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Toll-like Receptor 4 Activation Promotes Cardiac Arrhythmias By Decreasing The Transient Outward Potassium Current (ito) Through An Irf3-dependent And Myd88-independent Pathway.

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll-like receptors (TLRs) seem to be involved in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias, and the signaling pathway involved in these effects. Membrane potential was recorded in Wistar rat ventricle. Ca(2+) transients, as well as the L-type Ca(2+) current (ICaL) and the transient outward K(+) current (Ito), were recorded in isolated myocytes after 24 h exposure to the TLR4 agonist, lipopolysaccharide (LPS, 1 μg/ml). TLR4 stimulation in vitro promoted a cardiac electrical remodeling that leads to action potential prolongation associated with arrhythmic events, such as delayed afterdepolarization and triggered activity. After 24 h LPS incubation, Ito amplitude, as well as Kv4.3 and KChIP2 mRNA levels were reduced. The Ito decrease by LPS was prevented by inhibition of interferon regulatory factor 3 (IRF3), but not by inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4) or nuclear factor kappa B (NF-κB). Extrasystolic activity was present in 25% of the cells, but apart from that, Ca(2+) transients and ICaL were not affected by LPS; however, Na(+)/Ca(2+) exchanger (NCX) activity was apparently increased. We conclude that TLR4 activation decreased Ito, which increased AP duration via a MyD88-independent, IRF3-dependent pathway. The longer action potential, associated with enhanced Ca(2+) efflux via NCX, could explain the presence of arrhythmias in the LPS group.

Comparative Metagenomic Analysis Of Coral Microbial Communities Using A Reference-independent Approach.

By comparing the SEED and Pfam functional profiles of metagenomes of two Brazilian coral species with 29 datasets that are publicly available, we were able to identify some functions, such as protein secretion systems, that are overrepresented in the metagenomes of corals and may play a role in the establishment and maintenance of bacteria-coral associations. However, only a small percentage of the reads of these metagenomes could be annotated by these reference databases, which may lead to a strong bias in the comparative studies. For this reason, we have searched for identical sequences (99% of nucleotide identity) among these metagenomes in order to perform a reference-independent comparative analysis, and we were able to identify groups of microbial communities that may be under similar selective pressures. The identification of sequences shared among the metagenomes was found to be even better for the identification of groups of communities with similar niche requirements than the traditional analysis of functional profiles. This approach is not only helpful for the investigation of similarities between microbial communities with high proportion of unknown reads, but also enables an indirect overview of gene exchange between communities.

Detection Of Explosives On The Surface Of Banknotes By Raman Hyperspectral Imaging And Independent Component Analysis.

The aim of this study was to develop a methodology using Raman hyperspectral imaging and chemometric methods for identification of pre- and post-blast explosive residues on banknote surfaces. The explosives studied were of military, commercial and propellant uses. After the acquisition of the hyperspectral imaging, independent component analysis (ICA) was applied to extract the pure spectra and the distribution of the corresponding image constituents. The performance of the methodology was evaluated by the explained variance and the lack of fit of the models, by comparing the ICA recovered spectra with the reference spectra using correlation coefficients and by the presence of rotational ambiguity in the ICA solutions. The methodology was applied to forensic samples to solve an automated teller machine explosion case. Independent component analysis proved to be a suitable method of resolving curves, achieving equivalent performance with the multivariate curve resolution with alternating least squares (MCR-ALS) method. At low concentrations, MCR-ALS presents some limitations, as it did not provide the correct solution. The detection limit of the methodology presented in this study was 50μgcm(-2).

Model Independent Tests of Skyrmions and Their Holographic Cousins

We describe a new exact relation for large N(c) QCD for the long-distance behavior of baryon form factors in the chiral limit. This model-independent relation is used to test the consistency of the structure of several baryon models. All 4D semiclassical chiral soliton models satisfy the relation, as does the Pomarol-Wulzer holographic model of baryons as 5D Skyrmions. However, remarkably, we find that the holographic model treating baryons as instantons in the Sakai-Sugimoto model does not satisfy the relation.

Culture-Independent Assessment of Rhizobiales-Related Alphaproteobacteria and the Diversity of Methylobacterium in the Rhizosphere and Rhizoplane of Transgenic Eucalyptus

The rhizosphere is an ecosystem exploited by a variety of organisms involved in plant health and environmental sustainability. Abiotic factors influence microorganism-plant interactions, but the microbial community is also affected by expression of heterologous genes from host plants. In the present work, we assessed the community shifts of Alphaproteobacteria phylogenetically related to the Rhizobiales order (Rhizobiales-like community) in rhizoplane and rhizosphere soils of wild-type and transgenic eucalyptus. A greenhouse experiment was performed and the bacterial communities associated with two wild-type (WT17 and WT18) and four transgenic (TR-9, TR-15, TR-22, and TR-23) eucalyptus plant lines were evaluated. The culture-independent approach consisted of the quantification, by real-time polymerase chain reaction (PCR), of a targeted subset of Alphaproteobacteria and the assessment of its diversity using PCR-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Real-time quantification revealed a lesser density of the targeted community in TR-9 and TR-15 plants and diversity analysis by principal components analysis, based on PCR-DGGE, revealed differences between bacterial communities, not only between transgenic and nontransgenic plants, but also among wild-type plants. The comparison between clone libraries obtained from the transgenic plant TR-15 and wild-type WT17 revealed distinct bacterial communities associated with these plants. In addition, a culturable approach was used to quantify the Methylobacterium spp. in the samples where the identification of isolates, based on 16S rRNA gene sequences, showed similarities to the species Methylobacterium nodulans, Methylobacterium isbiliense, Methylobacterium variable, Methylobacterium fujisawaense, and Methylobacterium radiotolerans. Colonies classified into this genus were not isolated from the rhizosphere but brought in culture from rhizoplane samples, except for one line of the transgenic plants (TR-15). In general, the data suggested that, in most cases, shifts in bacterial communities due to cultivation of transgenic plants are similar to those observed when different wild-type cultivars are compared, although shifts directly correlated to transgenic plant cultivation may be found.

Endogenous opioids: role in prostaglandin-dependent and -independent fever

This study evaluated the participation of mu-opioid-receptor activation in body temperature (T-b) during normal and febrile conditions (including activation of heat conservation mechanisms) and in different pathways of LPS-induced fever. The intracerebroventricular treatment of male Wistar rats with the selective opioid mu-receptor-antagonist cyclic D-Phe-Cys-Try-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 0.1-1.0 mu g) reduced fever induced by LPS (5.0 mu g/kg) but did not change Tb at ambient temperatures of either 20 C or 28 C. The subcutaneous, intracerebroventricular, and intrahypothalamic injection of morphine (1.0 -10.0 mg/kg, 3.0 -30.0 mu g, and 1 -100 ng, respectively) produced a dose-dependent increase in Tb. Intracerebroventricular morphine also produced a peripheral vasoconstriction. Both effects were abolished by CTAP. CTAP (1.0 mu g icv) reduced the fever induced by intracerebroventricular administration of TNF-alpha (250 ng), IL-6 (300 ng), CRF (2.5 mu g), endothelin-1 (1.0 pmol), and macrophage inflammatory protein (500 pg) and the first phase of the fever induced by PGF(2 alpha) (500.0 ng) but not the fever induced by IL-1 beta (3.12 ng) or PGE(2) (125.0 ng) or the second phase of the fever induced by PGF(2 alpha). Morphine-induced fever was not modified by the cyclooxygenase (COX) inhibitor indomethacin (2.0 mg/kg). In addition, morphine injection did not induce the expression of COX-2 in the hypothalamus, and CTAP did not modify PGE2 levels in cerebrospinal fluid or COX-2 expression in the hypothalamus after LPS injection. In conclusion, our results suggest that LPS and endogenous pyrogens (except IL-1 beta and prostaglandins) recruit the opioid system to cause a mu-receptor-mediated fever.

Relaxation evoked by extracellular Ca(2+) in rat aorta is nerve-independent and involves sarcoplasmic reticulum and L-type Ca(2+) channell

The perivascular nerve network expresses a Ca(2+) receptor that is activated by high extracellular Ca(2+) concentrations and causes vasorelaxation in resistance arteries. We have verified the influence of perivascular nerve fibers on the Ca(2+)-induced relaxation in aortic rings. To test our hypothesis, either pre-contracted aortas isolated from rats after sensory denervation with capsaicin or aortic rings acutely denervated with phenol were stimulated to relax with increasing extracellular Ca(2+) concentration. We also studied the role of the endothelium on the Ca(2+)-induced relaxation, and we verified the participation of endothelial/nonendothelial nitric oxide and cyclooxygenise-arachidonic acid metabolites. Additionally, the role of the sarcoplasmic reticulum, K(+) channels and L-type Ca(2+) channels on the Ca(2+)-induced relaxation were evaluated. We have observed that the Ca(2+)-induced relaxation is completely nerve independent, and it is potentiated by endothelial nitric oxide (NO). In endothelium-denuded aortic rings, indomethacin and AH6809 (PGF(2 alpha) receptor antagonist) enhance the relaxing response to Ca(2+). This relaxation is inhibited by thapsigargin and verapamil, while was not altered by tetraethylammonium. In conclusion, we have shown that perivascular nervous fibers do not participate in the Ca(2+)-induced relaxation, which is potentiated by endothelial NO. In endothelium-denuded preparations, indomethacin and AH6809 enhance the relaxation induced by Ca(2+). The relaxing response to Call was impaired by verapamil and thapsigargin, revealing the importance of L-type Ca(2+) channels and sarcoplasmic reticulum in this response. (c) 2008 Elsevier Inc. All rights reserved.

ATP-induced apoptosis involves a Ca(2+)-independent phospholipase A(2) and 5-lipoxygenase in macrophages

Macrophages express P2X(7) and other nucleotide (P2) receptors, and display the phenomena of extracellular ATP (ATP(e))-induced P2X(7)-dependent membrane permeabilization and cell death by apoptosis and necrosis. P2X7 receptors also cooperate with toll-like receptors (TLRs) to induce inflammasome activation and IL-1 beta secretion. We investigated signaling pathways involved in the induction of cell death by ATP, in intraperitoneal murine macrophages. Apoptosis (hypodiploid nuclei) and necrosis (LDH release) were detected 6 h after an induction period of 20 min in the presence of ATP Apoptosis was blocked by caspase 3 and caspase 9 inhibitors and by cyclosporin A. The MAPK inhibitors PD-98059, SB-203580 and SB-202190 provoked no significant effect oil apoptosis, but SB-203580 blocked LDH release. Neither apoptosis nor necrosis was inhibited when both intra- and extracellular Ca(2+) were chelated during the induction period. Mepacrine, a generic PLA(2) inhibitor and BEL, an inhibitor of Ca(2+)-independent PLA(2) (iPLA(2)) blocked apoptosis, while pBPB and AACOOPF(3). inhibitors of secretory and Ca(2+)-dependent PLA(2) respectively, had no significant effect. Cycloxygenase inhibitors had no effect on apoptosis, while the inhibitors of lipoxygenase (LOX) and leukotriene biosynthesis nordihydroguaiaretic acid (NDGA), zileuton, AA-861, and MK-886 significantly decreased apoptosis. Neither NDGA nor MK-886 blocked apoptosis of 5-LOX(-/-) macrophages. CP-105696 and MK-571, antagonists of leukotriene receptors, had no significant effect on apoptosis. None of the inhibitors of PLA(2) and LOX/leukotriene pathway had a significant inhibitory effect on LDH release. Our results indicate that a Ca(2+) -independent step involving an iPLA(2) and 5-LOX are involved in the triggering of apoptosis but not necrosis by P2X7 in macrophages. (C) 2008 Elsevier Inc. All rights reserved.