Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. 3D reconstruction based on cryo-EM images and single-particle image processing revealed that in a relaxed state, myosin molecules undergo intramolecular head–head interactions, explaining why head activity switches off. The filament model obtained by rigidly docking a chicken smooth muscle myosin structure to the reconstruction was improved by flexibly fitting an atomic model built by mixing structures from different species to a tilt-corrected 2-nm 3D map of frozen-hydrated tarantula thick filament. We used heavy and light chain sequences from tarantula myosin to build a single-species homology model of two heavy meromyosin interacting-heads motifs (IHMs). The flexibly fitted model includes previously missing loops and shows five intramolecular and five intermolecular interactions that keep the IHM in a compact off structure, forming four helical tracks of IHMs around the backbone. The residues involved in these interactions are oppositely charged, and their sequence conservation suggests that IHM is present across animal species. The new model, PDB 3JBH, explains the structural origin of the ATP turnover rates detected in relaxed tarantula muscle by ascribing the very slow rate to docked unphosphorylated heads, the slow rate to phosphorylated docked heads, and the fast rate to phosphorylated undocked heads. The conservation of intramolecular interactions across animal species and presence of IHM in bilaterians suggest that a super-relaxed state should be maintained, as it plays a role in saving ATP in skeletal, cardiac, and smooth muscle.

A 63-day feeding trial was conducted in northern snakehead to observe the effects of a dietary soybean meal substitution on the microbiota community, morphology and inflammatory cytokine gene expression in the intestine. Four isonitrogenous and isoenergetic diets containing increasing levels of soybean meal were used to replace 0%, 25%, 50% and 75% of the defatted fishmeal (diets are referred to G1, G2, G3 and G4, respectively). Different dietary soybean meal substitutions significantly affected the intestinal microbiota composition. At the phylum level, Firmicutes abundance was the lowest in the G4 group, in contrast with Proteobacteria, Bacteroidetes and Planctomycetes. At the genus level, significantly lower abundance of Lactococcus, Geobacillus, Pseudomonas, Streptococcus, Bacillus and Acinetobacter,but higher abundance of Cetobacterium, Planctomyces, Shewanella, Thermomonas, Rubrivivax and Carnobacterium was observed in fish fed the G4 diet. With increased dietary soybean meal, the thickness of the muscularis, the height of the fold and the height of the microvillus in the distal intestine decreased, but the relative expression of IL-1β, IL-10 and IL-17F was significantly up-regulated. In conclusion, more emphasis should be placed on the functionality of intestinal microbiota and the pathogenesis of mucosal inflammation to assess the effects of diet and fish intestinal health through intestinal microbiota profiling.

Acyl homoserine lactone (AHL)-based quorum sensing commonly refers to cell density-dependent regulatory mechanisms found in bacteria. However, beyond bacteria, this cell-to-cell communication mechanism is poorly understood. Here we show that a methanogenic archaeon, Methanosaeta harundinacea 6Ac, encodes an active quorum sensing system that is used to regulate cell assembly and carbon metabolic flux. The methanogen 6Ac showed a cell density-dependent physiology transition, which was related to the AHL present in the spent culture and the filI gene-encoded AHL synthase. Through extensive chemical analyses, a new class of carboxylated AHLs synthesized by FilI protein was identified. These carboxylated AHLs facilitated the transition from a short cell to filamentous growth, with an altered carbon metabolic flux that favoured the conversion of acetate to methane and a reduced yield in cellular biomass. The transcriptomes of the filaments and the short cell forms differed with gene expression profiles consistent with the physiology. In the filaments, genes encoding the initial enzymes in the methanogenesis pathway were upregulated, whereas those for cellular carbon assimilation were downregulated. A luxI-luxR ortholog filI-filR was present in the genome of strain 6Ac. The carboxylated AHLs were also detected in other methanogen cultures and putative filI orthologs were identified in other methanogenic genomes as well. This discovery of AHL-based quorum sensing systems in methanogenic archaea implies that quorum sensing mechanisms are universal among prokaryotes.

In this work, we report the complete genome sequence of an obligate aceticlastic methanogen, Methanosaeta harundinacea 6Ac. Genome comparison indicated that the three cultured Methanosaeta spp., M. thermophila, M. concilii and M. harundinacea 6Ac, each carry an entire suite of genes encoding the proteins involved in the methyl-group oxidation pathway, a pathway whose function is not well documented in the obligately aceticlastic methanogens. Phylogenetic analysis showed that the methyl-group oxidation-involving proteins, Fwd, Mtd, Mch, and Mer from Methanosaeta strains cluster with the methylotrophic methanogens, and were not closely related to those from the hydrogenotrophic methanogens. Quantitative PCR detected the expression of all genes for this pathway, albeit ten times lower than the genes for aceticlastic methanogenesis in strain 6Ac. Western blots also revealed the expression of fwd and mch, genes involved in methyl-group oxidation. Moreover, 13C-labeling experiments suggested that the Methanosaeta strains might use the pathway as a methyl oxidation shunt during the aceticlastic metabolism. Because the mch mutants of Methanosarcina barkeri or M. acetivorans failed to grow on acetate, we suggest that Methanosaeta may use methyl-group oxidation pathway to generate reducing equivalents, possibly for biomass synthesis. An fpo operon, which encodes an electron transport complex for the reduction of CoM-CoB heterodisulfide, was found in the three genomes of the Methanosaeta strains. However, an incomplete protein complex lacking the FpoF subunit was predicted, as the gene for this protein was absent. Thus, F420H2 was predicted not to serve as the electron donor. In addition, two gene clusters encoding the two types of heterodisulfide reductase (Hdr), hdrABC, and hdrED, respectively, were found in the three Methanosaeta genomes. Quantitative PCR determined that the expression of hdrED was about ten times higher than hdrABC, suggesting that hdrED plays a major role in aceticlastic methanogenesis.

Interleukin-33 (IL-33) is a recently described member of the interleukin-1 (IL-1) family. It is produced by diverse cell types in response to a variety of stresses including hemorrhage and increased mechanical load. Though only relatively recently discovered, IL-33 has been shown to participate in several pathological processes including promoting type 2 T helper cell-associated autoimmune diseases. In contrast, IL-33 has been also found to have protective effects in cardiovascular diseases. Recent studies have illustrated that IL-33 attenuates cardiac fibrosis induced by increased cardiovascular load in mice (transaortic constriction). Since cardiac fibrosis is largely dependent on increased production of extracellular matrix by cardiac fibroblasts, we hypothesized that IL-33 directly inhibits pro-fibrotic activities of these cells. Experiments have been carried out with isolated rat cardiac fibroblasts to evaluate the effects of IL-33 on the modulation of cardiac fibroblast gene expression and function to test this hypothesis. The expression of the IL-33 receptor, interleukin-1 receptor-like 1 (ST2), was detected at the mRNA and protein levels in isolated adult rat cardiac fibroblasts. Subsequently, the effects of IL-33 treatment (0–100 ng/ml) on the expression of extracellular matrix proteins and pro-inflammatory cytokines/chemokines were examined as well as the effects on rat cardiac fibroblast activities including proliferation, collagen gel contraction and migration. While IL-33 did not directly inhibit collagen I and collagen III production, it yielded a dose-dependent increase in the expression of interleukin-6 and monocyte chemotactic protein-1. Treatment of rat cardiac fibroblasts with IL-33 also impaired the migratory activity of these cells. Further experiments illustrated that IL-33 rapidly activated multiple signaling pathways including extracellular signal-regulated kinases, p38 mitogen-activated protein kinase, c-Jun N-terminal kinases and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) in a dose-dependent manner. Experiments were carried out with pharmacological inhibitors to determine the role of specific signaling pathways in the response of fibroblasts to IL-33. These experiments illustrated that the activation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinases are critical to the increased production of interleukin-6 and monocyte chemotactic protein-1 in response to IL-33. These studies suggest that IL-33 has an important role in the modulation of fibroblast function and gene expression. Surprisingly, IL-33 had no effect on the expression of genes encoding extracellular matrix components or on proliferation, markers typical of fibrosis. The major effects of IL-33 detected in these studies included inhibition of cell migration and activation of cytokine/chemokine expression. The previously reported inhibition of cardiac fibrosis may include more complicated mechanisms that involve other cardiac cell types. Future studies aimed at determin...

Malignant gliomas are the most common primary brain tumors and are associated with aggressive growth, high morbidity, and mortality. Aberrant mesenchymal-epithelial transition factor (MET) activation occurs in approximately 30% of glioma patients and correlates with poor prognosis, elevated invasion, and increased drug resistance. Therefore, MET has emerged as an attractive target for glioma therapy. In this study, we developed a novel nanoinhibitor by conjugating MET-targeting cMBP peptides on the G4 dendrimer. Compared to the binding affinity of the free peptide ( K = 3.96 × 10 M), the binding affinity of the nanoinhibitor to MET increased 3 orders of magnitude to 1.32 × 10 M. This nanoinhibitor efficiently reduced the proliferation and invasion of human glioblastoma U87MG cells in vitro by blocking MET signaling with remarkably attenuated levels of phosphorylated MET ( pMET) and its downstream signaling proteins, such as pAKT and pERK1/2. Although no obvious therapeutic effect was observed after treatment with free cBMP peptide, in vivo T2-weighted magnetic resonance imaging (MRI) showed a significant delay in tumor growth after intravenous injection of the nanoinhibitor. The medium survival in mouse models was extended by 59%, which is similar to the effects of PF-04217903, a small molecule MET inhibitor currently in clinical trials. Immunoblotting studies of tumor homogenate verified that the nanoinhibitor restrained glioma growth by blocking MET downstream signaling. pMET and its downstream proteins pAKT and pERK1/2, which are involved in the survival and invasion of cancer cells, decreased in the nanoinhibitor-treated group by 44.2%, 62.2%, and 32.3%, respectively, compared with those in the control group. In summary, we developed a peptide-functionalized MET nanoinhibitor that showed extremely high binding affinity to MET and effectively inhibited glioma growth by blocking MET downstream signaling. To the best of our knowledge, this is the first report of therapeutic inhibition of glioma growth by blocking MET signaling with a novel nanoinhibitor. Compared to antibodies and chemical inhibitors in clinical trials, the nanoinhibitor blocks MET signaling and provides a new approach for the treatment of glioma with the advantages of high efficiency, affordability, and, most importantly, potentially reduced drug resistance.

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Increases in cardiovascular load (pressure overload) are known to elicit ventricular remodeling including cardiomyocyte hypertrophy and interstitial fibrosis. While numerous studies have focused on the mechanisms of myocyte hypertrophy, comparatively little is known regarding the response of the interstitial fibroblasts to increased cardiovascular load. Fibroblasts are the most numerous cell type in the mammalian myocardium and have long been recognized as producing the majority of the myocardial extracellular matrix. It is only now becoming appreciated that other aspects of fibroblast behavior are important to overall cardiac function. The present studies were performed to examine the temporal alterations in fibroblast activity in response to increased cardiovascular load. Rat myocardial fibroblasts were isolated at specific time-points (3, 7, 14, and 28 days) after induction of pressure overload by abdominal aortic constriction. Bioassays were performed to measure specific parameters of fibroblast function including remodeling and contraction of 3-dimensional collagen gels, migration, and proliferation. In addition, the expression of extracellular matrix receptors of the integrin family was examined. Myocardial hypertrophy and fibrosis were evident within 7 days after constriction of the abdominal aorta. Collagen gel contraction, migration, and proliferation were enhanced in fibroblasts from pressure-overloaded animals compared to fibroblasts from sham animals. Differences in fibroblast function and protein expression were evident within 7 days of aortic constriction, concurrent with the onset of hypertrophy and fibrosis of the intact myocardium. These data provide further support for the idea that rapid and dynamic changes in fibroblast phenotype accompany and contribute to the progression of cardiovascular disease.