Synthetic molecules derived from natural sugars with a positively charged amino group or ammonium salt and two lipophilic chains have been shown to inhibit TLR4 activation in vitro and in vivo. To ...characterize the mechanism of action of this class of molecules, we investigated possible interactions with the extracellular components that bind and shuttle endotoxin lipopolysaccharide (LPS) to TLR4, namely, LBP, CD14, and MD-2. Molecules that inhibited TLR4 activation inhibited LBP·CD14-dependent transfer of endotoxin monomers derived from aggregates of tritiated lipooligosaccharide (3HLOS) from Neisseria meninigitidis to MD-2·TLR4, resulting in a reduced level of formation of a (3HLOS·MD-2·TLR4ECD)2 (M r ∼ 190000) complex. This effect was due to inhibition of the transfer of 3HLOS from aggregates in solution to sCD14 with little or no effect on 3HLOS shuttling from 3HLOS·sCD14 to MD-2. These compounds also inhibited transfer of the 3HLOS monomer from full-length CD14 to a truncated, polyhistidine-tagged CD14. Dose-dependent inhibition of the transfer of 3HLOS between the two forms of CD14 was observed with each of three different synthetic compounds that inhibited TLR4 activation but not by another structurally related analogue that lacked TLR4 antagonistic activity. Saturation transfer difference (STD) NMR data showed direct binding to CD14 by the synthetic TLR4 antagonist mediated principally through the lipid chains of the synthetic compound. Taken together, our findings strongly suggest that these compounds inhibit TLR4 activation by endotoxin by competitively occupying CD14 and thereby reducing the level of delivery of activating endotoxin to MD-2·TLR4.
Vibrio harveyi is an important pathogen for aquatic fish and invertebrates, that can lead to significant economic losses in the aquaculture industry. Rapid and accurate serotyping is essential for ...the diagnosis and epidemiological investigation of bacterial pathogens. In Vibrio spp., serotyping is based on lipopolysaccharide or capsule polysaccharide loci. In this study, we screened and extracted the location and full-length sequences of 12 different lipopolysaccharide biosynthesis loci from all available V. harveyi genome sequences from public database. Herein their gene composition, homology and variation are discussed, and a database is developed to rapidly type V. harveyi isolates based on differences in lipopolysaccharide loci. This study further analyzses the relationship between lipopolysaccharide loci and genome characteristics. The identification and characterization of these lipopolysaccharide loci could help researchers understand the genetic, molecular, and basis serotype structural specificity of this pathogen, and also provide opportunities for further development and improvement of the molecular serotyping method, which would aid the diagnosis and clinical treatment of the disease caused by this pathogen.
•Identified the location and full length sequence of 12 different lipopolysaccharide biosynthesis loci for Vibrio harveyi.•Compared the gene composition, homologous and variation between these O-antigen encoding loci.•Developed a database which could be used to rapidly typing V. harveyi isolates based on the difference of lipopolysaccharide loci.•Analyzed the relationship between lipopolysaccharide synthesis loci and the genome characteristics.
Depression triggered by harmful stress during adolescence is a common problem that can affect mental health. To date, the mechanisms underlying this type of depression remain unclear. One mechanism ...for the promotion of depression by chronic stress in adulthood is the loss of hippocampal microglia. Since deleterious stress in adolescence also activates microglia, we investigated the dynamic changes of microglia in the hippocampus in mice exposed to chronic unpredictable stress (CUS) in adolescence. Our results showed that 12 days of CUS stimulation in adolescence induced typical depression-like behaviors in adult mice, which were accompanied by a significant decrease and dystrophy of microglia in the dentate gyrus of the hippocampus. Further analysis showed that this decrease in microglia was mediated by the initial response of microglia to unpredictable stress in the dentate gyrus of the hippocampus and their subsequent apoptosis. Blocking the initial response of microglia to unpredictable stress by pretreatment with minocycline was able to prevent apoptosis and microglial decline as well as the development of depression-like behaviors in adult mice induced by adolescent CUS. Moreover, administration of lipopolysaccharide (LPS) or macrophage-colony stimulatory factor (M-CSF), two drugs that reversed microglia decline in the dentate gyrus, ameliorated the depression-like behaviors induced by CUS stimulation in adolescence. These findings reveal a novel mechanism for the development of depression-like behaviors in animals triggered by deleterious stress in adolescence and suggest that reversing microglial decline in the hippocampus may be a hopeful strategy for the treatment of depression triggered by deleterious stress in adolescence.
•Detrimental stress exposure in adolescence induces depressive-like behaviors in mice.•Adolescent stress induces microglia loss and dystrophy in the dentate gyrus.•Adolescent stress-triggered microglial activation induces microglial apoptosis.•Microglial apoptosis mediates adolescent stress-induced dentate gyrus microglia loss.•Microglial activation reverses adolescent stress-induced depressive-like behavior.
Perceived chondroprotective benefits of extra-label bisphosphonates, such as clodronate disodium (CD), in juvenile horses led to widespread use despite little scientific understanding of ...intra-articular impacts. The objective was to determine the effects of CD on articular cartilage hypothesizing that administration of CD would decrease cartilage degradation following an acute inflammatory challenge by lipopolysaccharide (LPS). To test this, 32 yearling Quarter Horses were stratified by age (500 ± 13 d), BW (336 ± 26 kg), sex (n = 16 female; n = 16 male) and initial bone optical density into 4 treatment groups. The 140-d study consisted of 2 phases: phase 1 (d 0–83) emulated sales prep and phase 2 (d 84–140) mimicked early performance training. Horses were housed individually (3.6 m × 7.3 m) and fed to meet exercise requirements. Treatments consisted of control (CON; n = 8), single-dose (1X; n = 8; d 84), 2-doses (2X; n = 8; d 0, 84), and 4-doses (4X; n = 8; d 0, 42, 84, 126). All horses received iso-volumetric intramuscular injections of 1.8mg/kg BW clodronate disodium (OSPHOS®) or saline (placebo) on d 0, 42, 84, and 126. Post treatment administration on d 126, radial carpal joints of each horse were injected with 0.8 mL of either 0.5 ng sterile LPS derived from Escherichia coli O55:B5 or sterile lactated Ringer's solution as a contralateral control. Synovial fluid was collected before LPS injection(h 0) and at 6, 12, 24, and 336 h post induction. Carpal circumference (CC), heart rate (HR), respiration rate (RR), and rectal temperature (RT) were recorded before each collection. Synovial fluid was analyzed for carboxypropeptide of type II collagen (CPII) and collagenase cleavage neopeptide (C2C) by ELISA. Data were analyzed using PROC MIXED of SAS. Treatment had no effect on HR, RR, or RT. As expected, intra-articular LPS increased CC, CPII, C2C, and CPII:C2C (P ≤ 0.01). There was a trend for a treatment effect for CC (P = 0.09) where CC was greater in 2X and 4X compared with 1X with CON being intermediate. There tended to be a treatment by hour interaction in CPII (P = 0.06) where CPII was greater in 4X and 2X compared with 1X and CON at h 24 and 336. Likewise,there was a trend for a treatment effect onCPII:C2C (P = 0.06) with 2X and 4X having greater synthesis:degradation than 1X and CON. In conclusion, LPS administration resulted in an expected increase in carpal circumference. Trends of increasing CPII and CPII:C2C in the 2X and 4X treatment group indicate a net increase in articular cartilage synthesis following 2–4 doses of CD despite larger CC, suggesting a greater inflammatory response.
The present study aimed to investigate the overall changes in exosomal proteomes in metastatic and non‐metastatic non‐small‐cell lung cancers (NSCLC) and healthy human serum samples, and evaluate the ...potential of serum exosomal biomarkers to predict NSCLC metastasis. Tandem mass tags combined with multidimensional liquid chromatography and mass spectrometry analysis were used for screening the proteomic profiles of serum samples. Quantitative proteome, significant pathway, and functional categories of patients with metastatic and non‐metastatic NSCLC and healthy donors were investigated. In total, 552 proteins of the 628 protein groups identified were quantified. Bioinformatics analysis indicated that quantifiable proteins were mainly involved in multiple biological functions, metastasis‐related pathways. Moreover, lipopolysaccharide‐binding proteins (LBP) in the exosomes were found to be well distinguished between patients with metastatic and patients with non‐metastatic NSCLC. Area under the curve (AUC) was 0.803 with a sensitivity of 83.1% and a specificity of 67% (P < .0001). Circulating LBP were also well distinguishable between metastatic and non‐metastatic NSCLC, the AUC was 0.683 with a sensitivity of 79.5% and a specificity of 47.2% (P = .005). This novel study provided a reference proteome map for metastatic NSCLC. Patients with metastatic and non‐metastatic NSCLC differed in exosome‐related proteins in the serum. LBP might be promising and effective candidates of metastatic NSCLC.
This is the first study using the proteomics technique to find a diagnostic marker for metastatic NSCLC. It provides an objective basis for the early diagnosis, early treatment and prognosis of metastatic NSCLC, and provides a key point for the diagnosis of other cancerous solid tumors. It also provides a new idea for non‐invasive biomarkers for other metastatic cancers, and the clinical application of exosomes will have better prospects.
The role of vitamin D in innate immunity is increasingly recognized. Recent work has identified a number of tissues that express the enzyme 1alpha-hydroxylase and are able to activate vitamin D. This ...locally produced vitamin D is believed to have important immunomodulatory effects. In this paper, we show that primary lung epithelial cells express high baseline levels of activating 1alpha-hydroxylase and low levels of inactivating 24-hydroxylase. The result of this enzyme expression is that airway epithelial cells constitutively convert inactive 25-dihydroxyvitamin D(3) to the active 1,25-dihydroxyvitamin D(3). Active vitamin D that is generated by lung epithelium leads to increased expression of vitamin D-regulated genes with important innate immune functions. These include the cathelicidin antimicrobial peptide gene and the TLR coreceptor CD14. dsRNA increases the expression of 1alpha-hydroxylase, augments the production of active vitamin D, and synergizes with vitamin D to increase expression of cathelicidin. In contrast to induction of the antimicrobial peptide, vitamin D attenuates dsRNA-induced expression of the NF-kappaB-driven gene IL-8. We conclude that primary epithelial cells generate active vitamin D, which then influences the expression of vitamin D-driven genes that play a major role in host defense. Furthermore, the presence of vitamin D alters induction of antimicrobial peptides and inflammatory cytokines in response to viruses. These observations suggest a novel mechanism by which local conversion of inactive to active vitamin D alters immune function in the lung.
Toll-like receptor 4 (TLR4) activates two distinct signaling pathways inducing production of proinflammatory cytokines or type I interferons (IFNs), respectively. MyD88 and TIRAP/Mal are essential ...adaptor molecules for the former but not for the latter pathway. In contrast, TRIF/TICAM-1 and TRAM/TICAM-2 are essential for both. TIRAP is a sorting adaptor molecule recruiting MyD88 to activated TLR4 in the plasma membrane. TRAM is thought to bridge between TLR4 and TRIF by physical association. Little is known, however, how TRAM interacts with TLR4 or with TRIF during LPS response. Here, we show that TRAM recruits TRIF to the plasma membrane. Moreover, LPS induces upregulation of TLR4-association with TRAM and their subsequent translocation into endosome/lysosome. The internalized signaling complex consisting of TLR4 and TRAM colocalizes with TRAF3, a signaling molecule downstream of TRIF, in endosome/lysosome. These results suggest that TLR4 activates TRIF-signaling in endosome/lysosome after relocation from the cell surface.
Due to the high toxicity of bacterial lipopolysaccharide (LPS), resulting in sepsis and septic shock, two major causes of death worldwide, significant effort is directed toward the development of ...specific trace-level LPS detection systems. Here, we report sensitive, user-friendly, high-throughput LPS detection in a 96-well microplate using a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells that are transformed by a red fluorescent protein (mCherry) gene under the transcriptional control of an NF-κB response element. The recognition of LPS activates the biosensor cell, TLR4, and the co-receptor-induced NF-κB signaling pathway, which results in the expression of mCherry fluorescent protein. The novel cell-based biosensor detects LPS with specificity at low concentration. The cell-based biosensor was evaluated by testing LPS isolated from 14 bacteria. Of the tested bacteria, 13 isolated Enterobacteraceous LPSs with hexa-acylated structures were found to increase red fluorescence and one penta-acylated LPS from Pseudomonadaceae appeared less potent. The proposed biosensor has potential for use in the LPS detection in foodstuff and biological products, as well as bacteria identification, assisting the control of foodborne diseases.
Synthesis of the pentasaccharide repeating unit of the cell O-polysaccharide produced by Salmonella milwaukee O:43 strain (group U) has been achieved in very good yield adopting a convergent ...stereoselective 3 + 2 block glycosylation strategy. Thioglycosides and glycosyl trichloroacetimidate derivative were used as glycosyl donors in the presence of a combination of N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) as thiophilic activator and TMSOTf as trichloroacetimidate activator respectively. The stereochemical outcome of all glycosylation reactions was excellent.
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•Pentasaccharide of the cell wall of Salmonella milwaukee O:43 strain has been synthesized.•A convergent stereoselective 3 + 2 block glycosylation strategy has been used.•Thioglycosides and glycosyl trichloroacetimidate derivative were used as glycosyl donors.•A combination of NIS and TMSOTf has been used as thiophilic activator.•The stereochemical outcome of all glycosylation reactions was excellent.
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