XFEL and NMR Structures of Francisella Lipoprotein Reveal Conformational Space of Drug Target against Tularemia.

Francisella tularensis is the causative agent for the potentially fatal disease tularemia. The lipoprotein Flpp3 has been identified as a virulence determinant of tularemia with no sequence homology outside the Francisella genus. We report a room temperature structure of Flpp3 determined by serial femtosecond crystallography that exists in a significantly different conformation than previously described by the NMR-determined structure. Furthermore, we investigated the conformational space and energy barriers between these two structures by molecular dynamics umbrella sampling and identified three low-energy intermediate states, transitions between which readily occur at room temperature. We have also begun to investigate organic compounds in silico that may act as inhibitors to Flpp3. This work paves the road to developing targeted therapeutics against tularemia and aides in our understanding of the disease mechanisms of tularemia.

Effects of dietary plant meal and soya-saponin supplementation on intestinal and hepatic lipid droplet accumulation and lipoprotein and sterol metabolism in Atlantic salmon (Salmo salar L.).

Altered lipid metabolism has been shown in fish fed plant protein sources. The present study aimed to gain further insights into how intestinal and hepatic lipid absorption and metabolism are modulated by plant meal (PM) and soya-saponin (SA) inclusion in salmon feed. Post-smolt Atlantic salmon were fed for 10 weeks one of four diets based on fishmeal or PM, with or without 10 g/kg SA. PM inclusion resulted in decreased growth performance, excessive lipid droplet accumulation in the pyloric caeca and liver, and reduced plasma cholesterol levels. Intestinal and hepatic gene expression profiling revealed an up-regulation of the expression of genes involved in lipid absorption and lipoprotein (LP) synthesis (apo, fatty acid transporters, microsomal TAG transfer protein, acyl-CoA cholesterol acyltransferase, choline kinase and choline-phosphate cytidylyltransferase A), cholesterol synthesis (3-hydroxy-3-methylglutaryl-CoA reductase) and associated transcription factors (sterol regulatory element-binding protein 2 and PPARγ). SA inclusion resulted in reduced body pools of cholesterol and bile salts. The hepatic gene expression of the rate-limiting enzyme in bile acid biosynthesis (cytochrome P450 7A1 (cyp7a1)) as well as the transcription factor liver X receptor and the bile acid transporter abcb11 (ATP-binding cassette B11) was down-regulated by SA inclusion. A significant interaction was observed between PM inclusion and SA inclusion for plasma cholesterol levels. In conclusion, gene expression profiling suggested that the capacity for LP assembly and cholesterol synthesis was up-regulated by PM exposure, probably as a compensatory mechanism for excessive lipid droplet accumulation and reduced plasma cholesterol levels. SA inclusion had hypocholesterolaemic effects on Atlantic salmon, accompanied by decreased bile salt metabolism.

Soluble forms of Toll-like receptor (TLR)2 capable of modulating TLR2 signaling are present in human plasma and breast milk.

Dysregulation of the initial, innate immune response to bacterial infection may lead to septic shock and death. Toll-like receptors (TLRs) play a crucial role in this innate immune response, and yet the regulatory mechanisms controlling microbial-induced TLR triggering are still to be fully understood. We have therefore sought specific regulatory mechanisms that may modulate TLR signaling. In this study, we tested for the possible existence of a functionally active soluble form of TLR2. We demonstrated the existence of natural soluble forms of TLR2 (sTLR2), which we show to be capable of modulating cell activation. We found that blood monocytes released sTLR2 constitutively and that the kinetics of sTLR2 release increased upon cell activation. Analysis of cells expressing the human TLR2 cDNA or its c-myc-tagged version indicated that sTLR2 resulted from the posttranslational modification of the TLR2 protein in an intracellular compartment. Moreover, an intracellular pool of sTLR2 is maintained. sTLR2 was found naturally expressed in breast milk and plasma. Milk sTLR2 levels mirrored those of the TLR coreceptor soluble CD14. Depletion of sTLR2 from serum resulted in an increased cellular response to bacterial lipopeptide. Notably, serum sTLR2 was lower in tuberculosis patients. Coimmunoprecipitation experiments and computational molecular docking studies showed an interaction between sTLR2 and soluble CD14 in plasma and milk. These findings suggest the existence of a novel and specific innate immune mechanism regulating microbial-induced TLR triggering, and may lead to new therapeutics for the prevention and/or treatment of severe infectious diseases.

High-throughput fluorescence screening of antioxidative capacity in human serum.

Diphenylhexatriene-labeled phosphatidylcholine and propionic acid have been established as selective fluorescence markers for the continuous determination of oxidation processes in the lipid and aqueous phases of unfractionated human serum. Oxidation of the respective fluorophores leads to a decrease in fluorescence intensity from which the time-dependent degradation of the marker molecule can be determined. The lag times preceding the propagation of oxidation are representative for the antioxidative capacity of the system, which may be influenced by exogenous factors, e.g., the antioxidants from the diet. Supplementation of human serum by quercetin, rutin, vitamin E, vitamin C, or total apple phenolics in vitro led to a decrease in oxidizability depending on the oxidation marker and the hydrophobicity of the antioxidant. Quercetin and vitamin E showed a higher in vitro capacity of protecting lipoproteins against oxidation. In contrast, rutin and vitamin C were more efficient as inhibitors in the aqueous phase. The same effect on serum was found after dietary consumption of apples. This result is in line with the known observation that intake of plant polyphenols leads to an increase in serum levels of hydrophilic antioxidants.

Lp(a): a link between thrombosis and atherosclerosis.

Lipoprotein(a), or Lp(a), is a lipoprotein having lipid composition similar to that of LDL, but a protein moiety consisting of ApoB 100 linked by disulfide bridge to apo(a), a glycoprotein with structural similarity to plasminogen. Lp(a) can be both atherogenic and thrombogenic. These two actions are likely to be mutually operative, a fact that on a molar basis makes Lp(a) more of a cardiovascular pathogen than LDL.