IFNγ-IL12 axis regulates intercellular crosstalk in metabolic dysfunction-associated steatotic liver disease.

Obesity is a major cause of metabolic dysfunction-associated steatohepatitis (MASH) and is characterized by inflammation and insulin resistance. Interferon-γ (IFNγ) is a pro-inflammatory cytokine elevated in obesity and modulating macrophage functions. Here, we show that male mice with loss of IFNγ signaling in myeloid cells (Lyz-IFNγR2-/-) are protected from diet-induced insulin resistance despite fatty liver. Obesity-mediated liver inflammation is also attenuated with reduced interleukin (IL)-12, a cytokine primarily released by macrophages, and IL-12 treatment in vivo causes insulin resistance by impairing hepatic insulin signaling. Following MASH diets, Lyz-IFNγR2-/- mice are rescued from developing liver fibrosis, which is associated with reduced fibroblast growth factor (FGF) 21 levels. These results indicate critical roles for IFNγ signaling in macrophages and their release of IL-12 in modulating obesity-mediated insulin resistance and fatty liver progression to MASH. In this work, we identify the IFNγ-IL12 axis in regulating intercellular crosstalk in the liver and as potential therapeutic targets to treat MASH.

Polygala tenuifolia willd. Extract alleviates LPS-induced acute lung injury in rats via TLR4/NF-κB pathway and NLRP3 inflammasome suppression.

BACKGROUND: Acute lung injury (ALI) has received considerable attention in the field of critical care as it can lead to high mortality rates. Polygala tenuifolia, a traditional Chinese medicine with strong expectorant properties, can be used to treat pneumonia. Owing to the complexity of its composition, the main active ingredient is not yet known. Thus, there is a need to identify its constituent compounds and mechanism of action in the treatment of ALI using advanced technological means. PURPOSE: We investigated the anti-inflammatory mechanism and constituent compounds with regard to the effect of P. tenuifolia Willd. extract (EPT) in lipopolysaccharide (LPS)-induced ALI in vivo and in vitro. METHODS: The UHPLC-Q-Exactive Orbitrap MS technology was used to investigate the chemical profile of EPT. Network pharmacology was used to predict the targets and pathways of action of EPT in ALI, and molecular docking was used to validate the binding of polygalacic acid to Toll-like receptor (TLR) 4. The main compounds were determined using LC-MS. A rat model of LPS-induced ALI was established, and THP-1 cells were stimulated with LPS and adenosine triphosphate (ATP) to construct an in vitro model. Pathological changes were observed using hematoxylin and eosin staining, Wright-Giemsa staining, and immunohistochemistry. The expression of inflammatory factors (NE, MPO, Ly-6 G, TNF-α, IL-1ß, IL-6, and iNOS) was determined using enzyme-linked immunosorbent assay, real-time fluorescence quantitative polymerase chain reaction, and western blotting. The LPS + ATP-induced inflammation model in THP-1 cells was used to verify the in vivo experimental results. RESULTS: Ninety-nine compounds were identified or tentatively deduced from EPT. Using network pharmacology, we found that TLR4/NF-κB may be a relevant pathway for the prevention and treatment of ALI by EPT. Polygalacic acid in EPT may be a potential active ingredient. EPT could alleviate LPS-induced histopathological lung damage and reduce the wet/dry lung weight ratio in the rat model of ALI. Moreover, EPT decreased the white blood cell and neutrophil counts in the bronchoalveolar lavage fluid and decreased the expression of genes and proteins of relevant inflammatory factors (NE, MPO, Ly-6 G, TNF-α, IL-1ß, IL-6, and iNOS) in lung tissues. It also increased the expression of endothelial-type nitric oxide synthase expression. Western blotting confirmed that EPT may affect TLR4/NF-κB and NLRP3 signaling pathways in vivo. Similar results were obtained in THP-1 cells. CONCLUSION: EPT reduced the release of inflammatory factors by affecting TLR4/NF-κB and NLRP3 signaling pathways, thereby attenuating the inflammatory response of ALI. Polygalacic acid is the likely compounds responsible for these effects.

Aspirin eugenol ester affects ileal barrier function, inflammatory response and microbiota in broilers under lipopolysaccharide-induced immune stress conditions.

Aims: The aim of this study was to investigate the effects of aspirin eugenol ester (AEE) on ileal immune function in broilers under lipopolysaccharide (LPS)-induced immune stress. Methods: Two hundred and forty one-day-old male Arbor Acres chicks were randomly divided into four groups (saline, LPS, saline + AEE and LPS + AEE) with six replicates of ten broilers each. The saline group and LPS group were fed the normal diet, while the other two groups received normal diet plus 0.1 g/kg AEE. Broilers in the LPS and LPS + AEE groups were injected intraperitoneally with 0.5 mg/kg B.W LPS in saline for seven consecutive days beginning at 14 days of age, while broilers in the saline and saline + AEE groups were injected with saline only. Results: The results showed that AEE improved the ileal morphology and increased the ratio of villus height to crypt depth of immune-stressed broilers. LPS-induced immune stress significantly reduced the expression of the genes for the tight junction proteins occludin, zonula occludens-1 (ZO-1), claudin-1 and claudin-2, in the ileum, while AEE significantly up-regulated the expression of these genes. Compared with the saline group, the LPS-treated chickens showed significantly increased mRNA expression of the inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-10 (IL-10), cyclooxygenase-2 (COX-2), and microsomal Prostaglandin E Synthesase-1 (mPGES-1) in the ileum, while they were significantly decreased by AEE supplementation. In addition, analysis of the ileal bacterial composition showed that compared with saline and LPS + AEE groups, the proportion of Firmicutes and Lactobacillus in the LPS group was lower, while the proportion of Proteobacteria and Escherichia-Shigella was higher. Similarly, Line Discriminant Analysis Effect Size (LEfSe) analysis showed that compared with the LPS group, Brevibacillus was dominant in the saline group, while the LPS + AEE group was rich in Rhizobium, Lachnoclostridium, Ruminococcaceae, Faecalibacterium, Negativibacillus, Oscillospiraceae, and Flavonifractor. Conclusion: These results indicate that dietary supplementation with 0.1 g/kg AEE could protect the intestinal health by improving the intestinal villus morphology, enhancing the expression of tight junction genes and alleviating inflammation to resist the immune stress caused by LPS stimulation in broilers, and the mechanism may involve COX-2-related signal transduction and improved intestinal microbiota composition.

Preparation and Performance Evaluation of Castor Oil-Based Asphalt Regeneration Agent.

Regeneration agents play a critical role in modifying the mechanical properties and durability of RAP asphalt mixtures. This paper aimed to develop a castor oil-based asphalt regeneration agent. The effects of this regeneration agent on the pavement performance of laboratory-aged asphalt and an RAP asphalt mixture were comparatively studied by a series of laboratory tests. For the developed castor oil-based asphalt regeneration agent, the weight ratio of the castor oil to dibutyl phthalate was determined as 1:4. Moreover, the regeneration effectiveness of the castor oil-based regeneration agent was tested on three laboratory-aged asphalt binders and an RAP asphalt binder; the penetration, softening point and ductility of the RAP asphalt binder recovered to 83 dmm, 50.3 °C, and more than 100 cm, respectively. The optimum content of the regeneration agent was 5% by the weight of the aged asphalt binder. Furthermore, the castor oil-based regeneration agent could effectively restore the pavement performance of an RAP asphalt mixture. In this study, the RAP percentage can reach up to 60% by the weight of the HMA mixture using the castor oil-based asphalt regeneration agent according to the Chinese specification.

VGLL2 and TEAD1 fusion proteins drive YAP/TAZ-independent transcription and tumorigenesis by engaging p300.

Studies on Hippo pathway regulation of tumorigenesis largely center on YAP and TAZ, the transcriptional co-regulators of TEAD. Here, we present an oncogenic mechanism involving VGLL and TEAD fusions that is Hippo pathway-related but YAP/TAZ-independent. We characterize two recurrent fusions, VGLL2-NCOA2 and TEAD1-NCOA2, recently identified in spindle cell rhabdomyosarcoma. We demonstrate that, in contrast to VGLL2 and TEAD1, the fusion proteins are strong activators of TEAD-dependent transcription, and their function does not require YAP/TAZ. Furthermore, we identify that VGLL2 and TEAD1 fusions engage specific epigenetic regulation by recruiting histone acetyltransferase p300 to control TEAD-mediated transcriptional and epigenetic landscapes. We showed that small molecule p300 inhibition can suppress fusion proteins-induced oncogenic transformation both in vitro and in vivo. Overall, our study reveals a molecular basis for VGLL involvement in cancer and provides a framework for targeting tumors carrying VGLL, TEAD, or NCOA translocations.

Effect of dietary aspirin eugenol ester on the growth performance, antioxidant capacity, intestinal inflammation, and cecal microbiota of broilers under high stocking density.

This study was designed to examine the impact of aspirin eugenol ester (AEE) on the growth performance, serum antioxidant capacity, jejunal barrier function, and cecal microbiota of broilers raised under stressful high density (HD) stocking conditions compared with normal density broilers (ND). A total of 432 one-day-old AA+ male broilers were randomly divided into 4 groups: normal density (ND, 14 broilers /m2), high density (HD, 22 broilers /m2), ND + AEE, and HD + AEE. The results of the study revealed a significant decrease in the growth performance of broiler chickens as a result of HD stress (P < 0.05). The total antioxidant capacity (T-AOC) in serum demonstrated a significant decrease (P < 0.05) at both 28 and 35 d. Conversely, the serum level of malondialdehyde (MDA) exhibited a significant increase (P < 0.05). Dietary supplementation of AEE resulted in a significant elevation (P < 0.05) of serum GSH-PX, SOD and T-AOC activity at both 28 and 35 d. Moreover, exposure to HD stress resulted in a considerable reduction in the height of intestinal villi and mRNA expression of tight junction proteins in the jejunum, along with, a significant elevation in the mRNA expression of inflammatory cytokines (P < 0.05). However, the administration of AEE reversed the adverse effects of HD-induced stress on villus height and suppressed the mRNA expression of the pro-inflammatory genes, COX-2 and mPGES-1. Additionally, the exposure to HD stress resulted in a substantial reduction in the α-diversity of cecal microbiota and disruption in the equilibrium of intestinal microbial composition, with a notable decrease in the relative abundance of Bacteroides and Faecalibacterium (P < 0.05). In contrast, the addition of AEE to the feed resulted in a notable increase in the relative abundance of Phascolarctobacterium and enhanced microbial diversity (P < 0.05). The inclusion of AEE in the diet has been demonstrated to enhance intestinal integrity and growth performance of broilers by effectively mitigating disruptions in gut microbiota induced by HD stress.

Effects of Aspirin Eugenol Ester on Liver Oxidative Damage and Energy Metabolism in Immune-Stressed Broilers.

The aim of this study was to investigate the effects of aspirin eugenol ester (AEE) on liver oxidative damage and energy metabolism in immune-stressed broilers. In total, 312 broilers were divided into 4 groups (saline, LPS, SAEE, and LAEE). Broilers in the saline and LPS groups were fed a basal diet; the SAEE and LAEE groups had an added 0.01% AEE in their diet. Broilers in the LPS and LAEE groups were injected with lipopolysaccharides, while the saline and SAEE groups were injected with saline. Results showed that AEE increased the body weight, average daily gain, and average daily feed intake, as well as decreasing the feed conversion ratio of immune-stressed broilers. AEE protects against oxidative damage in immune-stressed broiler livers by elevating the total antioxidant capacity, superoxide dismutase activity, and glutathione S-transferase alpha 3 (GSTA3) and glutaredoxin 2 (GLRX2) expression, while decreasing malondialdehyde content. AEE lessened inflammation by reducing prostaglandin-F2α production and prostaglandin-endoperoxide synthase 2 (PTGS2) and interleukin-1beta (IL-1ß) expression. AEE decreased oxidative phosphorylation rates by increasing succinic acid levels and lowering both adenosine diphosphate (ADP) levels and ceroid lipofuscinosis neuronal 5 (CLN5) expression. AEE modulated the metabolism of phenylalanine, tyrosine, lipids, and cholesterol by reducing the phenyllactate and L-arogenate levels, lowering dopachrome tautomerase (DCT) and apolipoprotein A4 (APOA4) expression, and increasing phenylpyruvic acid and dopa decarboxylase (DDC) expression. In summary, AEE can effectively alleviate liver oxidative damage and energy metabolism disorders in immune-stressed broilers.

Decoupling subgenomes within hybrid lavandin provide new insights into speciation and monoterpenoid diversification of Lavandula.

Polyploidization and transposon elements contribute to shape plant genome diversity and secondary metabolic variation in some edible crops. However, the specific contribution of these variations to the chemo-diversity of Lamiaceae, particularly in economic shrubs, is still poorly documented. The rich essential oils (EOs) of Lavandula plants are distinguished by monoterpenoids among the main EO-producing species, L. angustifolia (LA), L. × intermedia (LX) and L. latifolia (LL). Herein, the first allele-aware chromosome-level genome was assembled using a lavandin cultivar 'Super' and its hybrid origin was verified by two complete subgenomes (LX-LA and LX-LL). Genome-wide phylogenetics confirmed that LL, like LA, underwent two lineage-specific WGDs after the γ triplication event, and their speciation occurred after the last WGD. Chloroplast phylogenetic analysis indicated LA was the maternal source of 'Super', which produced premium EO (higher linalyl/lavandulyl acetate and lower 1,8-cineole and camphor) close to LA. Gene expression, especially the monoterpenoid biosynthetic genes, showed bias to LX-LA alleles. Asymmetric transposon insertions in two decoupling 'Super' subgenomes were responsible for speciation and monoterpenoid divergence of the progenitors. Both hybrid and parental evolutionary analysis revealed that LTR (long terminal repeat) retrotransposon associated with AAT gene loss cause no linalyl/lavandulyl acetate production in LL, and multi-BDH copies retained by tandem duplication and DNA transposon resulted in higher camphor accumulation of LL. Advances in allelic variations of monoterpenoids have the potential to revolutionize future lavandin breeding and EO production.

MicroRNA-592 Inhibits the Growth of Ovarian Cancer Cells by Targeting ERBB3.

Objectives: Ovarian cancer is the most lethal gynecologic malignancy, and targeted therapy for different pathological types and molecular phenotypes is urgent to be studied. Studies have shown that MicroRNA-592 (miR-592) plays an important negative regulatory role in the occurrence of gastrointestinal malignancies, breast cancer, non-small cell lung cancer, and glioma, but the expression of miR-592 in ovarian cancer and the mechanism of action are still unclear. Methods: The expressions of miR-592 were examined by RT-PCR and Western Blot. Cell viability and migratory capacity were detected by CCK-8 and transwell assay. TargetScan (http://www.targetscan.org) was analyzed to predict potential targets of miR-592. Then Dual-luciferase reporter gene assay was performed to verify the targeting relationship between miR-592 and ERBB3. A mouse xenograft model was applied to confirm the effect of miR-592. Results: In our study, we found that the expression of miR-592 is reduced in epithelial ovarian cancer tissues. The exogenous expression of miR-592 inhibits the proliferation, migration, and invasion in epithelial ovarian cancer tumor cells. Furthermore, the exogenous expression of miR-592 inhibits tumor growth in the nude mouse xenograft model. Therefore, miR-592 may play a role of tumor suppressor miRNA in the occurrence and development of ovarian cancer. Further experiments demonstrated that tumor-related ERBB3 is a target gene mediated by miRNA-592. The dual-luciferase reporter system was used to identify miRNA-592 target genes; qPCR and Western Blot were used to detect the expression of ERBB3. Mechanical experiments confirmed that miRNA-592 negatively regulated ERBB3.Conclusion: Together, these findings identify a heretofore unrecognized link between miR-592 and ERBB3 and suggest that targeting on miR-592 warrants attention as a novel and potential therapeutic strategy for ovarian cancer.

Chromosome-level assembly of the Neolamarckia cadamba genome provides insights into the evolution of cadambine biosynthesis.

Neolamarckia cadamba (Roxb.), a close relative of Coffea canephora and Ophiorrhiza pumila, is an important traditional medicine in Southeast Asia. Three major glycosidic monoterpenoid indole alkaloids (MIAs), cadambine and its derivatives 3ß-isodihydrocadambine and 3ß-dihydrocadambine, accumulate in the bark and leaves, and exhibit antimalarial, antiproliferative, antioxidant, anticancer and anti-inflammatory activities. Here, we report a chromosome-scale N. cadamba genome, with 744.5 Mb assembled into 22 pseudochromosomes with contig N50 and scaffold N50 of 824.14 Kb and 29.20 Mb, respectively. Comparative genomic analysis of N. cadamba with Co. canephora revealed that N. cadamba underwent a relatively recent whole-genome duplication (WGD) event after diverging from Co. canephora, which contributed to the evolution of the MIA biosynthetic pathway. We determined the key intermediates of the cadambine biosynthetic pathway and further showed that NcSTR1 catalyzed the synthesis of strictosidine in N. cadamba. A new component, epoxystrictosidine (C27H34N2O10, m/z 547.2285), was identified in the cadambine biosynthetic pathway. Combining genome-wide association study (GWAS), population analysis, multi-omics analysis and metabolic gene cluster prediction, this study will shed light on the evolution of MIA biosynthetic pathway genes. This N. cadamba reference sequence will accelerate the understanding of the evolutionary history of specific metabolic pathways and facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding in plants.

Hepatocyte-Specific Deficiency of BAP31 Amplified Acetaminophen-Induced Hepatotoxicity via Attenuating Nrf2 Signaling Activation in Mice.

Liver-specific deficiency of B-cell receptor-associated protein 31 knockout mice (BAP31-LKO) and the littermates were injected with acetaminophen (APAP), markers of liver injury, and the potential molecular mechanisms were determined. In response to APAP overdose, serum aspartate aminotransferase and alanine aminotransferase levels were increased in BAP31-LKO mice than in wild-type controls, accompanied by enhanced liver necrosis. APAP-induced apoptosis and mortality were increased. Hepatic glutathione was decreased (1.60 ± 0.31 µmol/g tissue in WT mice vs. 0.85 ± 0.14 µmol/g tissue in BAP31-LKO mice at 6 h, p < 0.05), along with reduced glutathione reductase activity and superoxide dismutase; while malondialdehyde was significantly induced (0.41 ± 0.03 nmol/mg tissue in WT mice vs. 0.50 ± 0.05 nmol/mg tissue in BAP31-LKO mice for 6 h, p < 0.05). JNK signaling activation and APAP-induced hepatic inflammation were increased in BAP31-LKO mice. The mechanism research revealed that BAP31-deficiency decreased Nrf2 mRNA stability (half-life of Nrf2 mRNA decreased from ~1.3 h to ~40 min) and miR-223 expression, led to reduced nuclear factor erythroid 2-related factor 2 (Nrf2) signaling activation and antioxidant genes induction. BAP31-deficiency decreased mitochondrial membrane potentials, reduced mitochondria-related genes expression, and resulted in mitochondrial dysfunction in the liver. Conclusions: BAP31-deficiency reduced the antioxidant response and Nrf2 signaling activation via reducing Nrf2 mRNA stabilization, enhanced JNK signaling activation, hepatic inflammation, and apoptosis, amplified APAP-induced hepatotoxicity in mice.

Wnt/ß-catenin signaling pathway participates in the effect of miR-626 on oral squamous cell carcinoma by targeting RASSF4.

BACKGROUND: The role of miR-626 in oral squamous cell carcinoma (OSCC) was investigated by targeting RASSF4. METHODS: The miR-626 and RASSF4 expression was detected in normal oral mucosa or OSCC tissues and OSCC or normal cells. The methylation status of RASSF4 was analyzed using methylation-specific polymerase chain reaction (PCR). The cytoplasmic/nuclear ratios (C/N ratios) targeted by miR-626 were examined using microarray, followed by a dual-luciferase reporter assay. The subcellular localization of RASSF4 and miR-626 in OSCC cells was determined using RNA fluorescence in situ hybridization (FISH) and immunocytochemistry (ICC), respectively. Ca9-22 and HSC2 cells were divided into mock, inhibitor NC, miR-626 inhibitor, scramble, RASSF4 and miR-626 mimic + RASSF4 groups, and then CCK-8, Annexin V-FITC/PI, wound healing, Transwell, qRT-PCR and western blotting assays were performed. RESULTS: OSCC tissues and cells had increased miR-626 expression and decreased RASSF4 expression. Patients with RASSF4 methylation had lower RASSF4 expression than those without methylation. In addition, a negative correlation between miR-626 and RASSF4 was found in OSCC tissues, both of which were correlated with the pathological grade, pathological stage, lymph node metastasis and patient prognosis. MiR-626 targeted RASSF4 in OSCC cells. Overexpressed RASSF4 inhibited the proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) of OSCC cells, promoted cell apoptosis, and blocked the Wnt/ß-Catenin pathway, which was reversed by miR-626 overexpression. CONCLUSIONS: Inhibiting miR-626 can regulate the biological characteristics of OSCC cells, including proliferation, invasion, migration, EMT and apoptosis, by targeting RASSF4, which may be related to the Wnt/ß-Catenin pathway.

Clinical outcomes and safety of apatinib monotherapy in the treatment of patients with advanced epithelial ovarian carcinoma who progressed after standard regimens and the analysis of the VEGFR2 polymorphism.

The aims of the present study were to investigate the clinical outcomes and safety of apatinib monotherapy in the treatment of patients with advanced epithelial ovarian carcinoma (EOC) who have progressed after standard regimens, and to analyze the vascular endothelial growth factor receptor 2 (VEGFR2) rs2071559 polymorphism. A total of 118 patients with advanced EOC who received apatinib treatment were included in the study. Tumor response was evaluated using progression-free survival (PFS) and overall survival (OS) time, and safety data were documented. Additionally, peripheral blood and peripheral blood mononuclear cell (PBMC) specimens from the patients with EOC were collected to perform the genotyping of genetic polymorphism and assess the mRNA expression of VEGFR2, respectively. The objective response rate across the 118 patients with advanced EOC was 38.98%, the disease control rate was 63.56%, the median PFS time was 4.65 months and the median OS time was 15.10 months. Regarding the polymorphism analysis, the prevalence of rs2071559 in VEGFR2 among the 118 patients with advanced EOC was recorded as the TT genotype in 72 cases (61.02%), TC genotype in 41 cases (34.75%) and CC genotype in 5 cases (4.23%), and the minor allele frequency of rs2071559 was 0.22. The distribution of the three genotypes was in accordance with the Hardy-Weinberg equilibrium (P=0.781). TC and CC genotypes were merged in the subsequent analysis. The prognosis analyses suggested that the median PFS time of patients with the TC/CC genotype and the TT genotype was 3.10 and 5.40 months, respectively (P=0.015). Moreover, the median OS time of the two genotypes was 12.60 and 17.50 months, respectively (P=0.009). However, no association was noted between genotype status of the polymorphism and adverse reactions. Additionally, the mRNA expression analysis indicated that the mRNA expression levels of VEGFR2 in PBMC specimens were significantly different between TT and TC/CC genotypes (P<0.001). The present study suggested that the clinical outcomes of patients with advanced EOC, who progressed after standard regimens and received apatinib treatment, might be influenced by the VEGFR2 rs2071559 polymorphism.

Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.

Obesity-mediated inflammation is a major cause of insulin resistance, and macrophages play an important role in this process. The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum chaperone that modulates unfolded protein response (UPR), and mice with GRP78 heterozygosity were resistant to diet-induced obesity. Here, we show that mice with macrophage-selective ablation of GRP78 (Lyz- GRP78-/-) are protected from skeletal muscle insulin resistance without changes in obesity compared with wild-type mice after 9 wk of high-fat diet. GRP78-deficient macrophages demonstrated adapted UPR with up-regulation of activating transcription factor (ATF)-4 and M2-polarization markers. Diet-induced adipose tissue inflammation was reduced, and bone marrow-derived macrophages from Lyz- GRP78-/- mice demonstrated a selective increase in IL-6 expression. Serum IL-13 levels were elevated by >4-fold in Lyz- GRP78-/- mice, and IL-6 stimulated the myocyte expression of IL-13 and IL-13 receptor. Lastly, recombinant IL-13 acutely increased glucose metabolism in Lyz- GRP78-/- mice. Taken together, our data indicate that GRP78 deficiency activates UPR by increasing ATF-4, and promotes M2-polarization of macrophages with a selective increase in IL-6 secretion. Macrophage-derived IL-6 stimulates the myocyte expression of IL-13 and regulates muscle glucose metabolism in a paracrine manner. Thus, our findings identify a novel crosstalk between macrophages and skeletal muscle in the modulation of obesity-mediated insulin resistance.-Kim, J. H., Lee, E., Friedline, R. H., Suk, S., Jung, D. Y., Dagdeviren, S., Hu, X., Inashima, K., Noh, H. L., Kwon, J. Y., Nambu, A., Huh, J. R., Han, M. S., Davis, R. J., Lee, A. S., Lee, K. W., Kim, J. K. Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.

Multimerization and interaction of Toll and Spätzle in Drosophila.

The Toll family of receptors is required for innate immune response to pathogen-associated molecules, but the mechanism of signaling is not entirely clear. In Drosophila the prototypic Toll regulates both embryonic development and adult immune response. We demonstrate here that the host protein Spätzle can function as a ligand for Toll because Spätzle forms a complex with Toll in transgenic fly extracts and stimulates the expression of a Toll-dependent immunity gene, drosomycin, in adult flies. We also show that constitutively active mutants of Toll form multimers that contain intermolecular disulfide linkages. These disulfide linkages are critical for the activity of one of these mutant receptors, indicating that multimerization is essential for the constitutive activity. Furthermore, systematic mutational analysis revealed that a conserved cysteine-containing motif, different from the cysteines used for the intermolecular disulfide linkages, serves as a self-inhibitory module of Toll. Deleting or mutating this cysteine-containing motif leads to constitutive activity. This motif is located just outside the transmembrane domain and may provide a structural hindrance for multimerization and activation of Toll. Together, our results suggest that multimerization may be a regulated, essential step for Toll-receptor activation.

The Drosophila Toll-9 activates a constitutive antimicrobial defense.

The Toll family of transmembrane proteins participates in signaling infection during the innate immune response. We analyzed the nine Drosophila Toll proteins and found that wild-type Toll-9 behaves similar to gain-of-function Toll-1. Toll-9 activates strongly the expression of drosomycin, and utilizes similar signaling components to Toll-1 in activating the antifungal gene. The predicted protein sequence of Toll-9 contains a tyrosine residue in place of a conserved cysteine, and this residue switch is critical for the high activity of Toll-9. The Toll-9 gene is expressed in adult and larval stages prior to microbial challenge, and the expression correlates with the high constitutive level of drosomycin mRNA in the animals. The results suggest that Toll-9 is a constitutively active protein, and implies its novel function in protecting the host by maintaining a substantial level of antimicrobial gene products to ward off the continuous challenge of microorganisms.