Toll or Interleukin-1 Receptor (TIR) Domain-containing Adaptor Inducing Interferon-β (TRIF)-mediated Caspase-11 Protease Production Integrates Toll-like Receptor 4 (TLR4) Protein- and Nlrp3 Inflammasome-mediated Host Defense against Enteropathogens

Enteric pathogens represent a major cause of morbidity and mortality worldwide. Toll-like receptor (TLR) and inflammasome signaling are critical for host responses against these pathogens, but how these pathways are integrated remains unclear. Here, we show that TLR4 and the TLR adaptor TRIF are required for inflammasome activation in macrophages infected with the enteric pathogens Escherichia coli and Citrobacter rodentium. In contrast, TLR4 and TRIF were dispensable for Salmonella typhimurium-induced caspase-1 activation. TRIF regulated expression of caspase-11, a caspase-1-related protease that is critical for E. coli- and C. rodentium-induced inflammasome activation, but dispensable for inflammasome activation by S. typhimurium. Thus, TLR4- and TRIF-induced caspase-11 synthesis is critical for noncanonical Nlrp3 inflammasome activation in macrophages infected with enteric pathogens.     

Functional analysis of an unusual type IV pilus in the Gram‐positive Streptococcus sanguinis

Type IV pili (Tfp), which have been studied extensively in a few Gram‐negative species, are the paradigm of a group of widespread and functionally versatile nano‐machines. Here, we performed the most detailed molecular characterisation of Tfp in a Gram‐positive bacterium. We demonstrate that the naturally competent Streptococcus sanguinis produces retractable Tfp, which like their Gram‐negative counterparts can generate hundreds of piconewton of tensile force and promote intense surface‐associated motility. Tfp power ‘train‐like’ directional motion parallel to the long axis of chains of cells, leading to spreading zones around bacteria grown on plates. However, S. sanguinis Tfp are not involved in DNA uptake, which is mediated by a related but distinct nano‐machine, and are unusual because they are composed of two pilins in comparable amounts, rather than one as normally seen. Whole genome sequencing identified a locus encoding all the genes involved in Tfp biology in S. sanguinis. A systematic mutational analysis revealed that Tfp biogenesis in S. sanguinis relies on a more basic machinery (only 10 components) than in Gram‐negative species and that a small subset of four proteins dispensable for pilus biogenesis are essential for motility. Intriguingly, one of the piliated mutants that does not exhibit spreading retains microscopic motility but moves sideways, which suggests that the corresponding protein controls motion directionality. Besides establishing S. sanguinis as a useful new model for studying Tfp biology, these findings have important implications for our understanding of these widespread filamentous nano‐machines.     

Ultrathin Bilayer of Graphite/SiO2 as Solid Interface for Reviving Li Metal Anode

Lithium metal anodes are expected to drive practical applications that require high energy‐density storage. However, the direct use of metallic lithium causes safety concerns, low rate capabilities, and poor cycling performance due to unstable solid electrolyte interphase (SEI) and undesired lithium dendrite growth. To address these issues, a radio frequency sputtered graphite‐SiO₂ ultrathin bilayer on a Li metal chips is demonstrated, for the first time, as an effective SEI layer. This leads to a dendrite free uniform Li deposition to achieve a stable voltage profile and outstanding long hours plating/stripping compared to the bare Li. Compared to a bare Li anode, the graphite‐SiO₂ bilayer modified Li anode coupled with lithium nickel cobalt manganese oxide cathode (NMC111) and lithium titanate shows improved capacity retention, higher capacity at higher rates, longer cycling stability, and lower voltage hysteresis. Graphite acts as an electrical bridge between the plated Li and Li electrode, which lowers the impedance and buffers the volume expansion during Li plating/stripping. Adding an ultrathin SiO₂ layer facilitates Li‐ion diffusion and lithiation/delithiation, provides higher electrolyte affinity, higher chemical stability, and higher Young's modulus to suppress the Li dendrite growth.     

Scn3b knockout mice exhibit abnormal ventricular electrophysiological properties

We report for the first time abnormalities in cardiac ventricular electrophysiology in a genetically modified murine model lacking the Scn3b gene (Scn3b^−/−). Scn3b^−/− mice were created by homologous recombination in embryonic stem (ES) cells. RT-PCR analysis confirmed that Scn3b mRNA was expressed in the ventricles of wild-type (WT) hearts but was absent in the Scn3b^−/− hearts. These hearts also showed increased expression levels of Scn1b mRNA in both ventricles and Scn5a mRNA in the right ventricles compared to findings in WT hearts. Scn1b and Scn5a mRNA was expressed at higher levels in the left than in the right ventricles of both Scn3b^−/− and WT hearts. Bipolar electrogram and monophasic action potential recordings from the ventricles of Langendorff-perfused Scn3b^−/− hearts demonstrated significantly shorter ventricular effective refractory periods (VERPs), larger ratios of electrogram duration obtained at the shortest and longest S₁–S₂ intervals, and ventricular tachycardias (VTs) induced by programmed electrical stimulation. Such arrhythmogenesis took the form of either monomorphic or polymorphic VT. Despite shorter action potential durations (APDs) in both the endocardium and epicardium, Scn3b^−/− hearts showed ΔAPD₉₀ values that remained similar to those shown in WT hearts. The whole-cell patch-clamp technique applied to ventricular myocytes isolated from Scn3b^−/− hearts demonstrated reduced peak Na⁺ current densities and inactivation curves that were shifted in the negative direction, relative to those shown in WT myocytes. Together, these findings associate the lack of the Scn3b gene with arrhythmic tendencies in intact perfused hearts and electrophysiological features similar to those in Scn5a^+/− hearts.     

Chemical synthesis of scyllo-inosamine and catabolism studies in Sinorhizobium meliloti

Rhizopines such as scyllo-inosamine (SIA) and L-3-O-methyl-scyllo-inosamine (3-O-MSI) play an intricate role as nutritional mediators during the establishment of the symbiotic relationship between legumes and rhizobia. The mechanism of action is not well understood. One challenge is the availability of rhizopines, which occur in only minute amounts in plant nodules. We herewith report an efficient synthesis of scyllo-inosamine and its biochemical activity in specific bacteria. SIA was prepared in 7 steps and 32% overall yield from readily available myo-inositol. The chemically synthesized SIA was tested to determine whether it can serve as sole carbon and nitrogen source for Sinorhizobium meliloti wild-type strain L5-30 and for strains carrying mutations in the rhizopine degradation (moc) genes. The analysis of the phenotype of the mutant strains revealed that the moc genes previously shown to be essential for the breakdown of the rhizopines isolated from root nodules are also essential for the utilization of the chemically synthesized SIA.     

Temporal and Vertical Difference in Factors Limiting Growth Rate of Heterotrophic Bacteria in Lake Biwa

Abstract Dilution bioassays were performed to examine the seasonal and vertical difference in the relative importance of factors limiting growth of heterotrophic bacteria in Lake Biwa. The lake water diluted by 0.2 μm lake filtrate (1:6.6) was enriched either with glucose (C), inorganic phosphorus (P), ammonium nitrogen (N), amino acids (AA), or a combination of these, and incubated for 2 days at the depths where lake water was collected (2.5, 20 and 30 m depths). Experiments showed that at 2.5 m, P was the most deficient resource for bacterial growth, but the magnitude of P limitation depended on water temperature. Among others, amino acids showed a slight but significant stimulation of bacterial growth rates during the fall. At 20 and 30 m, however, growth stimulation by resource addition was rarely detected. Vertically reciprocal translocation experiments revealed that the growth rate was limited by low temperature rather than resource supply at the greater depths. The results support a simple view that bacterial growth rate is basically regulated by water temperature, but high growth rate is not realized in summer because of resource depletion. The present study suggests that both temperature and P supply play a crucial role in biogeochemical cycling of organic matter in Lake Biwa through the bacterial growth rate. Received: 10 March 1999; Accepted: 14 May 1999     

Inositol polyphosphate 5-phosphatases: lipid phosphatases with flair

Recent studies have identified the inositol polyphosphate 5-phosphatases as a large family of signal modifying enzymes comprising 10 mammalian and 4 yeast family members. A number of investigations including gene-targeted deletion of 5-phosphatases in mice have demonstrated that these enzymes regulate many important cellular events including hematopoietic cell proliferation and activation, insulin signaling, endocytosis, and actin polymerization.     

The structure and function of catalytic domains within inositol polyphosphate 5-phosphatases

Phosphoinositide signaling pathways regulate many essential cellular functions including proliferation, differentiation and survival, cytoskeletal organization, and vesicular trafficking. The inositol polyphosphate 5-phosphatases regulate the cellular levels of several bioactive phosphoinositide species. This review describes the structure and function of the 5-phosphatase and Sac1 catalytic domains of these enzymes. The crystal structure of the 5-phosphatase domain has been solved and shares homology with members of the AP endonuclease family. The phosphoinositide polyphosphatase activity of the Sac1 domain, found in some inositol polyphosphate 5-phosphatases, is defined by a motif, CX5 R(T/S), also found in both protein and lipid phosphatases.     

Skeletal muscle LIM protein 1 regulates integrin-mediated myoblast adhesion,spreading, and migration

The skeletal muscle LIM protein 1 (SLIM1) is highly expressed in skeletal and cardiac muscle, and itsexpression is downregulated significantly in dilated humancardiomyopathy. However, the function of SLIM1 is unknown. In thisstudy, we investigated the intracellular localization of SLIM1.Endogenous and recombinant SLIM1 localized to the nucleus, stressfibers, and focal adhesions in skeletal myoblasts plated onfibronectin, collagen, or laminin. However, after inhibition ofintegrin signaling either by plating on poly-L-lysine or bysoluble RGD peptide, SLIM1 localized diffusely in the cytosol, withdecreased nuclear expression. Disruption of the actin cytoskeleton bycytochalasin D did not inhibit nuclear localization of SLIM1 inintegrin-activated cells. Green fluorescent protein-tagged SLIM1shuttled in the nucleus of untransfected NIH 3T3 cells, in aheterokaryon fusion assay. Overexpression of SLIM1 in Sol8 myoblastsinhibited cell adhesion and promoted cell spreading and migration.These studies show SLIM1 localizes in an integrin-dependent manner tothe nucleus and focal adhesions where it functions downstream ofintegrin activation to promote cell spreading and migration.

     

Direct voltage control of signaling via P2Y1 and other Galphaq-coupled receptors

Emerging evidence suggests that Ca2+ release evoked by certain G-protein-coupled receptors can be voltage-dependent; however, the relative contribution of different components of the signaling cascade to this response remains unclear. Using the electrically inexcitable megakaryocyte as a model system, we demonstrate that inositol 1,4,5-trisphosphate-dependent Ca2+ mobilization stimulated by several agonists acting via Galphaq-coupled receptors is potentiated by depolarization and that this effect is most pronounced for ADP. Voltage-dependent Ca2+ release was not induced by direct elevation of inositol 1,4,5-trisphosphate, by agents mimicking diacylglycerol actions, or by activation of phospholipase Cgamma-coupled receptors. The response to voltage did not require voltage-gated Ca2+ channels as it persisted in the presence of nifedipine and was only weakly affected by the holding potential. Strong predepolarizations failed to affect the voltage-dependent Ca2+ increase; thus, an alteration of G-protein betagamma subunit binding is also not involved. Megakaryocytes from P2Y1(-/-) mice lacked voltage-dependent Ca2+ release during the application of ADP but retained this response after stimulation of other Galphaq-coupled receptors. Although depolarization enhanced Ca2+ mobilization resulting from GTPgammaS dialysis and to a lesser extent during AlF4- or thimerosal, these effects all required the presence of P2Y1 receptors. Taken together, the voltage dependence to Ca2+ release via Galphaq-coupled receptors is not due to control of G-proteins or down-stream signals but, rather, can be explained by a voltage sensitivity at the level of the receptor itself. This effect, which is particularly robust for P2Y1 receptors, has wide-spread implications for cell signaling.