The Strong Cell-based Hydrogen Peroxide Generation Triggered by Cold Atmospheric Plasma.

Hydrogen peroxide (H2O2) is an important signaling molecule in cancer cells. However, the significant secretion of H2O2 by cancer cells have been rarely observed. Cold atmospheric plasma (CAP) is a near room temperature ionized gas composed of neutral particles, charged particles, reactive species, and electrons. Here, we first demonstrated that breast cancer cells and pancreatic adenocarcinoma cells generated micromolar level H2O2 during just 1 min of direct CAP treatment on these cells. The cell-based H2O2 generation is affected by the medium volume, the cell confluence, as well as the discharge voltage. The application of cold atmospheric plasma (CAP) in cancer treatment has been intensively investigated over the past decade. Several cellular responses to CAP treatment have been observed including the consumption of the CAP-originated reactive species, the rise of intracellular reactive oxygen species, the damage on DNA and mitochondria, as well as the activation of apoptotic events. This is a new previously unknown cellular response to CAP, which provides a new prospective to understand the interaction between CAP and cells in vitro and in vivo. The short-lived reactive species in CAP may activate cells in vivo to generate long-lived reactive species such as H2O2, which may trigger immune attack on tumorous tissues via the H2O2-mediated lymphocyte activation

Developing an e-infrastructure for social science

We outline the aims and progress to date of the National Centre for e-Social Science e-Infrastructure project. We examine the challenges faced by the project, namely in ensuring outputs are appropriate to social scientists, managing the transition from research projects to service and embedding software and data within a wider infrastructural framework. We also provide pointers to related work where issues which have ramifications for this and similar initiatives are being addressed

Complexity of murine cardiomyocyte miRNA biogenesis, sequence variant expression and function

microRNAs (miRNAs) are critical to heart development and disease. Emerging research indicates that regulated precursor processing can give rise to an unexpected diversity of miRNA variants. We subjected small RNA from murine HL-1 cardiomyocyte cells to next generation sequencing to investigate the relevance of such diversity to cardiac biology. ∼40 million tags were mapped to known miRNA hairpin sequences as deposited in miRBase version 16, calling 403 generic miRNAs as appreciably expressed. Hairpin arm bias broadly agreed with miRBase annotation, although 44 miR* were unexpectedly abundant (>20% of tags); conversely, 33 -5p/-3p annotated hairpins were asymmetrically expressed. Overall, variability was infrequent at the 5' start but common at the 3' end of miRNAs (5.2% and 52.3% of tags, respectively). Nevertheless, 105 miRNAs showed marked 5' isomiR expression (>20% of tags). Among these was miR-133a, a miRNA with important cardiac functions, and we demonstrated differential mRNA targeting by two of its prevalent 5' isomiRs. Analyses of miRNA termini and base-pairing patterns around Drosha and Dicer cleavage regions confirmed the known bias towards uridine at the 5' most position of miRNAs, as well as supporting the thermodynamic asymmetry rule for miRNA strand selection and a role for local structural distortions in fine tuning miRNA processing. We further recorded appreciable expression of 5 novel miR*, 38 extreme variants and 8 antisense miRNAs. Analysis of genome-mapped tags revealed 147 novel candidate miRNAs. In summary, we revealed pronounced sequence diversity among cardiomyocyte miRNAs, knowledge of which will underpin future research into the mechanisms involved in miRNA biogenesis and, importantly, cardiac function, disease and therapy.This work was supported by by the Victor Chang Cardiac Research Institute and grants 573726, 573731 and 514904 from the National Health & Medical Research Council awarded to TP

The Functional DRD3 Ser9Gly Polymorphism (rs6280) Is Pleiotropic, Affecting Reward as Well as Movement

Abnormalities of motivation and behavior in the context of reward are a fundamental component of addiction and mood disorders. Here we test the effect of a functional missense mutation in the dopamine 3 receptor (DRD3) gene (ser9gly, rs6280) on reward-associated dopamine (DA) release in the striatum. Twenty-six healthy controls (HCs) and 10 unmedicated subjects with major depressive disorder (MDD) completed two positron emission tomography (PET) scans with [11C]raclopride using the bolus plus constant infusion method. On one occasion subjects completed a sensorimotor task (control condition) and on another occasion subjects completed a gambling task (reward condition). A linear regression analysis controlling for age, sex, diagnosis, and self-reported anhedonia indicated that during receipt of unpredictable monetary reward the glycine allele was associated with a greater reduction in D2/3 receptor binding (i.e., increased reward-related DA release) in the middle (anterior) caudate (p<0.01) and the ventral striatum (p<0.05). The possible functional effect of the ser9gly polymorphism on DA release is consistent with previous work demonstrating that the glycine allele yields D3 autoreceptors that have a higher affinity for DA and display more robust intracellular signaling. Preclinical evidence indicates that chronic stress and aversive stimulation induce activation of the DA system, raising the possibility that the glycine allele, by virtue of its facilitatory effect on striatal DA release, increases susceptibility to hyperdopaminergic responses that have previously been associated with stress, addiction, and psychosis

Slit2 prevents neutrophil recruitment and renal ischemia-reperfusion injury

Neutrophils recruited to the postischemic kidney contribute to the pathogenesis of ischemia-reperfusion injury (IRI), which is the most common cause of renal failure among hospitalized patients. The Slit family of secreted proteins inhibits chemotaxis of leukocytes by preventing activation of Rho-family GTPases, suggesting that members of this family might modulate the recruitment of neutrophils and the resulting IRI. Here, in static and microfluidic shear assays, Slit2 inhibited multiple steps required for the infiltration of neutrophils into tissue. Specifically, Slit2 blocked the capture and firm adhesion of human neutrophils to inflamed vascular endothelial barriers as well as their subsequent transmigration. To examine whether these observations were relevant to renal IRI, we administered Slit2 to mice before bilateral clamping of the renal pedicles. Assessed at 18 hours after reperfusion, Slit2 significantly inhibited renal tubular necrosis, neutrophil and macrophage infiltration, and rise in plasma creatinine. In vitro, Slit2 did not impair the protective functions of neutrophils, including phagocytosis and superoxide production, and did not inhibit neutrophils from killing the extracellular pathogen Staphylococcus aureus. In vivo, administration of Slit2 did not attenuate neutrophil recruitment or bacterial clearance in mice with ascending Escherichia coli urinary tract infections and did not increase the bacterial load in the livers of mice infected with the intracellular pathogen Listeriamonocytogenes. Collectively, these results suggest that Slit2 may hold promise as a strategy to combat renal IRI without compromising the protective innate immune response. Copyright © 2013 by the American Society of Nephrology

Establishment of a New Cell Line from Lepidopteran Epidermis and Hormonal Regulation on the Genes

When an insect molts, old cuticle on the outside of the integument is shed by apolysis and a new cuticle is formed under the old one. This process is completed by the epidermal cells which are controlled by 20-hydroxyecdysone (20E) and juvenile hormone. To understand the molecular mechanisms of integument remolding and hormonal regulation on the gene expression, an epidermal cell line from the 5th instar larval integument of Helicoverpa armigera was established and named HaEpi. The cell line has been cultured continuously for 82 passages beginning on June 30, 2005 until now. Cell doubling time was 64 h. The chromosomes were granular and the chromosome mode was from 70 to 76. Collagenase I was used to detach the cells from the flask bottom. Non-self pathogen AcMNPV induced the cells to apoptosis. The cell line was proved to be an epidermal cell line based on its unique gene expression pattern. It responded to 20E and the non-steroidal ecdysone agonist RH-2485. Its gene expression could be knocked down using RNA interference. Various genes in the cell line were investigated based on their response to 20E. This new cell line represents a platform for investigating the 20E signaling transduction pathway, the immune response mechanism in lepidopteran epidermis and interactions of the genes

Single Nucleotide Polymorphisms That Increase Expression of the Guanosine Triphosphatase RAC1 Are Associated With Ulcerative Colitis

BACKGROUND & AIMS: RAC1 is a GTPase that has an evolutionarily conserved role in coordinating immune defenses, from plants to mammals. Chronic inflammatory bowel diseases (IBD) are associated with dysregulation of immune defenses. We studied the role of RAC1 in IBD using human genetic and functional studies and animal models of colitis. METHODS: We used a candidate gene approach to HapMap-Tag single nucleotide polymorphisms (SNPs) in a discovery cohort; findings were confirmed in 2 additional cohorts. RAC1 mRNA expression was examined from peripheral blood cells of patients. Colitis was induced in mice with conditional disruption of Rac1 in phagocytes by administration of dextran sulphate sodium (DSS). RESULTS: We observed a genetic association between RAC1 with ulcerative colitis (UC) in a discovery cohort, 2 independent replication cohorts, and in combined analysis for the SNPs rs10951982 (Pcombined UC = 3.3 × 10–8, odds ratio [OR]=1.43 [1.26–1.63]) and rs4720672 (Pcombined UC=4.7 × 10–6, OR=1.36 [1.19–1.58]). Patients with IBD who had the rs10951982 risk allele had increased expression of RAC1, compared to those without this allele. Conditional disruption of Rac1 in macrophage and neutrophils of mice protected them against DSS-induced colitis. CONCLUSION: Studies of human tissue samples and knockout mice demonstrated a role for the GTPase RAC1 in the development of UC; increased expression of RAC1 was associated with susceptibility to colitis

Novel Patient Cell-Based HTS Assay for Identification of Small Molecules for a Lysosomal Storage Disease

Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs), inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS) assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA) activity found in patients with metachromatic leukodystrophy (MLD), a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t) cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS), detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S) cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC) acts as “plate fluorescence quencher” in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets

A propofol binding site on mammalian GABAA receptors identified by photolabeling

Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABA(A) receptors, but where it binds to this receptor is not known and has been a matter of some controversy. We have synthesized a novel propofol analogue photolabeling reagent that has a biological activity very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin which have been identified using X-ray crystallography. Using a combination of the protiated label and a deuterated version, and mammalian receptors labeled in intact membranes, we have identified a novel binding site for propofol in GABA(A) receptors consisting of both β(3) homopentamers and α(1)β(3) heteropentamers. The binding site is located within the β subunit, at the interface between the transmembrane domains and the extracellular domain, and lies close to known determinants of anesthetic sensitivity in transmembrane segments TM1 and TM2