Littérature scientifique sur le sujet « LptC protein »

ABSTRACT Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, including the OM protein complex LptD/LptE (formerly Imp/RlpB), the periplasmic LptA protein, the IM-associated cytoplasmic ATP binding cassette protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the proteins mentioned above leads to common phenotypes, including (i) the presence of abnormal membrane structures in the periplasm, (ii) accumulation of de novo-synthesized LPS in two membrane fractions with lower density than the OM, and (iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of the IM. Our results suggest that LptA, LptB, LptC, LptD, and LptE operate in the LPS assembly pathway and, together with other as-yet-unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space.

ABSTRACTThe assembly of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) requires the transenvelope Lpt (lipopolysaccharide transport) complex, made inEscherichia coliof seven essential proteins located in the inner membrane (IM) (LptBCFG), periplasm (LptA), and OM (LptDE). At the IM, LptBFG constitute an unusual ATP binding cassette (ABC) transporter, composed by the transmembrane LptFG proteins and the cytoplasmic LptB ATPase, which is thought to extract LPS from the IM and to provide the energy for its export across the periplasm to the cell surface. LptC is a small IM bitopic protein that binds to LptBFG and recruits LptA via its N- and C-terminal regions, and its role in LPS export is not completely understood. Here, we show that the expression level oflptBis a critical factor for suppressing lethality of deletions in the C-terminal region of LptC and the functioning of a hybrid Lpt machinery that carriesPa-LptC, the highly divergent LptC orthologue fromPseudomonas aeruginosa. We found that LptB overexpression stabilizes C-terminally truncated LptC mutant proteins, thereby allowing the formation of a sufficient amount of stable IM complexes to support growth. Moreover, the LptB level seems also critical for the assembly of IM complexes carryingPa-LptC which is otherwise defective in interactions with theE. coliLptFG components. Overall, our data suggest that LptB and LptC functionally interact and support a model whereby LptB plays a key role in the assembly of the Lpt machinery.IMPORTANCEThe asymmetric outer membrane (OM) of Gram-negative bacteria contains in its outer leaflet an unusual glycolipid, the lipopolysaccharide (LPS). LPS largely contributes to the peculiar permeability barrier properties of the OM that prevent the entry of many antibiotics, thus making Gram-negative pathogens difficult to treat. InEscherichia colithe LPS transporter (the Lpt machine) is made of seven essential proteins (LptABCDEFG) that form a transenvelope complex. Here, we show that increased expression of the membrane-associated ABC protein LptB can suppress defects of LptC, which participates in the formation of the periplasmic bridge. This reveals functional interactions between these two components and supports a role of LptB in the assembly of the Lpt machine.

Inexpensive liposome-polymer transfection complexes (LPTCs) were developed and used as for DNA or protein delivery. The particle sizes of the LPTCs were in the range of 212.2 to 312.1 nm, and the zetapotential was +38.7 mV. LPTCs condensed DNA and protected DNA from DNase I digestion and efficiently delivered LPTC/DNA complexes in Balb/3T3 cells. LPTCs also enhanced the cellular uptake of antigen in mouse macrophage cells and stimulated TNF-αrelease in naïve mice splenocytes, both indicating the potential of LPTCs as adjuvants for vaccines.In vivostudies were performed usingH. pylorirelative heat shock protein 60 as an antigen model. The vaccination of BALB/c mice with LPTC-complexed DNA and protein enhanced the humoral immune response. Therefore, we developed a DNA and protein delivery system using LPTCs that is inexpensive, and we successfully applied it to the development of a DNA and subunit vaccine.

The need for novel antibiotics has become imperative with the increasing prevalence of antibiotic resistance in Gram-negative bacteria in clinics. Acting as a permeability barrier, lipopolysaccharide (LPS) protects Gram-negative bacteria against drugs. LPS is synthesized in cells and transported to the outer membrane (OM) via seven lipopolysaccharide transport (Lpt) proteins (LptA–LptG). Of these seven Lpt proteins, LptC interacts with LptA to transfer LPS from the inner membrane (IM) to the OM, and assembly is aided by LptD/LptE. This interaction among the Lpt proteins is important for the biosynthesis of LPS; therefore, the Lpt proteins, which are significant in the assembly process of LPS, can be a potential target for new antibiotics. In this study, a yeast two-hybrid (Y2H) system was used to screen compounds that could block LPS transport by inhibiting LptA/LptC interaction, which finally disrupts the biosynthesis of the OM. We selected the compound IMB-0042 for this study. Our results suggest that IMB-0042 disrupts LptA/LptC interaction by binding to both LptA and LptC. Escherichia coli cells, when treated with IMB-0042, showed filament morphology, impaired OM integrity, and an accumulation of LPS in the periplasm. IMB-0042 inhibited the growth of Gram-negative bacteria and showed synergistic sensitization to other antibiotics, with low cytotoxicity. Thus, we successfully identified a potential antibacterial agent by using a Y2H system, which blocks the transport of LPS by targeting LptA/LptC interaction in Escherichia coli.

Gram-negative bacteria have a dense outer membrane (OM) coating of lipopolysaccharides, which is essential to their survival. This coating is assembled by the LPS (lipopolysaccharide) transport (Lpt) system, a coordinated seven-subunit protein complex that spans the cellular envelope. LPS transport is driven by an ATPase-dependent mechanism dubbed the “PEZ” model, whereby a continuous stream of LPS molecules is pushed from subunit to subunit. This review explores recent structural and functional findings that have elucidated the subunit-scale mechanisms of LPS transport, including the novel ABC-like mechanism of the LptB2FG subcomplex and the lateral insertion of LPS into the OM by LptD/E. New questions are also raised about the functional significance of LptA oligomerization and LptC. The tightly regulated interactions between these connected subcomplexes suggest a pathway that can react dynamically to membrane stress and may prove to be a valuable target for new antibiotic therapies for Gram-negative pathogens.

AbstractLipopolysaccharide (LPS) is an essential component of the outer membranes (OM) of most Gram-negative bacteria, which plays a crucial role in protection of the bacteria from toxic compounds and harsh conditions. The LPS is biosynthesized at the cytoplasmic side of inner membrane (IM), and then transported across the aqueous periplasmic compartment and assembled correctly at the outer membrane. This process is accomplished by seven LPS transport proteins (LptA-G), but the transport mechanism remains poorly understood. Here, we present findings by pull down assays in which the periplasmic component LptA interacts with both the IM complex LptBFGC and the OM complex LptDE in vitro, but not with complex LptBFG. Using purified Lpt proteins, we have successfully reconstituted the seven transport proteins as a complex in vitro. In addition, the LptC may play an essential role in regulating the conformation of LptBFG to secure the lipopolysaccharide from the inner membrane. Our results contribute to the understanding of lipopolysaccharide transport mechanism and will provide a platform to study the detailed mechanism of the LPS transport in vitro.

With the increasing resistance of many Gram-negative bacteria to existing classes of antibiotics, identifying new paradigms in antimicrobial discovery is an important research priority. Of special interest are the proteins required for the biogenesis of the asymmetric Gram-negative bacterial outer membrane (OM). Seven Lpt proteins (LptA to LptG) associate in most Gram-negative bacteria to form a macromolecular complex spanning the entire envelope, which transports lipopolysaccharide (LPS) molecules from their site of assembly at the inner membrane to the cell surface, powered by adenosine 5′-triphosphate hydrolysis in the cytoplasm. The periplasmic protein LptA comprises the protein bridge across the periplasm, which connects LptB2FGC at the inner membrane to LptD/E anchored in the OM. We show here that the naturally occurring, insect-derived antimicrobial peptide thanatin targets LptA and LptD in the network of periplasmic protein-protein interactions required to assemble the Lpt complex, leading to the inhibition of LPS transport and OM biogenesis inEscherichia coli.

The rise of antibiotic-resistant infections has been well documented and the need for novel antibiotics cannot be overemphasized. US FDA approved antibiotics target only a small fraction of bacterial cell wall or membrane components, well-validated antimicrobial targets. In this review, we highlight small molecules that inhibit relatively unexplored cell wall and membrane targets. Some of these targets include teichoic acids-related proteins (DltA, LtaS, TarG and TarO), lipid II, Mur family enzymes, components of LPS assembly (MsbA, LptA, LptB and LptD), penicillin-binding protein 2a in methicillin-resistant Staphylococcus aureus, outer membrane protein transport (such as LepB and BamA) and lipoprotein transport components (LspA, LolC, LolD and LolE). Inhibitors of SecA, cell division protein, FtsZ and compounds that kill persister cells via membrane targeting are also covered.

L’obiettivo di questa tesi è elucidare alcuni aspetti dell’interazione tra proteine che legano il lipopolisaccaride (LPS) batterico e il loro ligando naturale o ligandi di sintesi. LptC (Lipopolysaccharide transport C) è una proteina batterica che appartiene al sistema di trasporto Lpt, un sistema di 7 proteine essenziali che trasportano l’LPS sulla membrana esterna dei batteri Gram negativi dopo la sua biosintesi. Sebbene molti elementi della biosintesi dell’LPS siano stati elucidati, il preciso meccanismo di trasporto è ancora poco chiaro. Poiché LptC può essere considerata come proteina modello del sistema Lpt, in quanto presenta lo stesso folding delle altre proteine ed è la prima ad essere localizzata nel periplasma, abbiamo sviluppato ed ottimizzato un saggio di binding in vitro per studiare la sua interazione con l’LPS. Abbiamo ottenuto, per la prima volta, dettagliate informazioni sui parametri termodinamici e cinetici dell’interazione LptC-LPS. Abbiamo infatti dimostrato che in vitro il binding LptC-LPS è irreversibile con una Kd dell’ordine del μM. Considerando le analogie strutturali tra LptC e la proteina eucariotica CD14, appartenente al sistema recettoriale del TLR4, in modo analogo è stata studiata l’interazione di LptC con la molecola sintetica IAXO-102, un noto ligando di CD14. È emerso che IAXO-102 condivide lo stesso sito di legame dell’LPS e che l’interazione con la proteina è irreversibile con un’affinità inferiore a quella LptC-LPS. IAXO-102 può dunque essere considerato un prototipo per lo sviluppo di una nuova generazione di antibiotici che ha come target la biogenesi dell’LPS. L’LPS è in grado di interagire con molte altre proteine, tra le quali quelle del sistema dell’immunità innata (TLR4, CD14, MD-2). Il riconoscimento dell’LPS da parte di questi recettori induce una forte risposta infiammatoria che termina con la produzione di citochine pro-infiammatorie e fattori immunomodulatori. Questa reazione infiammatoria è utile all’organismo, ma quando si manifesta in modo eccessivamente potente e sregolato induce sepsi, processi infiammatori e sindromi autoimmuni per le quali non è ancora disponibile un trattamento farmacologico. Una possibile soluzione al problema consiste nella ricerca e nello sviluppo di composti in grado di modulare questa eccessiva attivazione. Nella seconda parte di questo lavoro, sono riportate le caratterizzazioni biologiche di alcuni composti di sintesi con caratteristiche chimiche differenti. Di tutti i composti è stata valutata la tossicità mediante saggio dell’MTT e l’attività modulatoria del pathway del TLR4 utilizzando cellule HEK stabilmente trasfettate con i geni del TLR4, CD14 ed MD-2. Ulteriori caratterizzazioni sono state effettuate sui composti più promettenti, effettuando saggi in vitro su cellule HEK trasfettate con il complesso umano o murino TLR4•MD-2 e saggi in vivo. Infine, abbiamo investigato la possibile correlazione tra le note proprietà anti-infiammatorie di alcuni composti naturali, come i composti fenolici presenti nell’olio di oliva, e il pathway del TLR4. L’obiettivo di questo lavoro è duplice: individuare un lead compound come possibile modulatore del TLR4, ma anche discriminare quali caratteristiche chimiche siano importanti per ottenere questo effetto. Inoltre, le informazioni ottenute potrebbero essere estremamente utili per guidare il rational design di altri modulatori del TLR4.<br>The purpose of this work is the elucidation of some aspects of the interaction between lipopolysaccharide (LPS) binding proteins and their natural ligand or synthetic compounds. LptC (Lipopolysaccharide transport C) is a bacterial protein belonging to Lpt complex, a molecular machinery composed of 7 essential proteins involved in the transport of LPS to the outer membrane in Gram negative bacteria after its biogenesis. Although many elements of LPS biosynthesis have been clarified, the precise mechanism of transport is still not completely understood. Since LptC can be considered as a model protein of Lpt complex, sharing the same folding of other proteins and being the first one in the periplasm, we have developed and optimized an in vitro binding assay to study its interaction with LPS. We have obtained, for the first time, detailed information about the thermodynamic and kinetic parameters of LptC-LPS binding. We have shown that the in vitro LptC-LPS binding is irreversible with a Kd of the order of μM. Considering the structural similarities between LptC and the eukaryotic protein CD14, belonging to TLR4 receptor system, the binding between LptC and the synthetic molecule iaxo-102, a known ligand of CD14, has been investigated. It is evident that iaxo-102 shares the same binding site of LPS and that the binding is irreversible with an affinity lower than that LptC-LPS. So, iaxo-102 can be considered as a lead compound for the development a new generation of antibiotics targeting the biogenesis of LPS. LPS also binds to other proteins, such as those of innate immunity TLR4, CD14 and MD-2. The LPS recognition by these receptors induces the production of pro-inflammatory cytokines and immunomodulators that trigger the inflammatory and immune responses. These reactions are useful for the organism, but when TLR4 activation is too strong or not well regulated induces sepsis, inflammation and autoimmune syndromes, which still lack a pharmacological treatment. A possible solution to solve this problem consists in the research and development of compounds which modulate this excessive activation. In the second part of thesis work, the biological characterization of some synthetic compounds, with different chemical features, have been reported. All compounds have been screened for their toxicity using MTT assay, and their modulatory activity on TLR4 pathway by using HEK cells stably transfected with TLR4, CD14 and MD-2 genes. The best compounds have been further characterized by in vitro assays on HEK cells transfected with the human or murine complex TLR4·MD-2 and in vivo studies. Finally, the possible correlation between the known anti-inflammatory properties of some natural compounds, such as the phenolic compounds of olive oil, and TLR4 activity has been investigated. The aim of this study is double: to find a lead compound active on TLR4 pathway, but also to discriminate which chemical features are important to obtain this effect. In addition, the information obtained could be very useful to guide the rational design of other TLR4 modulators.

Pseudomonas aeruginosais an opportunistic pathogen that infects cystic fibrosis (CF) patients contributing to their high morbidity and mortality. P. aeruginosaundergoes a phenotypic conversion in the CF lung, from nonmucoid to mucoid, by constitutively producing a polysaccharide called alginate. These mucoid strains often revert to nonmucoid in vitrodue to second-site suppressor mutations. We hypothesized that mapping these mutations would lead to the identification of novel genes involved in alginate production. In a previous study, a mucoid strain, PDO300 (PAOmucA22), was used to isolate suppressors of alginate phenotype (sap). One of the uncharacterized nonmucoid revertants, sap27, is the subject of this study. The mucoid phenotype in sap27was restored by pMO012217 from a minimal tiling path cosmid library. The cosmid pMO012217 harbors 18 P. aeruginosaopen reading frames (ORF). The cosmid was mutagenized with a transposon to map the contributing gene. It was mapped tolptD(PA0595) encoding lipopolysaccharide transport protein. E. coliLptD transports lipopolysaccharide to the outer leaflet of the outer membrane. The Alg+phenotype was restored upon complementation with P. aeruginosa lptDalone, suggesting that sap27likely harbor a chromosomal mutation inlptD. Sequencing analysis of sap27showed the presence of a mutation not in lptDbut in algO, which encodes a periplasmic protease protein. This suggests LptD is able to bypass analgO mutation by positively regulating alginate production. The lptD is a part of a three-gene operon lptD-surA-pdxA. SurA is an essential protein for survival in starvation and a major chaperone protein for all outer membrane proteins and PdxA is a NAD-dependent dehydrogenase and is involved in the vitamin B6biosynthetic pathway. Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6.P. aeruginosagrown in a media supplemented with PLP increased production of pyocyanin, a virulence factor. The PLP and aromatic amino acids are synthesized from a common precursor chorismic acid. We demonstrated an increase in pyocyanin production when the bacteria were cultured supplemented by the aromatic amino acids phenylalanine. We concluded that the lptDoperon plays a role in the P. aeruginosavirulence by regulating alginate and pyocyanin production.

Lo scopo del presente lavoro è la progettazione, la sintesi e la caratterizzazione di nuove small molecules, attive come ligandi di LPS (lipopolisaccaridi)-binding proteins. Gli LPS, o endotossine batteriche, sono macromolecole anfifiliche ubiquitarie sulla membrana esterna dei batteri Gram-negativi. Le proteine che legano gli LPS studiate nel corso di questo progetto di tesi di dottorato appartengono a due categorie: le proteine batteriche di trasporto Lpt e il sistema recettoriale TLR4, che comprende anche i co-recettori LBP, CD14, MD2. Le proteine Lpt, e in particolare la proteina LptC, sono responsabili del meccanismo di esportazione del LPS alla superficie cellulare, che è uno step fondamentale della via biosintetica dell’LPS. Pertanto, la biogenesi dell’LPS rappresenta un target ideale per lo sviluppo di nuovi antibiotici contro i batteri Gram-negativi. Inoltre, le strutture delle proteine Lpt sono state risolte, ma il meccanismo di trasporto è ancora da elucidare. Nel presente lavoro di tesi sono stati utilizzate diverse tecniche per studiare l'interazione tra LPS e LptC, con particolare attenzione agli studi di interazione via NMR. Inoltre, un nuovo LPS fluorescente è stato prodotto ed è stato utilizzato come tool per studi di interazione LPS-LptC con tecniche di fluorescenza. Sono state anche sviluppate alcune nuove molecole sintetiche. Questi glicolipidi sono stati progettati e sintetizzati per ottenere ligandi di LptC e, in prospettiva, potenziali antibiotici contro i batteri Gram-negativi. Il Toll-like receptor 4 (TLR4), il recettore dell'immunità innata, riconosce l’LPS aiutato da altre proteine (LBP, CD14 e MD-2) ed è responsabile dell'induzione della risposta infiammatoria. Molecole sintetiche in grado di modulare l'attività dei recettori dell’immunità innata sono un potente mezzo per studiare il sistema recettoriale TLR4 e hanno grande interesse farmacologico come adiuvanti vaccinali (agonisti), agenti antisepsi e anti-infiammatori (antagonisti). L’attività biologica di glicolipidi con una funzione amminica (IAXO-102) come antagonisti del TLR4 è stata chiaramente dimostrata dal nostro gruppo di ricerca. La sintesi di molecole derivate da IAXO-102, che mantengano l'attività biologica del precursore, è stato un obiettivo di questo lavoro. In particolare, sono state portate a termine le sintesi di sonde fluorescenti, utilizzate per studi di interazione, derivati zwitterionici e molecole dimeriche. Nei nostri laboratori sono stati ottenuti anche antagonisti anionici del TLR4 con una struttura chimica più simile a Lipide A. Lo scopo di questo lavoro è stato valutare, tramite esperimenti NMR, la loro capacità di legare co-recettore dell'immunità innata MD-2. Il carattere anfifilico degli analoghi sintetici del lipide A sintetizzati finora è spesso associato ad una bassa solubilità in acqua e a scarsa biodisponibilità. Invece, i composti attivi sul TLR4 di origine naturale hanno una migliore solubilità e biodisponibilità. La modifica chimica di queste strutture è molto utile per modulare l'attività biologica e per migliorare la specificità nei confronti del target. Di conseguenza, in una fase successiva di questo lavoro di tesi, è stata intrapresa la sintesi di nuove molecole con strutture chimiche ispirate ai modulatori naturali del TLR4. Recentemente è stato dimostrato che alcuni composti fenolici estratti da olio di oliva hanno una buona attività come antagonisti del TLR4. Pertanto, la sintesi di alcuni analoghi di queste molecole è stata eseguita per ottenere nuovi potenziali antagonisti del TLR4, con una migliore solubilità in acqua e una ridotta tossicità.<br>The purpose of this work is the design, synthesis and characterization of new small molecules, active as ligands of two different lipopolysaccharide (LPS)-binding proteins. LPS, or bacterial endotoxin, is an amphiphilic macromolecule ubiquitous on the outer membrane of Gram-negative bacteria. The LPS binding proteins studied during this thesis project belong to two classes: the bacterial proteins of the Lpt transport machinery and the mammalian TLR4 receptor system, including the co-receptors LBP, CD14, MD-2. Lpt proteins, and in particular the protein LptC, are responsible for the export mechanism of LPS to the cell surface of Gram negative bacteria, which is a fundamental step of the LPS biosynthetic pathway. Therefore, the LPS biogenesis represents an ideal target for development of novel antibiotics against Gram-negative bacteria. Moreover, the structures of Lpt proteins have been elucidated, but very little is known about the mechanism of LPS transport. In this thesis work different techniques were used to study the interaction between LPS and LptC, particularly NMR binding studies. Moreover, a new fluorescent LPS was produced and it was used as a tool to perform LPS-LptC interaction studies with fluorescence techniques. Some new synthetic molecules were also developed during this thesis. Glycolipidic small molecules were designed and synthesized in order to obtain LptC ligands and, in perspective, potential antibiotics against Gram-negative bacteria. Toll-like receptor 4 (TLR4), the innate immunity receptor, recognizes LPS, helped by other proteins (LBP, CD14 and MD-2), and it is responsible for the induction of inflammatory responses. Synthetic small molecules able to modulate innate immunity receptors activity are a powerful mean to study the TLR4 receptor system and have great pharmacological interest as vaccine adjuvants (agonists), antisepsis and anti-inflammatory agents (antagonists). Antagonist activity on TLR4 receptor system of amino glycolipids (IAXO-102) was clearly demonstrated by our research group. The synthesis of molecules derived from IAXO-102 which retain the biological activity of the precursor was a target of this work. In particular, the synthesis of fluorescent probes, used for binding studies, zwitterionic derivatives and dimeric molecules were performed. Anionic TLR4 antagonists with a chemical structure more similar to Lipid A were also obtained in our labs. The aim of this work was the evaluation via NMR binding experiments of their ability to bind the innate immunity co-receptor MD-2. The amphiphilic character of the synthetic lipid A analogues synthesized so far is often associated with low water solubility and poor bioavailability. In this respect, the natural TLR4-active compounds have better solubility and bioavailability. The chemical modification of these structures is very helpful to modulate their biological activity and to enhance target specificity. Consequently, in a later stage of this work, the synthesis of new small molecules with chemical structures inspired to natural TLR4 modulators was pursued. Very recently it was found that some phenolic compounds from olive oil extracts presented a good activity as TLR4 antagonists. The synthesis of some analogues of these molecules was performed to obtain new potential TLR4 antagonists with better water solubility and reduced toxicity.

碩士<br>國立臺灣大學<br>解剖學暨細胞生物學研究所<br>102<br>Previous studies have shown that Galanin modulated peripheral pain sensation via galanin receptor type 2 (GalR2). Following nerve injury, inflammation, spontaneous discharge and upregulation of pain related factors would involve in neuropathic pain development. To our knowledge, the correlation between median nerve demyelination and GalR2 and its substrate expression levels has not been documented; and yet the effect of GalR2 on medain neuropathic pain is not valid. Thus, using LPC treated median nerve injury model, we investigate the role of GalR2 and its pain corelated factors in the upper limb neuropathic pain. One week after LPC treatment of median nerve induced mechnical allodynia and thermal hyperalgesia. Immunohistochemistry analysis showed that GalR2-like immunoreactive (-LI) neurons were predominately in small-size DRG neurons of normal rats. However, one week after LPC treatment, GalR2-LI neurons not only increased in its percentage but also distributed in medium- and large-sized neurons. Moreover, to characterize GalR2-LI neurons in the DRG was using immunofluorescence double labeling for NF200, peripherin, pain-related factors including vanilloid receptor subtype 1 (VR1), P2X3, NPY, nNOS, Galanin, or MMP9. We found that the number and percentage of GalR2-LI neurons colocalized with NF200, P2X3, NPY, nNOS, Galanin and MMP9 were increased in the LPC-treated DRG. Furthermore, lidocaine pretreatment attenuated the number of upregulated GalR2-LI neurons in the LPC-treated DRG. Our study also found that one week afterLPC treatment, the number of GalR2-LI neurons in the cuneate nucleus of LPC treated rats was higer than that in the control group. The present results suggest that lidocaine pretreatment relieved the development of neuropathic pain partially pass through reducing GalR2 expression.

Food protein interaction with other biopolymers, such as tannic acids, within a food matrix can alter their structure and functionality. In this study, the nature of lentil protein-tannic acid (LPTA) interaction and its effect on in vitro peptic hydrolysis were investigated. In LPTA mixtures containing 1% w/v LP and 0.001% - 0.5% TA, a twenty-fold increase in particle size was observed in LPTA 0.5% compared to LPI, indicating aggregation. Static quenching of tryptophan residues within the protein hydrophobic folds were observed. Increasing TA content caused an overall increase in α-helix fractions. A 56.79% reduction in free amino nitrogen of LPTA 0.5%, relative to LPI, was observed after digestion. High molecular weight hydrolysates from LPTA 0.5% recorded slightly different amino acid profile. This study showed that 0.5% w/v TA induced protein aggregation, reduced lentil protein digestibility by hindering accessibility of pepsin to protein network, and modified amino acid profile.

The objective of this work presented in this paper is to study the performance of low pressure turbines in detail by extensive numerical simulations. The numerical flow simulations were conducted using the general purpose code ANSYS CFX. Particular attention is focused on the loss development in axial direction within the flow passage of the cascade. It is shown that modern CFD tools are able to break down the integral loss of the turbine profile into its components depending on attached and separated flow areas. In addition the numerical results allow to show the composition of the loss depending on the Reynolds number. The method of the analysis of axial loss development presented here allows for a much more comprehensive investigation and evaluation of the quality of the numerical results. For this reason the paper also demonstrates the capability of this method to quantify the influence of the axial velocity density ratio, the inflow turbulence level, the inflow angle and the Reynolds number on the loss configuration and the flow angle of the cascade as well as a comparison of steady state and transient results. The validation data of this LPT-Cascade have been obtained at the High Speed Cascade Wind Tunnel of the Institute of Jet Propulsion. For this purpose experiments were conducted within the range of Re2th = 40’000 to 400’000. To gather data at realistic engine operation conditions, the wind tunnel allows for an independent variation of Reynolds and Mach number. The experimental results presented herein contain detailed pressure measurements as well as measurements with 3-D-hot-wire anemometry. However, this paper shows only integral values of the experimental as well as the numerical results to protect the proprietary nature of the LPT-design.