The Transcriptional Corepressor NAB2 Inhibits NGF-induced Differentiation of PC12 Cells

The PC12 pheochromocytoma cell line responds to NGF by undergoing growth arrest and proceeding to differentiate toward a neuronal phenotype. Among the early genetic events triggered by NGF in PC12 cells are the rapid activation of the zinc finger transcription factor Egr1/NGFI-A, and a slightly delayed induction of NAB2, a corepressor that inhibits Egr1 transcriptional activity. We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF. Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection. Early events in the NGF signaling cascade, such as activation of MAP kinase and induction of immediate-early genes, were unaltered in the NAB2-overexpressing PC12 cell lines. However, induction of delayed NGF response genes such as TGF-beta 1 and MMP-3 was inhibited. Furthermore, NAB2 overexpression led to downregulation of p21WAF1, a molecule previously shown to play a pivotal role in the ability of PC12 cells to undergo growth arrest and commit to differentiation in response to NGF. Cotransfection with p21WAF1 restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF

NGAL: an emerging tool for predicting severity of AKI is easily detected by a clinical assay

Background\ud The authors have previously shown that urine neutrophil gelatinase-associated lipocalin (NGAL), measured by a research ELISA, is an early predictive biomarker of acute kidney injury (AKI) after cardiopulmonary bypass (CPB). The availability of a standardized clinical platform for NGAL measurements could revolutionize renal diagnostics, especially in intensive care situations.\ud \ud Methods\ud Objective\ud The first objective of the present study was to determine whether an NGAL immunoassay developed for a standardized clinical platform (ARCHITECT® analyzer, Abbott Diagnostics) correlates with the research-based assay. The second objective was to determine the utility of the standardized NGAL immunoassay as a predictive biomarker of AKI after CPB in a large prospective cohort.\ud \ud Design\ud Prospective cohort study\ud \ud Setting\ud Children's hospital at a large US academic medical center.\ud \ud Subjects\ud 196 children undergoing elective CPB for surgical correction or palliation of congenital heart lesions between January 2004 and June 2006.\ud \ud Intervention\ud None.\ud \ud Outcomes\ud The primary outcome variable was the development of AKI, defined as a 50% or greater increase in serum creatinine from baseline. Other outcomes included percent change in serum creatinine, days in AKI, dialysis requirement, length of hospital stay, and mortality.\ud \ud Results\ud In a pilot study with 136 urine samples (NGAL range, 0.3 to 815 ng/ml) and 6 calibration standards (NGAL range, 0 to 1000 ng/ml), NGAL measurements by research ELISA and by the ARCHITECT assay were highly correlated (r = 0.99). In a subsequent study, serial urine NGAL measurements were obtained by ARCHITECT assay. Of the 196 children undergoing CPB, AKI developed in 99 patients (51%), but the diagnosis using serum creatinine was delayed by 2 to 3 d after CPB. In contrast, mean urine NGAL levels increased 15-fold within 2 h and by 25-fold at 4 and 6 h after CPB. For the 2-h urine NGAL measurement, the area under the curve was 0.95, sensitivity was 0.82, and the specificity was 0.90 for prediction of AKI using a cutoff value of 100 ng/ml. The 2-h urine NGAL levels correlated with severity and duration of AKI, length of stay, dialysis requirement, and death.\ud \ud Conclusions\ud Accurate measurements of urine NGAL are obtained using the ARCHITECT® platform. Urine NGAL is an early predictive biomarker of AKI severity after CPB

Haploinsufficiency at the Nkx3.1 locus A paradigm for stochastic, dosage-sensitive gene regulation during tumor initiation

AbstractTumorigenesis requires sequential accumulation of multiple genetic lesions. In the prostate, tumor initiation is often linked to loss of heterozygosity at the Nkx3.1 locus. In mice, loss of even one Nkx3.1 allele causes prostatic epithelial hyperplasia and eventual prostatic intraepithelial neoplasia (PIN) formation. Here we demonstrate that Nkx3.1 allelic loss extends the proliferative stage of regenerating luminal cells, leading to epithelial hyperplasia. Microarray analysis identified Nkx3.1 target genes, many of which show exquisite dosage sensitivity. The number of Nkx3.1 alleles determines the relative probabilities of stochastic activation or inactivation of a given target gene. Thus, loss of a single Nkx3.1 allele likely results in hyperplasia and PIN by increasing the probability of completely inactivating select Nkx3.1-regulated pathways within a subset of affected cells

A model of toxic neuropathy in Drosophila reveals a role for MORN4 in promoting axonal degeneration

Axonal degeneration is a molecular self-destruction cascade initiated following traumatic, toxic, and metabolic insults. Its mechanism underlies a number of disorders including hereditary and diabetic neuropathies and the neurotoxic side effects of chemotherapy drugs. Molecules that promote axonal degeneration could represent potential targets for therapy. To identify such molecules, we designed a screening platform based on intoxication of Drosophila larvae with paclitaxel (taxol), a chemotherapeutic agent that causes neuropathy in cancer patients. In Drosophila, taxol treatment causes swelling, fragmentation, and loss of axons in larval peripheral nerves. This axonal loss is not due to apoptosis of neurons. Taxol-induced axonal degeneration in Drosophila shares molecular execution mechanisms with vertebrates, including inhibition by both NMNAT (nicotinamide mononucleotide adenylyltransferase) expression and loss of wallenda/DLK (dual leucine zipper kinase). In a pilot RNAi-based screen we found that knockdown of retinophilin (rtp), which encodes a MORN (membrane occupation and recognition nexus) repeat-containing protein, protects axons from degeneration in the presence of taxol. Loss-of-function mutants of rtp replicate this axonal protection. Knockdown of rtp also delays axonal degeneration in severed olfactory axons. We demonstrate that the mouse ortholog of rtp, MORN4, promotes axonal degeneration in mouse sensory axons following axotomy, illustrating conservation of function. Hence, this new model can identify evolutionarily conserved genes that promote axonal degeneration, and so could identify candidate therapeutic targets for a wide-range of axonopathies

Geographically Based Hydrogen Consumer Demand and Infrastructure Analysis: Final Report

In FY 2004 and 2005, NREL developed a proposed minimal infrastructure to support nationwide deployment of hydrogen vehicles by offering infrastructure scenarios that facilitated interstate travel. This report identifies key metropolitan areas and regions on which to focus infrastructure efforts during the early hydrogen transition

Treating Proximal Tibial Growth Plate Injuries Using Poly(Lactic-co-Glycolic Acid) Scaffolds

Growth plate fractures account for nearly 18.5% of fractures in children. Depending on the type and severity of the injury, inhibited bone growth or angular deformity caused by bone forming in place of the growth plate can occur. The current treatment involves removal of the bony bar and replacing it with a filler substance, such as a free fat graft. Unfortunately, reformation of the bony bar frequently occurs, preventing the native growth plate from regenerating. The goal of this pilot study was to determine whether biodegradable scaffolds can enhance native growth plate regeneration following a simulated injury that resulted in bony bar formation in the proximal tibial growth plate of New Zealand white rabbits. After removing the bony bar, animals received one of the following treatments: porous poly(lactic-co-glycolic acid) (PLGA) scaffold; PLGA scaffold loaded with insulin-like growth factor I (IGF-I); PLGA scaffold loaded with IGF-I and seeded with autogenous bone marrow cells (BMCs) harvested at the time of implantation; or fat graft (as used clinically). The PLGA scaffold group showed an increased chondrocyte population and a reduced loss of the remaining native growth plate compared to the fat graft group (the control group). An additional increase in chondrocyte density was seen in scaffolds loaded with IGF-I, and even more so when BMCs were seeded on the scaffold. While there was no significant reduction in the angular deformation of the limbs, the PLGA scaffolds increased the amount of cartilage and reduced the amount of bony bar reformation

Hydrogen Resource Assessment: Hydrogen Potential from Coal, Natural Gas, Nuclear, and Hydro Power

This paper estimates the quantity of hydrogen that could be produced from coal, natural gas, nuclear, and hydro power by county in the United States. The study estimates that more than 72 million tonnes of hydrogen can be produced from coal, natural gas, nuclear, and hydro power per year in the country (considering only 30% of their total annual production). The United States consumed about 396 million tonnes of gasoline in 2007; therefore, the report suggests the amount of hydrogen from these sources could displace about 80% of this consumption

Gene therapy targeting SARM1 blocks pathological axon degeneration in mice

Axonal degeneration (AxD) following nerve injury, chemotherapy, and in several neurological disorders is an active process driven by SARM1, an injury-activated NADase. Axons of SARM1-null mice exhibit greatly delayed AxD after transection and in models of neurological disease, suggesting that inhibiting SARM1 is a promising strategy to reduce pathological AxD. Unfortunately, no drugs exist to target SARM1. We, therefore, developed SARM1 dominant-negatives that potently block AxD in cellular models of axotomy and neuropathy. To assess efficacy in vivo, we used adeno-associated virus-mediated expression of the most potent SARM1 dominant-negative and nerve transection as a model of severe AxD. While axons of vehicle-treated mice degenerate rapidly, axons of mice expressing SARM1 dominant-negative can remain intact for \u3e10 d after transection, similar to the protection observed in SARM1-null mice. We thus developed a novel in vivo gene therapeutic to block pathological axon degeneration by inhibiting SARM1, an approach that may be applied clinically to treat manifold neurodegenerative diseases characterized by axon loss

Regional Consumer Hydrogen Demand and Optimal Hydrogen Refueling Station Siting

Using a GIS approach to spatially analyze key attributes affecting hydrogen market transformation, this study proposes hypothetical hydrogen refueling station locations in select subregions to demonstrate a method for determining station locations based on geographic criteria

Abnormal microglia and enhanced inflammation-related gene transcription in mice with conditional deletion of Ctcf in Camk2a-Cre-expressing neurons

CCCTC-binding factor (CTCF) is an 11 zinc finger DNA-binding domain protein that regulates gene expression by modifying 3D chromatin structure. Human mutations inCTCFcause intellectual disability and autistic features. Knocking outCtcfin mouse embryonic neurons is lethal by neonatal age, but the effects of CTCF deficiency in postnatal neurons are less well studied. We knocked outCtcfpostnatally in glutamatergic forebrain neurons under the control ofCamk2a-Cre. CtcfloxP/loxP;Camk2a-Cre+(CtcfCKO) mice of both sexes were viable and exhibited profound deficits in spatial learning/memory, impaired motor coordination, and decreased sociability by 4 months of age.CtcfCKO mice also had reduced dendritic spine density in the hippocampus and cerebral cortex. Microarray analysis of mRNA fromCtcfCKO mouse hippocampus identified increased transcription of inflammation-related genes linked to microglia. Separate microarray analysis of mRNA isolated specifically fromCtcfCKO mouse hippocampal neurons by ribosomal affinity purification identified upregulation of chemokine signaling genes, suggesting crosstalk between neurons and microglia inCtcfCKO hippocampus. Finally, we found that microglia inCtcfCKO mouse hippocampus had abnormal morphology by Sholl analysis and increased immunostaining for CD68, a marker of microglial activation. Our findings confirm thatCtcfKO in postnatal neurons causes a neurobehavioral phenotype in mice and provide novel evidence that CTCF depletion leads to overexpression of inflammation-related genes and microglial dysfunction.SIGNIFICANCE STATEMENTCCCTC-binding factor (CTCF) is a DNA-binding protein that organizes nuclear chromatin topology. Mutations inCTCFcause intellectual disability and autistic features in humans. CTCF deficiency in embryonic neurons is lethal in mice, but mice with postnatal CTCF depletion are less well studied. We find that mice lackingCtcfinCamk2a-expressing neurons (CtcfCKO mice) have spatial learning/memory deficits, impaired fine motor skills, subtly altered social interactions, and decreased dendritic spine density. We demonstrate thatCtcfCKO mice overexpress inflammation-related genes in the brain and have microglia with abnormal morphology that label positive for CD68, a marker of microglial activation. Our findings suggest that inflammation and dysfunctional neuron–microglia interactions are factors in the pathology of CTCF deficiency.</jats:p