as skeletal muscle dysfunction, often characterize these patients and severely limit life expectancy. Despite considerable research efforts, the molecular basis of muscle degeneration in COPD is still a matter of intense debate. In this study, we have applied a network biology approach to model the relationship between muscle molecular and physiological response to training and systemic inflammatory mediators. Our model shows that failure to co-ordinately activate expression of several tissue remodelling and bioenergetics pathways is a specific landmark of COPD diseased muscles. Our findings also suggest that this phenomenon may be linked to an abnormal expression of a number of histone modifiers, which we discovered correlate with oxygen utilization. These observations raised the interesting possibility that cell hypoxia may be a key factor driving skeletal muscle degeneration in COPD patients

Short-Term Environmental Enrichment Enhances Adult Neurogenesis, Vascular Network and Dendritic Complexity in the Hippocampus of Type 1 Diabetic Mice

Background: Several brain disturbances have been described in association to type 1 diabetes in humans. In animal models, hippocampal pathological changes were reported together with cognitive deficits. The exposure to a variety of environmental stimuli during a certain period of time is able to prevent brain alterations and to improve learning and memory in conditions like stress, aging and neurodegenerative processes. Methodology/Principal Findings: We explored the modulation of hippocampal alterations in streptozotocin-induced type 1 diabetic mice by environmental enrichment. In diabetic mice housed in standard conditions we found a reduction of adult neurogenesis in the dentate gyrus, decreased dendritic complexity in CA1 neurons and a smaller vascular fractional area in the dentate gyrus, compared with control animals in the same housing condition. A short exposure-10 days- to an enriched environment was able to enhance proliferation, survival and dendritic arborization of newborn neurons, to recover dendritic tree length and spine density of pyramidal CA1 neurons and to increase the vascular network of the dentate gyrus in diabetic animals. Conclusions/Significance: The environmental complexity seems to constitute a strong stimulator competent to rescue th

The Effect of a DNA Repair Gene on Cellular Invasiveness: Xrcc3 Over-Expression in Breast Cancer Cells

Over-expression of DNA repair genes has been associated with resistance to radiation and DNA-damage induced by chemotherapeutic agents such as cisplatin. More recently, based on the analysis of genome expression profiling, it was proposed that over-expression of DNA repair genes enhances the invasive behaviour of tumour cells. In this study we present experimental evidence utilizing functional assays to test this hypothesis. We assessed the effect of the DNA repair proteins known as X-ray complementing protein 3 (XRCC3) and RAD51, to the invasive behavior of the MCF-7 luminal epithelial-like and BT20 basal-like triple negative human breast cancer cell lines. We report that stable or transient over-expression of XRCC3 but not RAD51 increased invasiveness in both cell lines in vitro. Moreover, XRCC3 over-expressing MCF-7 cells also showed a higher tumorigenesis in vivo and this phenotype was associated with increased activity of the metalloproteinase MMP-9 and the expression of known modulators of cell-cell adhesion and metastasis such as CD44, ID-1, DDR1 and TFF1. Our results suggest that in addition to its' role in facilitating repair of DNA damage, XRCC3 affects invasiveness of breast cancer cell lines and the expression of genes associated with cell adhesion and invasion