Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner

Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1α (IRE1α)/X-box binding protein-1. PA-induced EVs were enriched in C16:0 ceramide in an IRE1α-dependent manner, and activated macrophage chemotaxis via formation of sphingosine-1-phosphate (S1P) from C16:0 ceramide. This chemotaxis was blocked by sphingosine kinase inhibitors and S1P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C16:0 ceramide. PA induces C16:0 ceramide-enriched EV release in an IRE1α-dependent manner. The ceramide metabolite, S1P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions.     

Contrasting genetic metrics and patterns among naturalized rainbow trout (Oncorhynchus mykiss) in two Patagonian lakes differentially impacted by trout aquaculture

Different pathways of propagation and dispersal of non‐native species into new environments may have contrasting demographic and genetic impacts on established populations. Repeated introductions of rainbow trout (Oncorhynchus mykiss) to Chile in South America, initially through stocking and later through aquaculture escapes, provide a unique setting to contrast these two pathways. Using a panel of single nucleotide polymorphisms, we found contrasting genetic metrics and patterns among naturalized trout in Lake Llanquihue, Chile's largest producer of salmonid smolts for nearly 50 years, and Lake Todos Los Santos (TLS), a reference lake where aquaculture has been prohibited by law. Trout from Lake Llanquihue showed higher genetic diversity, weaker genetic structure, and larger estimates for the effective number of breeders (N_b) than trout from Lake TLS. Trout from Lake TLS were divergent from Lake Llanquihue and showed marked genetic structure and a significant isolation‐by‐distance pattern consistent with secondary contact between documented and undocumented stocking events in opposite shores of the lake. Multiple factors, including differences in propagule pressure, origin of donor populations, lake geomorphology, habitat quality or quantity, and life history, may help explain contrasting genetic metrics and patterns for trout between lakes. We contend that high propagule pressure from aquaculture may not only increase genetic diversity and N_b via demographic effects and admixture, but also may impact the evolution of genetic structure and increase gene flow, consistent with findings from artificially propagated salmonid populations in their native and naturalized ranges.     

Epidemiological study of the relationship between sleep disturbances and somatic and psychological complaints among the Japanese general population

Sleep and Biological Rhythms - There have been only a few studies on the relationship between sleep disturbances and somatic and psychological complaints in Japanese people. In this study, we...     

Intramolecular Binding of the Rad9 C Terminus in the Checkpoint Clamp Rad9-Hus1-Rad1 Is Closely Linked with Its DNA Binding

The human checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded onto chromatin by its loader complex, Rad17-RFC, following DNA damage. The 120-amino acid (aa) stretch of the Rad9 C terminus (C-tail) is unstructured and projects from the core ring structure (CRS). Recent studies showed that 9-1-1 and CRS bind DNA independently of Rad17-RFC. The DNA-binding affinity of mutant 9^ΔC-1-1, which lacked the Rad9 C-tail, was much higher than that of wild-type 9-1-1, suggesting that 9-1-1 has intrinsic DNA binding activity that manifests in the absence of the C-tail. C-tail added in trans interacted with CRS and prevented it from binding to DNA. We narrowed down the amino acid sequence in the C-tail necessary for CRS binding to a 15-aa stretch harboring two conserved consecutive phenylalanine residues. We prepared 9-1-1 mutants containing the variant C-tail deficient for CRS binding, and we demonstrated that the mutant form restored DNA binding as efficiently as 9^ΔC-1-1. Furthermore, we mapped the sequence necessary for TopBP1 binding within the same 15-aa stretch, demonstrating that TopBP1 and CRS share the same binding region in the C-tail. Indeed, we observed their competitive binding to the C-tail with purified proteins. The importance of interaction between 9-1-1 and TopBP1 for DNA damage signaling suggests that the competitive interactions of TopBP1 and CRS with the C-tail will be crucial for the activation mechanism.     

Coupling effects of distal loops on structural stability and enzymatic activity of Escherichia coli dihydrofolate reductase revealed by deletion mutants

Residues distal from the active site in dihydrofolate reductase (DHFR) have regulatory roles in catalytic reaction and also folding stability. The couplings of the distal residues to the ones in the active site have been analyzed using site-directed mutants. To expand our understanding of the structural and functional influences of distal residue mutation, we explored the structural stability and enzymatic activity of deletion mutants. Deletion has greater structural and dynamical impacts on the corresponding part than site-directed mutation does. Thus, deletion amplifies the effects caused by distal mutations, which should make the mutual couplings among the distant residues more apparent. We focused on residues 52, 67, 121, and 145 in the four distinct loops of DHFR. All the single-residue deletion mutants showed marked reduction in stability, except for Δ52 in an αC–βC loop. Double deletion mutants showed that the loop αC–βC has nonadditive couplings with the βF–βG and βG–βH loops regarding stability. Single deletion to the loops αC–βC or βC–βD resulted in considerable activity reduction, demonstrating that the loops couple to the residues near the active site. The four loops were shown to be functionally interdependent from the double deletion experiments.     

Light activates H2 15O flow in rice: Detailed monitoring using a positron-emitting tracer imaging system (PETIS)

Water (H₂ ¹⁵O) translocation from the roots to the top of rice plants ( Oryza saliva L. cv. Nipponbare) was visualized over time by a positron-emitting tracer imaging system (PETIS). H₂ ¹⁵O flow was activated 8 min after plants were exposed to bright light (1 500 μmol m⁻² s⁻¹). When the light was subsequently removed, the flow gradually slowed and completely stopped after 12 min. In plants exposed to low light (500 μmol m⁻² s⁻¹), H₂ ¹⁵O flow was activated more slowly, and a higher translocation rate of H₂ ¹⁵O was observed in the same low light at the end of the next dark period. NaCl (80 m M ) and methylmercury (1 m M ) directly suppressed absorption of H₂ ¹⁵O by the roots, while methionine sulfoximine (1 m M ), abscisic acid (10 μ M ) and carbonyl cyanide m -chlorophenylhydrazone (10 m M ) were transported to the leaves and enhanced stomatal closure, reducing H₂ ¹⁵O translocation.     

Differences in Ca2+ mobilization induced by alpha-adrenergic agonist and phosphatidic acid in cultured hepatocytes

In an attempt to elucidate the relationship between phosphatidylinositol breakdown and alpha-adrenergic responses, effects of phosphatidic acid and phosphatidylinositol related metabolites on Ca²⁺ mobilization and glucose output in cultured hepatocytes were examined. Norepinephrine induced the net ⁴⁵Ca²⁺ efflux from preloaded cells and stimulated glucose output via alpha-adrenergic receptor stimulation, whereas phosphatidic acid caused ⁴⁵Ca²⁺ uptake to cells and did not stimulate glucose output. Myo-inositol-monophosphate, diglyceride and arachidonic acid, which are released by phosphatidylinositol breakdown, had no effect on ⁴⁵Ca²⁺ efflux and glucose output in cells. These results suggest that phosphatidic acid and phosphatidylinositol related metabolites can not mimic the alpha-adrenergic actions in cultured hepatocytes.