Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2

NADPH oxidase (Nox4) produces reactive oxygen species (ROS) that are important for vascular smooth muscle cell (SMC) behavior, but the potential impact of Nox4 in stem cell differentiation is unknown. When mouse embryonic stem (ES) cells were plated on collagen IV-coated dishes/flasks, a panel of SMC-specific genes was significantly and consistently upregulated. Nox4 expression was markedly correlated with such a gene induction as confirmed by real-time PCR, immunofluorescence, and Western blot analysis. Overexpression of Nox4 specifically resulted in increased SMC marker production, whereas knockdown of Nox4 induced a decrease. Furthermore, SMC-specific transcription factors, including serum response factor (SRF) and myocardin were activated by Nox4 gene expression. Moreover, Nox4 was demonstrated to drive SMC differentiation through generation of H(2)O(2). Confocal microscopy analysis indicates that SRF was translocated into the nucleus during SMC differentiation in which SRF was phosphorylated. Additionally, autosecreted transforming growth factor (TGF)-beta(1) activated Nox4 and promoted SMC differentiation. Interestingly, cell lines generated from stem cells by Nox4 transfection and G418 selection displayed a characteristic of mature SMCs, including expression of SMC markers and cells with contractile function. Thus we demonstrate for the first time that Nox4 is crucial for SMC differentiation from ES cells, and enforced Nox4 expression can maintain differentiation status and functional features of stem cell-derived SMCs, highlighting its impact on vessel formation in vivo and vascular tissue engineering in the future.

HIV-1 Nef limits communication between linker of activated T cells and SLP-76 to reduce formation of SLP-76-signaling microclusters following TCR stimulation

Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCR-CD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nef-expressing and HIV-1-infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef's ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76-signaling MCs.

The variability in nutrient composition of Anaerobic Digestate granules produced from high shear granulation

This study investigates the production of organic fertilizer using Anaerobic Digestate (as a nutrient source) and limestone powder as the raw materials. A two-level factorial experimental design was used to determine the influence of process variables on the nutrient homogeneity within the granules. Increasing the liquid-to-solid ratio during granulation resulted in increased granule nutrient homogeneity. Increasing the processing time and the impeller speed were also found to increase the nutrient homogeneity. In terms of nutrients release into deionized water, the granules effectively released both potassium and phosphate into solution. © 2012 Elsevier Ltd.

Generation of scaled protogalactic seed magnetic fields in laser-produced shock waves

The standard model for the origin of galactic magnetic fields is through the amplification of seed fields via dynamo or turbulent processes to the level consistent with present observations. Although other mechanisms may also operate, currents from misaligned pressure and temperature gradients (the Biermann battery process) inevitably accompany the formation of galaxies in the absence of a primordial field. Driven by geometrical asymmetries in shocks associated with the collapse of protogalactic structures, the Biermann battery is believed to generate tiny seed fields to a level of about 10 gauss (refs 7, 8). With the advent of high-power laser systems in the past two decades, a new area of research has opened in which, using simple scaling relations, astrophysical environments can effectively be reproduced in the laboratory. Here we report the results of an experiment that produced seed magnetic fields by the Biermann battery effect. We show that these results can be scaled to the intergalactic medium, where turbulence, acting on timescales of around 700 million years, can amplify the seed fields sufficiently to affect galaxy evolution.

Lipoxins attenuate renal fibrosis by inducing let-7c and suppressing TGFβR1

Lipoxins, which are endogenously produced lipid mediators, promote the resolution of inflammation, and may inhibit fibrosis, suggesting a possible role in modulating renal disease. Here, lipoxin A4 (LXA4) attenuated TGF-ß1-induced expression of fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1 in cultured human proximal tubular epithelial (HK-2) cells through a mechanism involving upregulation of the microRNA let-7c. Conversely, TGF-ß1 suppressed expression of let-7c. In cells pretreated with LXA4, upregulation of let-7c persisted despite subsequent stimulation with TGF-ß1. In the unilateral ureteral obstruction model of renal fibrosis, let-7c upregulation was induced by administering an LXA4 analog. Bioinformatic analysis suggested that targets of let-7c include several members of the TGF-ß1 signaling pathway, including the TGF-ß receptor type 1. Consistent with this, LXA4-induced upregulation of let-7c inhibited both the expression of TGF-ß receptor type 1 and the response to TGF-ß1. Overexpression of let-7c mimicked the antifibrotic effects of LXA4 in renal epithelia; conversely, anti-miR directed against let-7c attenuated the effects of LXA4. Finally, we observed that several let-7c target genes were upregulated in fibrotic human renal biopsies compared with controls. In conclusion, these results suggest that LXA4-mediated upregulation of let-7c suppresses TGF-ß1-induced fibrosis and that expression of let-7c targets is dysregulated in human renal fibrosis.

Self-organized electromagnetic field structures in laser-produced counter-streaming plasmas

Self-organization(1,2) occurs in plasmas when energy progressively transfers from smaller to larger scales in an inverse cascade(3). Global structures that emerge from turbulent plasmas can be found in the laboratory(4) and in astrophysical settings; for example, the cosmic magnetic field(5,6,) collisionless shocks in supernova remnants(7) and the internal structures of newly formed stars known as Herbig-Haro objects(8). Here we show that large, stable electromagnetic field structures can also arise within counter-streaming supersonic plasmas in the laboratory. These surprising structures, formed by a yet unexplained mechanism, are predominantly oriented transverse to the primary flow direction, extend for much larger distances than the intrinsic plasma spatial scales and persist for much longer than the plasma kinetic timescales. Our results challenge existing models of counter-streaming plasmas and can be used to better understand large-scale and long-time plasma self-organization.

Stimulation of cholesteryl ester synthesis in human monocyte-derived macrophages by low-density lipoproteins from type 1 (insulin-dependent) diabetic patients:the influence of non-enzymatic glycosylation of low-density lipoproteins

Diabetes mellitus is an independent risk factor in the development of atherosclerosis. In this study we aimed to demonstrate whether there is an abnormal interaction between low-density lipoproteins from diabetic patients and human macrophages. We measured cholesteryl ester synthesis and cholesteryl ester accumulation in human monocyte-derived macrophages (obtained from non-diabetic donors) incubated with low density lipoproteins from Type 1 (insulin-dependent) diabetic patients in good or fair glycaemic control. Low density lipoproteins from the diabetic patients stimulated more cholesteryl ester synthesis than low density lipoproteins from non-diabetic control subjects (7.19 +/- 1.19 vs 6.11 +/- 0.94 nmol/mg cell protein/20 h, mean +/- SEM, p less than 0.05). The stimulation of cholesteryl ester synthesis by low density lipoproteins isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (p less than 0.02). There were no significant differences in the lipid composition of low density lipoproteins between the diabetic and control groups. Non-enzymatic glycosylation of low density lipoproteins was higher in the diabetic group (p less than 0.01) and correlated significantly with cholesteryl ester synthesis (r = 0.58). Similarly, low-density lipoproteins obtained from non-diabetic subjects and glycosylated in vitro stimulated more cholesteryl ester synthesis in macrophages than control low density lipoproteins. The increase in cholesteryl ester synthesis and accumulation by cells exposed to low density lipoproteins from diabetic patients seems to be mediated by an increased uptake of these lipoproteins by macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

SMART Arm with outcome-triggered electrical stimulation:a pilot randomized clinical trial

The SMART (SensoriMotor Active Rehabilitation Training) Arm is a nonrobotic device designed to allow stroke survivors with severe paresis to practice reaching. It can be used with or without outcome-triggered electrical stimulation (OT-stim) to augment movement. The aim of this study was to evaluate the efficacy of SMART Arm training when used with or without OT-stim, in addition to usual care, as compared with usual care alone during inpatient rehabilitation.

Biochemical and physiochemical characteristics of a pre-B cell stimulating factor produced by the plasmacytoma cell line LICR LON HMY2

We have characterized the pre-B cell colony stimulating activity (pre-B cell CSA) from LICR LON HMY2 conditioned medium (CM) by a variety of biochemical techniques. Pre-B cell CSA was found to be associated with a heat stable glycoprotein which has an isoelectric point of 8.3 and a mol. wt, as determined by polyacrylamide gel electrophoresis, of 28-32 kD. The relationship of this activity to previously described factors acting on cells of the B cell lineage is discussed.

Histamine H3 receptor-mediated inhibition of sympathetically evoked mydriasis in rats

This study was designed to determine if the histamine H3 receptor agonist R-alpha-methylhistamine would play a role in modulation of sympathetically evoked mydriasis in anesthetized rats, and if so, to ascertain the specific receptor subtype(s) involved. Reproducible frequency-response curves of pupillary dilation were generated by stimulation of the cervical preganglionic sympathetic nerve (1-32 Hz). Systemic administration of R-alpha-methylhistamine (0.3-3.0 mg kg(-1)) produced a dose-related inhibition of the evoked mydriasis. The greatest inhibition was seen at lower frequency levels, with about 43% depression observed at 2 Hz. The specific histamine H3 receptor antagonist, clobenpropit (3.0 mg kg(-1), i.v.), blocked the inhibitory effect of R-alpha-methylhistamine, whereas neither the histamine H2 receptor antagonist, cimetidine (5.0 mg kg(-1), i.v.), nor the histamine H1 receptor antagonist, chlorpheniramine (0.5 mg kg(-1), i.v.), was effective. The histamine H2 receptor agonist, dimaprit (10 mg kg(-1), i.v.), was also without effect on the evoked mydriasis. R-alpha-methylhistamine (3.0 mg kg(-1)) did not inhibit phenylephrine-induced mydriasis. These results support the conclusion that R-alpha-methylhistamine produces inhibition of sympathetically evoked mydriasis via histamine H3 receptor stimulation, presumably by an action on presynaptic histamine H3 receptors.

Speed of processing in the primary motor cortex: A continuous theta burst stimulation study

‘Temporally urgent’ reactions are extremely rapid, spatially precise movements that are evoked following discrete stimuli. The involvement of primary motor cortex (M1) and its relationship to stimulus intensity in such reactions is not well understood. Continuous theta burst stimulation (cTBS) suppresses focal regions of the cortex and can assess the involvement of motor cortex in speed of processing. The primary objective of this study was to explore the involvement of M1 in speed of processing with respect to stimulus intensity. Thirteen healthy young adults participated in this experiment. Behavioral testing consisted of a simple button press using the index finger following median nerve stimulation of the opposite limb, at either high or low stimulus intensity. Reaction time was measured by the onset of electromyographic activity from the first dorsal interosseous (FDI) muscle of each limb. Participants completed a 30 min bout of behavioral testing prior to, and 15 min following, the delivery of cTBS to the motor cortical representation of the right FDI. The effect of cTBS on motor cortex was measured by recording the average of 30 motor evoked potentials (MEPs) just prior to, and 5 min following, cTBS. Paired t-tests revealed that, of thirteen participants, five demonstrated a significant attenuation, three demonstrated a significant facilitation and five demonstrated no significant change in MEP amplitude following cTBS. Of the group that demonstrated attenuated MEPs, there was a biologically significant interaction between stimulus intensity and effect of cTBS on reaction time and amplitude of muscle activation. This study demonstrates the variability of potential outcomes associated with the use of cTBS and further study on the mechanisms that underscore the methodology is required. Importantly, changes in motor cortical excitability may be an important determinant of speed of processing following high intensity stimulation.