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Other Publications (55)

Articles by Leslie Smith in JoVE

 JoVE Biology

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

1Department of Biochemistry and Molecular Biology, Knight Cancer Institute, Oregon Health & Science University


JoVE 4400

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Other articles by Leslie Smith on PubMed

Intravenous Infusions and Mastoiditis

Protective Vasomotor Effects of in Vivo Recombinant Endothelial Nitric Oxide Synthase Gene Expression in a Canine Model of Cerebral Vasospasm

Post-subarachnoid hemorrhage (SAH) cerebral vasospasm is a potentially devastating condition whose pathogenesis involves impaired nitric oxide (NO) bioavailability. We aimed to determine whether recombinant endothelial NO synthase (eNOS) gene expression may protect vasomotor function and prevent vasospasm in a canine experimental SAH model.

Using Beowulf Clusters to Speed Up Neural Simulations

Simulation of large neural systems on PCs requires large amounts of memory, and takes a long time. Parallel computers can speed them up. A new form of parallel computer, the Beowulf cluster, is an affordable version. Event-driven simulation and processor farming are two ways of exploiting this parallelism in neural simulations.

Long-term Vitamin C Treatment Increases Vascular Tetrahydrobiopterin Levels and Nitric Oxide Synthase Activity

In cultured endothelial cells, the antioxidant, L-ascorbic acid (vitamin C), increases nitric oxide synthase (NOS) enzyme activity via chemical stabilization of tetrahydrobiopterin. Our objective was to determine the effect of vitamin C on NOS function and tetrahydrobiopterin metabolism in vivo. Twenty-six to twenty-eight weeks of diet supplementation with vitamin C (1%/kg chow) significantly increased circulating levels of vitamin C in wild-type (C57BL/6J) and apolipoprotein E (apoE)--deficient mice. Measurements of NOS enzymatic activity in aortas of apoE-deficient mice indicated a significant increase in total NOS activity. However, this increase was mainly due to high activity of inducible NOS, whereas eNOS activity was reduced. Significantly higher tetrahydrobiopterin levels were detected in aortas of apoE-deficient mice. Long-term treatment with vitamin C restored endothelial NOS activity in aortas of apoE-deficient mice, but did not affect activity of inducible NOS. In addition, 7,8-dihydrobiopterin levels, an oxidized form of tetrahydrobiopterin, were decreased and vascular endothelial function of aortas was significantly improved in apoE-deficient mice. Interestingly, vitamin C also increased tetrahydrobiopterin and NOS activity in aortas of C57BL/6J mice. In contrast, long-term treatment with vitamin E (2000 U/kg chow) did not affect vascular NOS activity or metabolism of tetrahydrobiopterin. In vivo, beneficial effect of vitamin C on vascular endothelial function appears to be mediated in part by protection of tetrahydrobiopterin and restoration of eNOS enzymatic activity.

Characterization of a Novel Epigenetic Effect of Ionizing Radiation: the Death-inducing Effect

The detrimental effects associated with exposure to ionizing radiation have long been thought to result from the direct targeting of the nucleus leading to DNA damage; however, the emergence of concepts such as radiation-induced genomic instability and bystander effects have challenged this dogma. After cellular exposure to ionizing radiation, we have isolated a number of clones of Chinese hamster-human hybrid GM10115 cells that demonstrate genomic instability as measured by chromosomal destabilization. These clones show dynamic and persistent generation of chromosomal rearrangements multiple generations after the original insult. We hypothesize that these unstable clones maintain this delayed instability phenotype by secreting factors into the culture medium. To test this hypothesis we transferred filtered medium from unstable cells to unirradiated GM10115 cells. No GM10115 cells were able to survive this medium. This phenomenon by which GM10115 cells die when cultured in medium from chromosomally unstable GM10115 clones is the death-inducing effect. Medium transfer experiments indicate that a factor or factors is/are secreted by unstable cells within 8 h of growth in fresh medium and result in cell killing within 24 h. These factors are stable at ambient temperature but do not survive heating or freezing, and are biologically active when diluted with fresh medium. We present the initial description and characterization of the death-inducing effect. This novel epigenetic effect of radiation has implications for radiation risk assessment and for health risks associated with radiation exposure.

Protective Effect of Chronic Vitamin C Treatment on Endothelial Function of Apolipoprotein E-deficient Mouse Carotid Artery

Endothelium-dependent relaxations are impaired in carotid artery of apolipoprotein E-deficient (apoE-/-) mice. This impairment seems to be due to increased formation of superoxide anions and inactivation of endothelial nitric oxide (NO). In the present study, we tested hypothesis that chronic treatment with vitamin C may prevent endothelial dysfunction by increasing release of NO from endothelial cells. C57BL/6 and apoE-/- mice were treated for 26 weeks with Western-type fat diet with and without 1% vitamin C. Vasomotor function of isolated carotid arteries was studied by video dimension analyzer. Expression of endothelial NO synthase (eNOS) and platelet-endothelial cell adhesion molecule-1 (PECAM-1) protein were evaluated by Western blotting. Levels of cGMP and cAMP were measured by radioimmunoassay. In apoE-/- mice, vitamin C significantly augmented relaxations to acetylcholine (10-9-10-5 mol/l), but did not affect relaxations to NO donor diethylammonium-(Z)-1-(N,N-diethylamino) diazen-1-1,2-diolate (DEA-NONOate; 10-9-10-5 mol/l). In contrast, vitamin C reduced relaxations to acetylcholine and DEA-NONOate in C57BL/6 mice. Interestingly, vitamin C significantly increased basal cGMP levels in C57BL/6 mice but did not affect cGMP formation in apoE-/-. Vitamin C treatment did not affect expression of eNOS protein, whereas elevated expression of PECAM-1 protein in apoE-/- mice was returned to normal level. Our findings demonstrate that chronic treatment with vitamin C prevents endothelial dysfunction of carotid artery induced by hypercholesterolemia. This effect seems to be mediated by preservation of NO bioavailability in endothelial cells.

Radiation-induced Genomic Instability: Radiation Quality and Dose Response

Genomic instability is a term used to describe a phenomenon that results in the accumulation of multiple changes required to convert a stable genome of a normal cell to an unstable genome characteristic of a tumor. There has been considerable recent debate concerning the importance of genomic instability in human cancer and its temporal occurrence in the carcinogenic process. Radiation is capable of inducing genomic instability in mammalian cells and instability is thought to be the driving force responsible for radiation carcinogenesis. Genomic instability is characterized by a large collection of diverse endpoints that include large-scale chromosomal rearrangements and aberrations, amplification of genetic material, aneuploidy, micronucleus formation, microsatellite instability, and gene mutation. The capacity of radiation to induce genomic instability depends to a large extent on radiation quality or linear energy transfer (LET) and dose. There appears to be a low dose threshold effect with low LET, beyond which no additional genomic instability is induced. Low doses of both high and low LET radiation are capable of inducing this phenomenon. This report reviews data concerning dose rate effects of high and low LET radiation and their capacity to induce genomic instability assayed by chromosomal aberrations, delayed lethal mutations, micronuclei and apoptosis.

Pediatric-onset Primary Biliary Cirrhosis

Unlike other autoimmune liver diseases, primary biliary cirrhosis (PBC) has not been reported in childhood. We report 2 cases of PBC diagnosed at 16 and 15 years of age, respectively. The first girl was noted to have increased liver enzyme levels at 16 years of age. Antimitochondrial antibody (AMA) was strongly positive, and serum quantitative immunoglobulin M level was 8.26 g/L (normal, 0.6-3 g/L). A liver biopsy specimen showed stage II PBC. Despite treatment with ursodeoxycholic acid, she developed progressive cholestasis, intractable pruritus, and a significant sensory neuropathy and weight loss eventually requiring liver transplantation. Her mother had PBC/autoimmune overlap syndrome and underwent successful liver transplantation at 34 years of age. The second girl had persistently elevated liver enzyme levels following cholecystectomy at 15 years of age for symptomatic cholelithiasis. Endoscopic retrograde cholangiopancreatography showed no abnormalities. AMA was positive at 1:160, and serum quantitative immunoglobulin was 6.96 g/L. A liver biopsy specimen showed stage II PBC, and her liver enzyme levels almost normalized after starting treatment with ursodeoxycholic acid. In conclusion, we present 2 liver biopsy-confirmed cases of pediatric-onset AMA-positive PBC. With increased awareness of early-onset PBC, further pediatric cases may be discovered.

Mechanisms of Cell Death Associated with Death-inducing Factors from Genomically Unstable Cell Lines

We recently described a unique non-targeted effect of ionizing radiation whereby growth medium from two clones of GM10115 cells exhibiting radiation-induced chromosomal instability was cytotoxic to parental GM10115 cells. We termed this the death-inducing effect (DIE). The goal of the present study was to determine how DIE killed cells. Our hypothesis was that DIE medium contained either a secreted factor(s) from unstable clones or products from dead/dying cells that were cytotoxic to parental cells. First, we investigated the apoptotic characteristics of our unstable clones by Annexin V binding and TUNEL assays. Both the parental GM10115 cells and cells from the unstable clone LS12 had a low background (approximately 2%) level of apoptosis. The unstable Fe-10-3 clone showed a high spontaneous level of apoptosis, indicating major differences in the spontaneously occurring levels of apoptosis. We then analyzed how medium from these unstable clones killed cells by investigating the induction of DNA breaks, micronucleus formation and apoptosis induction in cells exposed to medium from unstable clones. Medium from unstable clones was capable of eliciting DNA double-strand breaks and increased apoptosis. Increased micronucleus frequencies were also observed in cells exposed to medium from either unstable clone, indicating a role of mitotis-linked cell death in DIE. These data suggest that DIE most likely results from cytotoxic factors secreted into the culture medium that can cause DNA double-strand breaks in recipient cells. These breaks can then lead to mitotis-linked cell death, as measured by micronuclei, or apoptosis, which accounts for the DIE.

In Vivo Expression and Function of Recombinant GTPCH I in the Rabbit Carotid Artery

Tetrahydrobiopterin (BH4) is an essential co-factor for endothelial nitric oxide synthase enzymatic activity. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme in BH4 synthesis. This study set out to test the hypothesis that in vivo gene transfer of GTPCH I to endothelial cells could increase bioavailability of BH4, enhance biosynthesis of nitric oxide and thereby enhance endothelium-dependent relaxations mediated by nitric oxide. In vivo gene transfer was carried out by adenovirus (Ad)-mediated delivery into rabbit carotid arteries. Each artery was transduced by 20-min intraluminal incubation of 10(9) plaque-forming units of Ad-encoding GTPCH I (AdGTPCH) or beta-galactosidase as a control. The rabbits were euthanized 72 h later, and vasomotor function of isolated arteries was assessed by isometric force recording. GTPCH I enzymatic activity, BH4, and oxidized biopterin levels were detected with the use of HPLC, and cGMP was measured with the use of radioimmunoassay. Expression of recombinant proteins was detected predominantly in endothelial cells. Both GTPCH I activity and BH4 levels were increased in arteries transduced with AdGTPCH. However, contraction to phenylephrine (10(-5) to 10(-9) M), endothelium-dependent relaxation to acetylcholine (10(-5) to 10(-9) M) and cGMP levels were not significantly affected by increased expression of GTPCH I. Our results suggest that expression of GTPCH I in vascular endothelium in vivo increases intracellular concentration of BH4. However, under physiological conditions, it appears that this increase does not affect nitric oxide production in endothelial cells of the carotid artery.

Ionizing Radiation Induces Delayed Hyperrecombination in Mammalian Cells

Exposure to ionizing radiation can result in delayed effects that can be detected in the progeny of an irradiated cell multiple generations after the initial exposure. These effects are described under the rubric of radiation-induced genomic instability and encompass multiple genotoxic endpoints. We have developed a green fluorescence protein (GFP)-based assay and demonstrated that ionizing radiation induces genomic instability in human RKO-derived cells and in human hamster hybrid GM10115 cells, manifested as increased homologous recombination (HR). Up to 10% of cells cultured after irradiation produce mixed GFP(+/-) colonies indicative of delayed HR or, in the case of RKO-derived cells, mutation and deletion. Consistent with prior studies, delayed chromosomal instability correlated with delayed reproductive cell death. In contrast, cells displaying delayed HR showed no evidence of delayed reproductive cell death, and there was no correlation between delayed chromosomal instability and delayed HR, indicating that these forms of genome instability arise by distinct mechanisms. Because delayed hyperrecombination can be induced at doses of ionizing radiation that are not associated with significantly reduced cell viability, these data may have important implications for assessment of radiation risk and understanding the mechanisms of radiation carcinogenesis.

The Effective Source Area of 90Sr for a Stream Near Chernobyl, Ukraine

Remediation of streams impacted by non-point source contaminants requires an understanding of both the areas within a watershed that are contributing contamination to streams and the pathways of contaminant migration to streams. From 1998 to 2002, we studied the migration of 90Sr in the Borschi watershed, a small (8.5 km2) catchment three km south of the Chernobyl Nuclear Power Plant, Ukraine. Fuel particles, distributed in a heterogeneous pattern across the watershed, are weathering and releasing 90Sr from the fuel matrix. Depletion of (90)Sr, evaluated in comparison to the immobile fission product europium-154, is occurring in the channel and wetland sediment. Channel sediments are uniformly depleted in 90Sr with depth. In wetland sediments, there is a zone of depletion in the top 10 cm and a zone of accumulation at depths from 10 to 25 cm. Estimates of 90Sr depletion are used to map the effective source area that has contributed (90)Sr loading to the main channel. The effective source area includes channel bottom sediments, a wetland in the central region of the watershed, and periodically flooded soils surrounding the wetland. The total depletion from the effective source area is estimated to be 36 +/- 7 x 10(10) Bq. Based on observations of stream flow rate and water quality in 1999-2001, the annual 90Sr removal rate from the watershed is estimated to be 1.4 +/-0.2 x 10(10) or 1.5% of the inventory per year. When extrapolated over a 15-year period following the Chernobyl accident, the last value is in reasonable agreement with the estimated depletion of the source area based on 90Sr/154Eu ratios. The 90Sr yearly leaching rate considering the whole watershed is 0.2% while the 90Sr leaching rate considering the effective source area is an order of magnitude higher. Most of the 90Sr release in the watershed has originated from an effective source area of 0.62 km2, or 7% of the watershed area.

Mechanisms of Aging-induced Impairment of Endothelium-dependent Relaxation: Role of Tetrahydrobiopterin

Oxidative stress has been implicated as an important mechanism of vascular endothelial dysfunction induced by aging. Previous studies suggested that tetrahydrobiopterin (BH4), an essential cofactor of endothelial NO synthase, could be a molecular target for oxidation. We tested the hypothesis that oxidative stress, in particular oxidation of BH4, may contribute to attenuation of endothelium-dependent relaxation in aged mice. Vasomotor function of isolated carotid arteries was studied using a video dimension analyzer. Vascular levels of BH4 and its oxidation products were measured via HPLC. In aged mice (age, 95 +/- 2 wk), endothelium-dependent relaxation to ACh (10(-5) to 10(-9) M) as well as endothelium-independent relaxation to the NO donor diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, 10(-5) to 10(-9) M) were significantly reduced compared with relaxation detected in young mice (age, 23 +/- 0.5 wk). Incubation of aged mouse carotid arteries with the cell-permeable SOD mimetic Mn(III)tetra(4-benzoic acid)porphyrin chloride normalized relaxation to ACh and DEA-NONOate. Furthermore, production of superoxide anion in aorta and serum levels of amyloid P component, which is the murine analog of C-reactive protein, was increased in old mice. In aorta, neither the concentration of BH4 nor the ratio of reduced BH4 to the oxidation products were different between young and aged mice. Our results demonstrate that in mice, aging impairs relaxation mediated by NO most likely by increased formation of superoxide anion. Oxidation of BH4 does not appear to be an important mechanism underlying vasomotor dysfunction in aged mouse arteries.

Transplantation of Circulating Endothelial Progenitor Cells Restores Endothelial Function of Denuded Rabbit Carotid Arteries

Circulating endothelial progenitor cells (EPCs) play an important role in repair of injured vascular endothelium and neovascularization. The present study was designed to determine the effect of EPCs transplantation on the regeneration of endothelium and recovery of endothelial function in denuded carotid arteries.

Robust Sound Onset Detection Using Leaky Integrate-and-fire Neurons with Depressing Synapses

A biologically inspired technique for detecting onsets in sound is presented. Outputs from a cochlea-like filter are spike coded, in a way similar to the auditory nerve (AN). These AN-like spikes are presented to a leaky integrate-and-fire neuron through a depressing synapse. Onsets are detected with essentially zero latency relative to these AN spikes. Onset detection results for a tone burst, musical sounds and the DARPA/NIST TIMIT speech corpus are presented.

Ionizing Radiation Induces Frequent Translocations with Delayed Replication and Condensation

Certain chromosome rearrangements display a significant delay in replication timing that is associated with a delay in mitotic chromosome condensation. Chromosomes with delay in replication timing/delay in mitotic chromosome condensation participate in frequent secondary rearrangements, indicating that cells with delay in replication timing/delay in mitotic chromosome condensation display chromosomal instability. In this report, we show that exposing cell lines or primary blood lymphocytes to ionizing radiation results in chromosomes with the delay in replication timing/delay in mitotic chromosome condensation phenotype, and that the delay in replication timing/delay in mitotic chromosome condensation phenotype occurs predominantly on chromosome translocations. In addition, exposing mice to ionizing radiation also induces cells with delay in replication timing/delay in mitotic chromosome condensation chromosomes that persist for as long as 2 years. Cells containing delay in replication timing/delay in mitotic chromosome condensation chromosomes frequently display hyperdiploid karyotypes, indicating that delay in replication timing/delay in mitotic chromosome condensation is associated with aneuploidy. Finally, using a chromosome engineering strategy, we show that only a subset of chromosome translocations displays delay in replication timing/delay in mitotic chromosome condensation. Our results indicate that specific chromosome rearrangements result in the generation of the delay in replication timing/delay in mitotic chromosome condensation phenotype and that this phenotype occurs frequently in cells exposed to ionizing radiation both in vitro and in vivo.

Strenuous Physical Exercise Inhibits Granulocyte Activation Induced by High Altitude

To test the hypothesis of whether strenuous physical exercise inhibits neutrophils that can get activated by hypobaric hypoxia, we analyzed the effects of both high altitude and strenuous exercise alone and in combination on potentially cytotoxic functions of granulocytes in healthy volunteers (n = 12 men; average age 27.6 yr; range 24-38 yr). To this end, a field study was prospectively performed with an open-labeled within-subject design comprising three protocols. Protocol I (high altitude) involved a helicopter ascent, overnight stay at 3,196 m, and descent on the following day. Protocol II (physical exercise) involved hiking below an altitude of 2,100 m with repetitive ascents amounting to a total ascent to that of protocol III. Protocol III (combination of physical exercise and high altitude) involved climbing from 1,416 to 3,196 m, stay overnight, and descent on the following day. In protocol I, number of granulocytes did not change, but potentially cytotoxic functions of cells (CD18 expression and superoxide production) were early and significantly upregulated. In protocol II, subjects developed granulocytosis, but functions of cells were inhibited. In protocol III, granulocytosis occurred at higher values than those observed under protocol II. Potentially cytotoxic functions of cells, however, were strongly inhibited again. In conclusion, high altitude alone, even moderate in extent, can activate potentially cytotoxic functions of circulating granulocytes. Strenuous physical exercise strongly inhibits this activation, which may give protection from an otherwise inflammatory injury.

The Hyperbilirubinemic Gunn Rat is Resistant to the Pressor Effects of Angiotensin II

ANG II induces vasoconstriction, at least in part, by stimulating NADPH oxidase and generating reactive oxygen species. ANG II also induces heme oxygenase activity, and bilirubin, a product of such activity, possesses antioxidant properties. We hypothesized that bilirubin, because of its antioxidant properties, may reduce the pressor and prooxidant effects of ANG II. Our in vivo studies used the hyperbilirubinemic Gunn rat which is deficient in the enzyme uridine diphosphate glucuronosyl transferase, the latter enabling the excretion of bilirubin into bile. ANG II (0.5 mg x kg(-1) x day(-1)) or saline vehicle was administered by osmotic minipump to control and Gunn rats for 4 wk. The rise in systolic blood pressure induced by ANG II, as observed in control rats, was markedly reduced in Gunn rats, the latter approximately 50% less at 3 and 4 wk after the initiation of ANG II infusion. The chronic administration of ANG II also impaired endothelium-dependent relaxation responses in control rats but not in Gunn rats. As assessed by the tetrahydrobiopterin/dihydrobiopterin ratio, ANG II induced oxidative stress in the aorta in control rats but not in Gunn rats. Heightened generation of superoxide anion in aortic rings in ANG II-infused rats and by vascular smooth muscle cells exposed to ANG II was normalized by bilirubin in vitro. We conclude that the pressor and prooxidant effects of ANG II are attenuated in the hyperbilirubinemic Gunn rat, an effect which, we speculate, may reflect, at least in part, the scavenging of superoxide anion by bilirubin.

Expression and Function of Recombinant S1179D Endothelial NO Synthase in Human Pial Arteries

Mutation of serine 1179 to aspartate on the endothelial NO synthase (eNOS) increases NO production in the absence of stimulation by agonists. The present study was designed to determine the effect of recombinant S1179DeNOS gene expression on the vasomotor function of human pial arteries.

In Vivo Expression of Recombinant Vascular Endothelial Growth Factor in Rabbit Carotid Artery Increases Production of Superoxide Anion

Vascular endothelial growth factor (VEGF) is one of the most important pro-angiogenic cytokines. Ability of VEGF to stimulate formation of superoxide anion in vivo has not been studied. We hypothesized that in vivo expression of recombinant VEGF in the rabbit carotid artery increases production of superoxide anion.

Precision Constrained Stochastic Resonance in a Feedforward Neural Network

Stochastic resonance (SR) is a phenomenon in which the response of a nonlinear system to a subthreshold information-bearing signal is optimized by the presence of noise. By considering a nonlinear system (network of leaky integrate-and-fire (LIF) neurons) that captures the functional dynamics of neuronal firing, we demonstrate that sensory neurons could, in principle harness SR to optimize the detection and transmission of weak stimuli. We have previously characterized this effect by use of signal-to-noise ratio (SNR). Here in addition to SNR, we apply an entropy-based measure (Fisher information) and compare the two measures of quantifying SR. We also discuss the performance of these two SR measures in a full precision floating point model simulated in Java and in a precision limited integer model simulated on a field programmable gate array (FPGA). We report in this study that stochastic resonance which is mainly associated with floating point implementations is possible in both a single LIF neuron and a network of LIF neurons implemented on lower resolution integer based digital hardware. We also report that such a network can improve the SNR and Fisher information of the output over a single LIF neuron.

Variation in Apoptosis Profiles in Radiation-induced Genomically Unstable Cell Lines

Delayed reproductive cell death or lethal mutations in the survivors of irradiated cells is a well-characterized end point associated with radiation-induced genomic instability. Although the mechanism for this delayed lethality has not been identified, it is thought to be a means of eliminating cells that have sustained extensive damage, thus preventing tissue disruption after radiation exposure. In this study we have tested the hypothesis that delayed reproductive cell death in chromosomally unstable GM10115 clones is due to persistently increased levels of apoptosis. Evidence for differences in apoptosis in two representative genomically unstable clones after irradiation is presented. In addition, one of the unstable clones was found to have abnormal levels of apoptosis after radiation exposure. An understanding of apoptosis in genomically unstable clones may provide insight into the maintenance of genomic instability and the mechanism by which genomically unstable cells evade cell death, potentially contributing to carcinogenesis.

Engineering Translocations with Delayed Replication: Evidence for Cis Control of Chromosome Replication Timing

Certain chromosome rearrangements, found in cancer cells or in cells exposed to ionizing radiation, exhibit a chromosome-wide delay in replication timing (DRT) that is associated with a delay in mitotic chromosome condensation (DMC). We have developed a chromosome engineering strategy that allows the generation of chromosomes with this DRT/DMC phenotype. We found that approximately 10% of inter-chromosomal translocations induced by two distinct mechanisms, site-specific recombination mediated by Cre or non-homologous end joining of DNA double-strand breaks induced by I-Sce1, result in DRT/DMC. Furthermore, on certain balanced translocations only one of the derivative chromosomes displays the phenotype. Finally, we show that the engineered DRT/DMC chromosomes acquire gross chromosomal rearrangements at an increased rate when compared with non-DRT/DMC chromosomes. These results indicate that the DRT/DMC phenotype is not the result of a stochastic process that could occur at any translocation breakpoint or as an epigenetic response to chromosome damage. Instead, our data indicate that the replication timing of certain derivative chromosomes is regulated by a cis-acting mechanism that delays both initiation and completion of DNA synthesis along the entire length of the chromosome. Because chromosomes with DRT/DMC are common in tumor cells and in cells exposed to ionizing radiation, we propose that DRT/DMC represents a common mechanism responsible for the genomic instability found in cancer cells and for the persistent chromosomal instability associated with cells exposed to ionizing radiation.

Role of Endothelial NO Synthase Phosphorylation in Cerebrovascular Protective Effect of Recombinant Erythropoietin During Subarachnoid Hemorrhage-induced Cerebral Vasospasm

In the present study, the effect of subarachnoid hemorrhage (SAH) on the phosphorylation of endothelial NO synthase (eNOS) and the ability of recombinant erythropoietin (Epo) to augment this vasodilator mechanism in the spastic arteries were studied.

Anaerobic Degradation of Naphthalene in a Fluvial Aquifer: a Radiotracer Study

A radiotracer study was conducted in a creosote-contaminated aquifer beneath the Fraser River, British Columbia Canada to investigate the in situ degradation of naphthalene. The groundwater is anaerobic, with abundant methane, ferrous iron and carbon dioxide. This study followed earlier work at the site where the contaminant distribution could only be explained by invoking a mass loss through degradation, even though extensive field and laboratory microcosm studies closer to the source zone onshore could not confirm degradation. Accordingly, 14C-naphthalene was injected into the aquifer offshore, further from the source zone where modeling suggested degradation was occurring. During the 230-day monitoring period, 14CO2 was detected, confirming the degradation of the radio-labeled naphthalene tracer. A zero-order degradation rate of naphthalene of 5 microg/L-day was estimated based on the decrease in 14C-naphthalene concentration with time. While the degradation pathway could not be determined from the radiotracer study alone, the geochemistry of the site suggests that either iron reduction or methanogenesis is the terminal electron accepting processes responsible for naphthalene oxidation.

In Vivo Stimulatory Effect of Erythropoietin on Endothelial Nitric Oxide Synthase in Cerebral Arteries

The discovery of tissue protective effects of erythropoietin has stimulated significant interest in erythropoietin (Epo) as a novel therapeutic approach to vascular protection. The present study was designed to determine the cerebral vascular effects of recombinant Epo in vivo. Recombinant adenoviral vectors (10(9) plaque-forming units/animal) encoding genes for human erythropoietin (AdEpo) and beta-galactosidase (AdLacZ) were injected into the cisterna magna of rabbits. After 48 h, basilar arteries were harvested for analysis of vasomotor function, Western blotting, and measurement of cGMP levels. Gene transfer of AdEpo increased the expressions of recombinant Epo and its receptor in the basilar arteries. Arteries exposed to recombinant Epo demonstrated attenuation of contractile responses to histamine (10(-9) to 10(-5) mol/l) (P < 0.05, n = 5). Endothelium-dependent relaxations to acetylcholine (10(-9) to 10(-5) mol/l) were significantly augmented (P < 0.05, n = 5), whereas endothelium-independent relaxations to a nitric oxide (NO) donor 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt remained unchanged in AdEpo-transduced basilar arteries. Transduction with AdEpo increased the protein expression of endothelial NO synthase (eNOS) and phosphorylated the S1177 form of the enzyme. Basal levels of cGMP were significantly elevated in arteries transduced with AdEpo consistent with increased NO production. Our studies suggest that in cerebral circulation, Epo enhances endothelium-dependent vasodilatation mediated by NO. This effect could play an important role in the vascular protective effect of Epo.

A Tool for Synthesizing Spike Trains with Realistic Interference

Spike detection and spike sorting techniques are often difficult to assess because of the lack of ground truth data (i.e., spike timings for each neuron). This is particularly important for in vitro recordings where the signal to noise ratio is poor (as is the case for multi-electrode arrays at the bottom of a cell culture dish). We present an analysis of the transmission of intracellular signals from neurons to an extracellular electrode, and a set of MATLAB functions based on this analysis. These produce realistic signals from neighboring neurons as well as interference from more distant neurons, and Gaussian noise. They thus generate realistic but controllable synthetic signals (for which the ground truth is known) for assessing spike detection and spike sorting techniques. They can also be used to generate realistic (non-Gaussian) background noise. We use signals generated in this way to compare two automated spike-sorting techniques. The software is available freely on the web.

Essential Role of Endothelial Nitric Oxide Synthase in Vascular Effects of Erythropoietin

Erythropoietin (EPO) fosters tissue oxygenation by stimulating erythropoiesis. More recently, EPO has been recognized as a tissue-protective cytokine. In this study, we tested the hypothesis that endothelial NO synthase (eNOS) plays a key role in the vascular protective effect of EPO. A murine model of wire-induced injury of carotid artery was used to examine the effect of EPO on endothelial repair and arterial wall architecture. Recombinant human EPO (1000 U/kg, SC, biweekly) was administered for 2 weeks in wild-type and eNOS-deficient mice after which reactivity of isolated carotid arteries was studied in vitro, and the vasculature was histologically assessed. Injured arteries exhibited impairment of endothelium-dependent relaxations to acetylcholine (P<0.05). This was associated with increased medial cross-sectional area (P<0.05). EPO upregulated expression of phosphorylated Ser1177-eNOS and normalized the vasodilator response to acetylcholine (P<0.05). Furthermore, EPO prevented the injury-induced increase in medial cross-sectional area (P<0.05). The vascular protective effects of EPO were abolished in eNOS-deficient mice. Most notably, EPO significantly increased systolic blood pressure and enhanced medial thickening of injured carotid arteries in eNOS-deficient mice (P<0.05). Our results demonstrate that EPO prevents aberrant remodeling of the injured carotid artery. The protective effects of EPO are critically dependent on activation of eNOS.

Endothelial Progenitor Cells Stimulate Cerebrovascular Production of Prostacyclin by Paracrine Activation of Cyclooxygenase-2

In the present study we hypothesized that endothelial progenitor cells (EPCs) enhance production of vasoprotective substances in cerebral arteries. Isolated mononuclear cells from rabbit peripheral blood were cultured in endothelial growth medium (EGM-2) for 7 days to yield EPCs. Rabbit basilar arteries were exposed to autologous EPCs ( approximately 5x10(5) cells) in vitro or in vivo. Twenty-four hours after intracisternal delivery of autologous EPCs, basilar arteries were isolated and expression of vasoregulatory proteins, production of prostacyclin (PGI(2)), and cAMP were determined. Arteries transplanted with EPCs demonstrated increased protein expression of cyclooxygenase-2 and PGI(2) in adventitia, media, and endothelium. Furthermore, production of PGI(2) and arterial content of cAMP, second messenger for PGI(2), were significantly augmented after transplantation of EPCs. In contrast, production of thromboxane A(2) was significantly reduced, whereas production of prostaglandin E(2) remained unchanged. The increased production of PGI(2) and arterial content of cAMP were inhibited only by a selective cyclooxygenase-2 inhibitor, NS-398. In vitro or in vivo treatment of basilar artery with conditioned media from EPCs also caused increase in cyclooxygenase-2 and PGI(2) synthase protein expression associated with elevation of cAMP. Our results suggest that in cerebral arteries, paracrine effect of EPCs promotes vasoprotection by increasing PGI(2) production and intracellular concentration of cAMP. This effect appears to be mediated by activation of arachidonic acid metabolism via stimulation of cyclooxygenase-2/PGI(2) synthase pathway.

Data Integration in EHealth: a Domain/disease Specific Roadmap

The paper documents a series of data integration workshops held in 2006 at the UK National e-Science Centre, summarizing a range of the problem/solution scenarios in multi-site and multi-scale data integration with six HealthGrid projects using schizophrenia as a domain-specific test case. It outlines emerging strategies, recommendations and objectives for collaboration on shared ontology-building and harmonization of data for multi-site trials in this domain.

Decaying Invertebrate Carcasses Increase Growth of Aedes Triseriatus (Diptera: Culicidae) when Leaf Litter Resources Are Limiting

Treeholes are detritus-based communities, and resource quantity and quality play a large role in structuring such communities. The primary resource is leaf litter, but decaying invertebrates also are a resource to treehole inhabitants. These communities are subject to a variety of disturbances, which may affect resources or cause widespread mortality. When dead inhabitants decay, they provide a potentially high-quality resource to survivors or subsequent colonists. We predicted that variation in decaying larvae (0, 7.3, and 29.2 mg/liter) and leaf litter (1, 5, and 10 g/liter) would influence the performance of populations of Aedes triseriatus (Say), the eastern treehole mosquito. We tested this prediction in field mesocosms, which were subjected to a freezing event causing widespread mortality of the scirtid beetle Helodes pulchella Guerin. We then added a cohort of first instar mosquitoes to mesocosms, and we monitored their development from March until June 2005. At the highest leaf litter level, survival, adult mass, and time to complete development were unaffected by decaying scirtids, and they were different from treatments with lower levels of leaf litter. In treatments with 1 and 5 g/liter leaf litter and decaying scirtids, mosquito survival and adult mass were higher than in treatments with 1 and 5 g/liter leaf litter and no decaying scirtids. At 5 g/liter leaf litter, a higher mass of dead scirtids was required to significantly increase adult mass. Faster decay of carcasses and release of limiting nutrients likely spur growth of microorganisms, upon which mosquitoes feed. Invertebrate populations in high-disturbance communities may be subject to high mortality, and mosquitoes hatching after the disturbance will benefit, but only when other resources are limiting.

An Antisense Transcript Spanning the CGG Repeat Region of FMR1 is Upregulated in Premutation Carriers but Silenced in Full Mutation Individuals

Expansion of the polymorphic CGG repeats within the 5'-UTR of the FMR1 gene is associated with variable transcriptional regulation of FMR1. Here we report a novel gene, ASFMR1, overlapping the CGG repeat region of FMR1 and transcribed in the antisense orientation. The ASFMR1 transcript is spliced, polyadenylated and exported to the cytoplasm. Similar to FMR1, ASFMR1 is upregulated in individuals with premutation alleles and is not expressed from full mutation alleles. Moreover, it exhibits premutation-specific alternative splicing. Taken together, these observations suggest that in addition to FMR1, ASFMR1 may contribute to the variable phenotypes associated with the CGG repeat expansion.

Neural Network Based Pattern Matching and Spike Detection Tools and Services--in the CARMEN Neuroinformatics Project

In the study of information flow in the nervous system, component processes can be investigated using a range of electrophysiological and imaging techniques. Although data is difficult and expensive to produce, it is rarely shared and collaboratively exploited. The Code Analysis, Repository and Modelling for e-Neuroscience (CARMEN) project addresses this challenge through the provision of a virtual neuroscience laboratory: an infrastructure for sharing data, tools and services. Central to the CARMEN concept are federated CARMEN nodes, which provide: data and metadata storage, new, thirdparty and legacy services, and tools. In this paper, we describe the CARMEN project as well as the node infrastructure and an associated thick client tool for pattern visualisation and searching, the Signal Data Explorer (SDE). We also discuss new spike detection methods, which are central to the services provided by CARMEN. The SDE is a client application which can be used to explore data in the CARMEN repository, providing data visualization, signal processing and a pattern matching capability. It performs extremely fast pattern matching and can be used to search for complex conditions composed of many different patterns across the large datasets that are typical in neuroinformatics. Searches can also be constrained by specifying text based metadata filters. Spike detection services which use wavelet and morphology techniques are discussed, and have been shown to outperform traditional thresholding and template based systems. A number of different spike detection and sorting techniques will be deployed as services within the CARMEN infrastructure, to allow users to benchmark their performance against a wide range of reference datasets.

Local and Regional Factors Influence the Structure of Treehole Metacommunities

Abiotic and biotic factors in a local habitat may strongly impact the community residing within, but spatially structured metacommunities are also influenced by regional factors such as immigration and colonization. We used three years of monthly treehole census data to evaluate the relative influence of local and regional factors on our study system.

The Deployed Electronic Medical Record

This article reviews the current state of the electronic medical record in the deployed environment, with a discussion of challenges faced in the course of mission execution. Focus discussion includes current system architecture, system integration, interoperability, networking, and security concerns. The Department of Defense electronic medical documentation system does function, and records care from the point of injury through enduring care within the Veterans Health Administration. However, there is a high cost in dollars and man-hours, which should be aggressively addressed and improved.

Preparation of Human Mitochondrial Single-stranded DNA-binding Protein

Defects in mtDNA replication are the principle cause of severe, heritable metabolic disorders classified as mitochondrial diseases. In vitro analysis of the biochemical mechanisms of mtDNA replication has proven to be a powerful tool for understanding the origins of mitochondrial disease. Mitochondrial single-stranded DNA-binding protein (mtSSB) is an essential component of the mtDNA replication machinery. To facilitate ongoing biochemical studies, a recombinant source of mtSSB is needed to avoid the time and expense of human tissue culture. This chapter focuses on the subcloning, purification, and initial functional validation of the recombinant human mitochondrial single-stranded DNA-binding protein. The cDNA encoding the mature form of the human mtSSB protein was amplified from a HeLa cDNA library, and recombinant human mtSSB was overproduced in Escherichia coli. A procedure was developed to rapidly purify milligram quantities of homogenous, nuclease-free mtSSB that avoids DNA-cellulose chromatography. We show that, similar to E. coli SSB, human mtSSB assembles into a tetramer and binds single-stranded oligonucleotides in a 4-to-1 protein:oligonucleotide molar ratio.

Anatomic Stem Design Reduces Risk of Thin Cement Mantles in Primary Hip Replacement

To analyse the influence of femoral stem design in the lateral plane (anatomic vs. straight) on the cement mantle quality.

A New Spike Detection Algorithm for Extracellular Neural Recordings

Signals from extracellular electrodes in neural systems record voltages resulting from activity in many neurons. Detecting action potentials (spikes) in a small number of specific (target) neurons is difficult because many neurons, both near and more distant, contribute to the signal at the electrode. We consider some nearby neurons as target neurons (providing a signal) and all the other contributions to the signal as noise. A new algorithm for spike detection has been developed: this applies a cepstrum of bispectrum (CoB) estimated inverse filter to provide blind equalization. This technique is based on higher order statistics, and seeks to find a sequence of event times or delta sequence. We show that the CoB-based technique can achieve a 98% hit rate on an extracellular signal containing three spike trains at up to 0 dB SNR. Threshold setting for this technique is discussed, and we show the application of the technique to some real signals. We compare performance with four established techniques and report that the CoB-based algorithm performs best.

Brain-derived Neurotrophic Factor Stimulates Production of Prostacyclin in Cerebral Arteries

The role of brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin receptor kinase B, in control of cerebral circulation is poorly understood. The present study was designed to investigate the cerebral vascular effects of BDNF in vivo.

Neuromorphic Systems: Past, Present and Future

Neuromorphic systems are implementations in silicon of elements of neural systems. The idea of electronic implementation is not new, but modern microelectronics has provided opportunities for producing systems for both sensing and neural modelling that can be mass produced straightforwardly. We review the the history of neuromorphic systems, and discuss the range of neuromorphic systems that have been developed. We discuss recent ideas for overcoming some of the problems, particularly providing effective adaptive synapses in large numbers.

Erythropoietin Increases Expression and Function of Vascular Copper- and Zinc-containing Superoxide Dismutase

Previous studies have shown that treatment with erythropoietin (EPO) exerts vascular protective effects. The exact mechanisms responsible for these effects are not completely understood. In the present study, we hypothesized that EPO stimulates expression and activity of copper- and zinc-containing superoxide dismutase (SOD1), thus protecting vascular tissue from oxidative stress induced by excessive concentrations of superoxide anions. EPO treatment of wild-type mice for 2 weeks (1000 U/kg, SC, biweekly) significantly increased aortic expression of SOD1. This effect resulted in a significant reduction of superoxide anion concentrations in aorta of treated mice. The ability of EPO to reduce vascular production of superoxide anions was abolished in SOD1-deficient mice. In a mouse model of wire-induced injury of the common carotid artery, treatment of wild-type mice with EPO prevented pathological remodeling, whereas the vascular effect of EPO was absent in SOD1-deficient mice. Our findings demonstrate that treatment with EPO increases vascular expression of SOD1. This effect appears to be an important molecular mechanism underlying vascular protection by EPO.

PGC1beta Mediates PPARgamma Activation of Osteoclastogenesis and Rosiglitazone-induced Bone Loss

Long-term usage of rosiglitazone, a synthetic PPARgamma agonist, increases fracture rates among diabetic patients. PPARgamma suppresses osteoblastogenesis while activating osteoclastogenesis, suggesting that rosiglitazone decreases bone formation while sustaining or increasing bone resorption. Using mouse models with genetically altered PPARgamma, PGC1beta, or ERRalpha, here we show that PGC1beta is required for the resorption-enhancing effects of rosiglitazone. PPARgamma activation indirectly induces PGC1beta expression by downregulating beta-catenin and derepressing c-jun. PGC1beta, in turn, functions as a PPARgamma coactivator to stimulate osteoclast differentiation. Complementarily, PPARgamma also induces ERRalpha expression, which coordinates with PGC1beta to enhance mitochondrial biogenesis and osteoclast function. ERRalpha knockout mice exhibit osteoclast defects, revealing ERRalpha as an important regulator of osteoclastogenesis. Strikingly, PGC1beta deletion in osteoclasts confers complete resistance to rosiglitazone-induced bone loss. These findings identify PGC1beta as an essential mediator for the PPARgamma stimulation of osteoclastogenesis by targeting both PPARgamma itself and ERRalpha, thus activating two distinct transcriptional programs.

An Autosomal Locus That Controls Chromosome-wide Replication Timing and Mono-allelic Expression

Mammalian DNA replication initiates at multiple sites along chromosomes at different times, following a temporal replication program. Homologous alleles typically replicate synchronously; however, mono-allelically expressed genes such as imprinted genes, allelically excluded genes and genes on the female X chromosome replicate asynchronously. We have used a chromosome engineering strategy to identify a human autosomal locus that controls this replication timing program in cis. We show that Cre/loxP-mediated rearrangements at a discrete locus at 6q16.1 result in delayed replication of the entire chromosome. This locus displays asynchronous replication timing that is coordinated with other mono-allelically expressed genes on chromosome 6. Characterization of this locus revealed mono-allelic expression of a large intergenic non-coding RNA, which we have named asynchronous replication and autosomal RNA on chromosome 6, ASAR6. Finally, disruption of this locus results in the activation of the previously silent alleles of linked mono-allelically expressed genes. We previously found that chromosome rearrangements involving eight different autosomes display delayed replication timing, and that cells containing chromosomes with delayed replication timing have a 30-80-fold increase in the rate at which new gross chromosomal rearrangements occurred. Taken together, these observations indicate that human autosomes contain discrete cis-acting loci that control chromosome-wide replication timing, mono-allelic expression and the stability of entire chromosomes.

Characteristic Immune, Apoptosis and Inflammatory Gene Profiles Associated with Intestinal Acute Cellular Rejection in Formalin-fixed Paraffin-embedded Mucosal Biopsies

Small bowel transplantation (SBT) is becoming a preferred treatment for patients with irreversible intestinal failure. Despite continuous improvement of immunosuppression, SBT is plagued by a high incidence of acute cellular rejection (ACR) that is frequently intractable. Therefore, there is a need for reliable detection markers and novel immunosuppressive strategies that can achieve better control of ACR. We hypothesized that particular transcriptomes provide critical regulation of the intragraft immune response. The aim of our study was to detect potential molecular biomarkers for identifying ACR in minute mucosal biopsies. We examined 30 intestinal mucosal biopsies (AR/NR; 17/13) obtained from recipients after SBT or multivisceral transplantation. We utilized TaqMan® Gene Signature Arrays (immune, inflammation and apoptosis) and investigated the expression of 280 genes. As one of our validations, we performed immunohistochemistry for selected targets. We detected 252 mRNAs in total, 92 of which were found with significantly different expression levels between the AR and NR groups. Immunohistochemistry showed significantly increased staining for IL1R2, ICAM1, GZMB, and CCL3 (P < 0.05) during ACR. For the first time, we characterize the potential molecular changes that are associated with modulation of histological appearances of intestinal ACR. These differences in transcriptome patterns can be used to identify robust biomarkers and potential novel therapeutic targets for immunosuppressive agents.

Activation of Peroxisome Proliferator-activated Receptor-{delta} Enhances Regenerative Capacity of Human Endothelial Progenitor Cells by Stimulating Biosynthesis of Tetrahydrobiopterin

The mechanisms underlying the regenerative capacity of endothelial progenitor cells (EPCs) are not fully understood. We hypothesized that biosynthesis of tetrahydrobiopterin is an important mechanism responsible for the stimulatory effects of peroxisome proliferator-activated receptor-δ (PPARδ) activation on regenerative function of human EPCs. Treatment of human EPCs with a selective PPARδ agonist GW501516 for 24 hours increased the levels of mRNA, protein, and enzymatic activity of GTP cyclohydrolase I (GTPCH I), as well as the production of tetrahydrobiopterin. The effects of GW501516 were mediated by suppression of PTEN expression, thereby increasing phosphorylation of AKT. The AKT signaling also mediated GW501516-induced phosphorylation of endothelial NO synthase. In addition, activation of PPARδ significantly enhanced proliferation of EPCs. This effect was abolished by the GTPCH I inhibitor, 2,4-diamino-6-hydroxypyrimidine, or genetic inactivation of GTPCH I with small interfering RNA but not by inhibition of endothelial NO synthase with N(G)-nitro-l-arginine methyl ester. Supplementation of NO did not reverse 2,4-diamino-6-hydroxypyrimidine-inhibited 5-bromodeoxyuridine incorporation. Furthermore, transplantation of human EPCs stimulated re-endothelialization in a mouse model of carotid artery injury. Pretreatment of EPCs with GW501516 significantly enhanced the ability of transplanted EPCs to repair denuded endothelium. GTPCH I-small interfering RNA transfection significantly inhibited in vivo regenerative capacity of EPCs stimulated with GW501516. Thus, in human EPCs, activation of PPARδ stimulates expression and activity of GTPCH I and biosynthesis of tetrahydrobiopterin via PTEN-AKT signaling pathway. This effect enhances the regenerative function of EPCs.

Differential Effects of ENOS Uncoupling on Conduit and Small Arteries in GTP-cyclohydrolase I-deficient Hph-1 Mice

In the present study, we used the hph-1 mouse, which displays GTP-cyclohydrolase I (GTPCH I) deficiency, to test the hypothesis that loss of tetrahydrobiopterin (BH(4)) in conduit and small arteries activates compensatory mechanisms designed to protect vascular wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and BH(4) levels were reduced in the aortas and small mesenteric arteries of hph-1 mice. However, the BH(4)-to-7,8-dihydrobiopterin ratio was significantly reduced only in hph-1 aortas. Furthermore, superoxide anion and 3-nitrotyrosine production were significantly enhanced in aortas but not in small mesenteric arteries of hph-1 mice. In contrast to the aorta, protein expression of copper- and zinc-containing superoxide dismutase (CuZnSOD) was significantly increased in small mesenteric arteries of hph-1 mice. Protein expression of catalase was increased in both aortas and small mesenteric arteries of hph-1 mice. Further analysis of endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP) signaling demonstrated that protein expression of phosphorylated Ser(1177)-eNOS as well as basal cGMP levels and hydrogen peroxide was increased in hph-1 aortas. Increased production of hydrogen peroxide in hph-1 mice aortas appears to be the most likely mechanism responsible for phosphorylation of eNOS and elevation of cGMP. In contrast, upregulation of CuZnSOD and catalase in resistance arteries is sufficient to protect vascular tissue from increased production of reactive oxygen species generated by uncoupling of eNOS. The results of our study suggest that anatomical origin determines the ability of vessel wall to cope with oxidative stress induced by uncoupling of eNOS.

Memory Efficient On-line Streaming for Multichannel Spike Train Analysis

Rapid advances in multichannel neural signal recording technologies in recent years have spawned broad applications in neuro-prostheses and neuro-rehabilitation. The dramatic increase in data bandwidth and volume associated with multichannel recording requires a significant computational effort which presents major design challenges for brain-machine interface (BMI) system in terms of power dissipation and hardware area. In this paper, we present a streaming method for implementing real-time memory efficient neural signal processing hardware. This method exploits the pseudo-stationary property of neural signals and, thus, eliminates the need of temporal storage in batch-based processing. The proposed technique can significantly reduce memory size and dynamic power while effectively maintaining the accuracy of algorithms. The streaming kernel is robust when compared to the batch processing over a range of BMI benchmark algorithms. The advantages of the streaming kernel when implemented on field-programmable gate array (FPGA) devices are also demonstrated.

Frequent Aneuploidy Among Normal Human Hepatocytes

Murine hepatocytes become polyploid and then undergo ploidy reversal and become aneuploid in a dynamic process called the ploidy conveyor. Although polyploidization occurs in some types of human cells, the degree of aneuploidy in human hepatocytes is not known. We isolated hepatocytes derived from healthy human liver samples and determined chromosome number and identity using traditional karyotyping and fluorescence in situ hybridization. Similar to murine hepatocytes, human hepatocytes are highly aneuploid. Moreover, imaging studies revealed multipolar spindles and chromosome segregation defects in dividing human hepatocytes. Aneuploidy therefore does not necessarily predispose liver cells to transformation but might promote genetic diversity among hepatocytes.

Stream-based Hebbian Eigenfilter for Real-time Neuronal Spike Discrimination

Principal component analysis (PCA) has been widely employed for automatic neuronal spike sorting. Calculating principal components (PCs) is computationally expensive, and requires complex numerical operations and large memory resources. Substantial hardware resources are therefore needed for hardware implementations of PCA. General Hebbian algorithm (GHA) has been proposed for calculating PCs of neuronal spikes in our previous work, which eliminates the needs of computationally expensive covariance analysis and eigenvalue decomposition in conventional PCA algorithms. However, large memory resources are still inherently required for storing a large volume of aligned spikes for training PCs. The large size memory will consume large hardware resources and contribute significant power dissipation, which make GHA difficult to be implemented in portable or implantable multi-channel recording micro-systems.

Loss of Maternal CTCF is Associated with Peri-implantation Lethality of Ctcf Null Embryos

CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5-E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16-32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development.

Modeling of Strategies for Performance Monitoring of Groundwater Contamination at Sites Underlain by Fractured Bedrock

A three dimensional flow and transport modeling using FRAC3DVS was undertaken to examine factors which influence plume detection in a performance monitoring network for a site where an unconfined aquifer composed of uniform unconsolidated sediments overlies fractured bedrock. The bedrock is assumed to contain a fracture system with three orthogonal fracture sets embedded in a low permeable homogeneous rock matrix. A dissolved phase, non-reactive contaminant is released from a source zone located at the ground surface. The processes which influence plume geometry, and probabilities of plume detection for a performance monitoring network located between the contaminant source and a downstream compliance boundary, are evaluated. Factors considered include the hydraulic conductivity of the unconfined aquifer, the geometric properties of the fracture network and the matrix permeability of the bedrock, and the contaminant detection threshold concentration. The simulations demonstrate that the character of the fracture network not only controls contaminant transport and plume detection in the bedrock but also influences plume detection in the overlying unconfined aquifer. The ratio of the hydraulic conductivity of the unconfined aquifer to the effective hydraulic conductivity of the fractured bedrock, and the contaminant detection threshold concentration, are principal factors influencing detection probability in the performance monitoring network. Results suggest that in many instances encountered in field practice, the unconfined aquifer and fractured bedrock should be viewed as an integrated hydrogeologic system from a monitoring perspective.

A Neurally Inspired Musical Instrument Classification System Based Upon the Sound Onset

Physiological evidence suggests that sound onset detection in the auditory system may be performed by specialized neurons as early as the cochlear nucleus. Psychoacoustic evidence shows that the sound onset can be important for the recognition of musical sounds. Here the sound onset is used in isolation to form tone descriptors for a musical instrument classification task. The task involves 2085 isolated musical tones from the McGill dataset across five instrument categories. A neurally inspired tone descriptor is created using a model of the auditory system's response to sound onset. A gammatone filterbank and spiking onset detectors, built from dynamic synapses and leaky integrate-and-fire neurons, create parallel spike trains that emphasize the sound onset. These are coded as a descriptor called the onset fingerprint. Classification uses a time-domain neural network, the echo state network. Reference strategies, based upon mel-frequency cepstral coefficients, evaluated either over the whole tone or only during the sound onset, provide context to the method. Classification success rates for the neurally-inspired method are around 75%. The cepstral methods perform between 73% and 76%. Further testing with tones from the Iowa MIS collection shows that the neurally inspired method is considerably more robust when tested with data from an unrelated dataset.

Uncoupling of ENOS Causes Superoxide Anion Production and Impairs NO Signaling in the Cerebral Microvessels of Hph-1 Mice

In this study, we used the GTP cyclohydrolase I-deficient mice, i.e., hyperphenylalaninemic (hph-1) mice, to test the hypothesis that the loss of tetrahydrobiopterin (BH(4)) in cerebral microvessels causes endothelial nitric oxide synthase (eNOS) uncoupling, resulting in increased superoxide anion production and inhibition of endothelial nitric oxide signaling. Both homozygous mutant (hph-1(-/-)) and heterozygous mutant (hph-1(+/-) mice) demonstrated reduction in GTP cyclohydrolase I activity and reduced bioavailability of BH(4). In the cerebral microvessels of hph-1(+/-) and hph-1(-/-) mice, increased superoxide anion production was inhibited by supplementation of BH(4) or NOS inhibitor- L- N(G) -nitro arginine-methyl ester, indicative of eNOS uncoupling. Expression of 3-nitrotyrosine was significantly increased, whereas NO production and cGMP levels were significantly reduced. Expressions of antioxidant enzymes namely copper and zinc superoxide dismutase, manganese superoxide dismutase, and catalase were not affected by uncoupling of eNOS. Reduced levels of BH(4), increased superoxide anion production, as well as inhibition of NO signaling were not different between the microvessels of male and female mice. The results of our study are the first to demonstrate that, regardless of gender, reduced BH(4) bioavailability causes eNOS uncoupling, increases superoxide anion production, inhibits eNOS/cGMP signaling, and imposes significant oxidative stress in the cerebral microvasculature.

The Recipe for Success? Invest in Your Team

Creating, Documenting and Sharing Network Models

As computational neuroscience matures, many simulation environments are available that are useful for neuronal network modeling. However, methods for successfully documenting models for publication and for exchanging models and model components among these projects are still under development. Here we briefly review existing software and applications for network model creation, documentation and exchange. Then we discuss a few of the larger issues facing the field of computational neuroscience regarding network modeling and suggest solutions to some of these problems, concentrating in particular on standardized network model terminology, notation, and descriptions and explicit documentation of model scaling. We hope this will enable and encourage computational neuroscientists to share their models more systematically in the future.

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