Enlighten-Research publications by members of the University of Glasgow http://eprints.gla.ac.uk Minimally invasive surgery with thrombolysis in intracerebral haemorrhage evacuation (MISTIE III): a randomised, controlled, open-label phase 3 trial with blinded endpoint

Prolific generation of NO by inducible nitric oxide synthase (iNOS) can cause unintended injury to host cells during glomerulonephritis and other inflammatory diseases. While much is known about the mechanisms of iNOS induction, few transcriptional repressors have been found. We explored the role of signal transducers and activators of transcription 3 (STAT3) proteins in interleukin (IL)-1beta- and lipopolysaccharide (LPS)+interferon (IFN)-gamma-mediated iNOS induction in murine mesangial cells. Both stimuli induced rapid phosphorylation of STAT3 and sequence-specific STAT3 DNA-binding activity. Supershift assays with a STAT3 element probe demonstrated that nuclear factor kappaB (NF-kappaB) p65 and p50 complexed with STAT3 in the DNA-protein complex. The direct interaction of STAT3 and NF-kappaB p65 was verified in vivo by co-immunoprecipitation and in vitro by pull-down assays with glutathione S-transferase-NF-kappaB p65 fusion protein and in vitro -translated STAT3alpha. Overexpression of STAT3 dramatically inhibited IL-1beta- or LPS+IFN-gamma-mediated induction of iNOS promoter-luciferase constructs that contained the wild-type iNOS promoter or ones harbouring mutated STAT-binding elements. In tests of indirect inhibitory effects of STAT3, overexpression of STAT3 dramatically inhibited the activity of an NF-kappaB-dependent promoter devoid of STAT-binding elements without affecting NF-kappaB DNA-binding activity. Thus STAT3, via direct interactions with NF-kappaB p65, serves as a dominant-negative inhibitor of NF-kappaB activity to suppress indirectly cytokine induction of the iNOS promoter in mesangial cells. These results provide a new model for the termination of NO production by activated iNOS following exposure to pro-inflammatory stimuli.

Protein phosphorylation regulates a series of important biological processes in eukaryotes. However, the phosphorylation sites found up to now are far below than that actually exists in proteins due to the extreme complexity of the proteome sample. Here a new reversed-phase-reversed-phase liquid chromatography (RP-RPLC) approach was developed for multidimensional separation of phosphopeptides. In this approach, a large number of fractions were collected from the first dimensional RPLC separation at high pH. And then these fractions were pooled every two fractions with equal time interval, one from the early eluted section and another one from the later eluted section. The pooled fractions were finally submitted to RPLC-tandem mass spectrometry (MS/MS) analysis at low pH. It was found the resulting 2D separation was highly orthogonal and yielded more than 30% phosphopeptide identifications over the conventional RP-RPLC approach. This study provides a powerful approach for efficient separation of phosphopeptides and global phosphorylation analysis, where the orthogonality of 2D separation is greatly improved and the first dimensional separation is of high resolution.

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Significant hematoma expansion (HE) affects one‐fifth of people within 24 hours after acute intracerebral hemorrhage (ICH), and its prevention is an appealing treatment target. Although the computed tomography (CT)‐angiography spot sign predicts HE, only a minority of ICH patients receive contrast injection. Conversely, noncontrast CT (NCCT) is used to diagnose nearly all ICH, so NCCT markers represent a widely available alternative for prediction of HE. However, different NCCT signs describe similar features, with lack of consensus on the optimal image acquisition protocol, assessment, terminology, and diagnostic criteria. In this review, we propose practical guidelines for detecting, interpreting, and reporting NCCT predictors of HE. ANN NEUROL 2019;86:480–492

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BackgroundEmerging evidence indicates that reactive microglia-initiated inflammatory responses are responsible for secondary damage after primary traumatic spinal cord injury (SCI); epidermal growth factor receptor (EGFR) signaling may be involved in cell activation. In this report, we investigate the influence of EGFR signaling inhibition on microglia activation, proinflammatory cytokine production, and the neuronal microenvironment after SCI.MethodsLipopolysaccharide-treated primary microglia/BV2 line cells and SCI rats were used as model systems. Both C225 and AG1478 were used to inhibit EGFR signaling activation. Cell activation and EGFR phosphorylation were observed after fluorescent staining and western blot. Production of interleukin-1beta (IL-1β) and tumor necrosis factor alpha (TNFα) was tested by reverse transcription PCR and ELISA. Western blot was performed to semi-quantify the expression of EGFR/phospho-EGFR, and phosphorylation of Erk, JNK and p38 mitogen-activated protein kinases (MAPK). Wet-dry weight was compared to show tissue edema. Finally, axonal tracing and functional scoring were performed to show recovery of rats.ResultsEGFR phosphorylation was found to parallel microglia activation, while EGFR blockade inhibited activation-associated cell morphological changes and production of IL-1β and TNFα. EGFR blockade significantly downregulated the elevated MAPK activation after cell activation; selective MAPK inhibitors depressed production of cytokines to a certain degree, suggesting that MAPK mediates the depression of microglia activation brought about by EGFR inhibitors. Subsequently, seven-day continual infusion of C225 or AG1478 in rats: reduced the expression of phospho-EGFR, phosphorylation of Erk and p38 MAPK, and production of IL-1β and TNFα; lessened neuroinflammation-associated secondary damage, like microglia/astrocyte activation, tissue edema and glial scar/cavity formation; and enhanced axonal outgrowth and functional recovery.ConclusionsThese findings indicate that inhibition of EGFR/MAPK suppresses microglia activation and associated cytokine production; reduces neuroinflammation-associated secondary damage, thus provides neuroprotection to SCI rats, suggesting that EGFR may be a therapeutic target, and C225 and AG1478 have potential for use in SCI treatment.

Abstract. The inducible nitric oxide synthase (iNOS) gene plays an important role in renal diseases. Transcription is the principal mode of regulation. This study explores the role of acetylation in cytokine-mediated iNOS induction in cultured murine mesangial cells and RAW 264.7 cells. Nitric oxide production was measured by the Griess reaction. The activity of the iNOS promoter and a nuclear factor-B (NF-B) element promoter were assessed in transient transfection assays. Gel shift and supershift assays were used to identify NF-B in nuclear extracts. Protein-protein interactions were assayed by co-immunoprecipitation and GST pull-down assays. Treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and overexpression of HDAC isoforms were used to assess the impact of acetylation status on iNOS and NF-B element promoter activity. TSA inhibited induction of endogenous NO production and iNOS as well as NF-B element promoter activity in response to interleukin-1␤ (IL-1␤) or lipopolysaccharide (LPS) ϩ interferon-␥ (IFN-␥) in both cell types without altering NF-B DNA binding activity. Overexpression of specific HDAC isoforms enhanced cytokine induction of both the iNOS and the NF-B element promoter. HDAC2 and NF-B p65 co-immunoprecipitated from mesangial cell nuclear extracts, and in vitro translated HDAC2 specifically interacted with an NF-B p65 GST fusion protein.Hyperacetylation diminishes cytokine induction of iNOS transcription activity, at least partially, by limiting the functional efficacy of NF-B. The specific recruitment of HDAC2 to NF-B at target promoters and the consequent effects on acetylation status may play an important role in regulating iNOS as well as other NF-B-dependent genes involved in inflammation.