Phosphoglycerol-type wall and lipoteichoic acids are enantiomeric polymers differentiated by the stereospecific glycerophosphodiesterase GlpQ.

The cell envelope of Gram-positive bacteria generally comprises two types of polyanionic polymers linked to either peptidoglycan (wall teichoic acids; WTA) or to membrane glycolipids (lipoteichoic acids; LTA). In some bacteria, including Bacillus subtilis strain 168, both WTA and LTA are glycerolphosphate polymers yet are synthesized through different pathways and have distinct but incompletely understood morphogenetic functions during cell elongation and division. We show here that the exolytic sn-glycerol-3-phosphodiesterase GlpQ can discriminate between B. subtilis WTA and LTA. GlpQ completely degraded unsubstituted WTA, which lacks substituents at the glycerol residues, by sequentially removing glycerolphosphates from the free end of the polymer up to the peptidoglycan linker. In contrast, GlpQ could not degrade unsubstituted LTA unless it was partially precleaved, allowing access of GlpQ to the other end of the polymer, which, in the intact molecule, is protected by a connection to the lipid anchor. Differences in stereochemistry between WTA and LTA have been suggested previously on the basis of differences in their biosynthetic precursors and chemical degradation products. The differential cleavage of WTA and LTA by GlpQ reported here represents the first direct evidence that they are enantiomeric polymers: WTA is made of sn-glycerol-3-phosphate, and LTA is made of sn-glycerol-1-phosphate. Their distinct stereochemistries reflect the dissimilar physiological and immunogenic properties of WTA and LTA. It also enables differential degradation of the two polymers within the same envelope compartment in vivo, particularly under phosphate-limiting conditions, when B. subtilis specifically degrades WTA and replaces it with phosphate-free teichuronic acids.

Exploration of key residues and conformational change of anti-terminator protein GlpP for ligand and RNA binding.

Anti-terminator protein GlpP regulates gene expression of glycerol uptake operon at post-transcriptional level in a number of bacteria. By now, the molecular dynamics details of ligand and RNA binding by GlpP are still obscure. In this study, we employed the molecular dynamic (MD) simulation and constructed a functional verification platform of GlpP to resolve these puzzles. By combining molecular docking, MD simulation and alanine scanning mutagenesis, a ligand binding pocket consisting of R14, R104 and R157 was identified. Among these residues with positive charge, R14 was dominant for binding glycerol-3-phosphate (G3P). Moreover, the "parallel to crossed" conformational change of the predicted RNA binding region was observed in MD simulation. In this process, the interaction between R104 and E129 was crucial to trigger the conformational change. To further verify this speculation, three ligand independent mutants were obtained by error-prone PCR. The MD simulation indicated that the conformational change happened in all the three mutants, confirming the "parallel to crossed" conformational change endowed GlpP the activity of binding RNA. In recent years, as a potable biological part, anti-terminator was more and more widely used to regulate gene expression in metabolic engineering and synthetic biology. The work in this study deepened our understanding to the typical anti-terminator GlpP, contributing to the further engineering and application of this type of regulator.

Hidden complexity in membrane permeabilization behavior of antimicrobial polycations.

A promising alternative to classical antibiotics are antimicrobial peptides and their synthetic mimics (smAMPs) that supposedly act directly on membranes. For a more successful design of smAMPs, we need to know how the type of interaction with the membrane determines the type of membrane perturbation. How this, in turn, transfers into selectivity and microbial killing activity is largely unknown. Here, we characterize the action of two smAMPs: MM:CO (a copolymer of hydrophobic cyclooctyl subunits and charged ß-monomethyl-α-aminomethyl subunits) and the highly charged poly-NM (a homopolymer of α-aminomethyl subunits). By thorough characterization of vesicle leakage experiments, we elucidate complex membrane perturbation behavior in zwitterionic or negatively charged vesicles. Vesicle leakage data does not entirely agree with the growth inhibition of microbes. Our ensemble of advanced membrane permeabilization approaches clarifies these discrepancies. Long cumulative leakage kinetics show that the two smAMPs act either by transient leakage or by rare stochastic leakage events that occur at charge neutralization in the sample. We determine the strengths of individual leakage events induced by the smAMPs in membranes of various compositions. These strengths indicate changes in leakage mechanism over time and concentration range. Thus, our sophisticated analysis of vesicle leakage experiments reveals a fine-tuned flexibility in membrane permeabilization mechanisms. These details are indispensable in judging and designing membrane-active compounds.

Physicochemical and Antimicrobial Properties of Thermosensitive Chitosan Hydrogel Loaded with Fosfomycin.

Thermosensitive chitosan hydrogels-renewable, biocompatible materials-have many applications as injectable biomaterials for localized drug delivery in the treatment of a variety of diseases. To combat infections such as Staphylococcus aureus osteomyelitis, localized antibiotic delivery would allow for higher doses at the site of infection without the risks associated with traditional antibiotic regimens. Fosfomycin, a small antibiotic in its own class, was loaded into a chitosan hydrogel system with varied beta-glycerol phosphate (ß-GP) and fosfomycin (FOS) concentrations. The purpose of this study was to elucidate the interactions between FOS and chitosan hydrogel. The Kirby Bauer assay revealed an unexpected concentration-dependent inhibition of S. aureus, with reduced efficacy at the high FOS concentration but only at the low ß-GP concentration. No effect of FOS concentration was observed for the planktonic assay. Rheological testing revealed that increasing ß-GP concentration increased the storage modulus while decreasing gelation temperature. NMR showed that FOS was removed from the liquid portion of the hydrogel by reaction over 12 h. SEM and FTIR confirmed gels degraded and released organophosphates over 5 days. This work provides insight into the physicochemical interactions between fosfomycin and chitosan hydrogel systems and informs selection of biomaterial components for improving infection treatment.

Effect of dexamethasone as osteogenic supplementation in in vitro osteogenic differentiation of stem cells from human exfoliated deciduous teeth.

In in vitro culture systems, dexamethasone (DEX) has been applied with ascorbic acid (ASC) and ß-glycerophosphate (ßGLY) as culture media supplementation to induce osteogenic differentiation of mesenchymal stem cells. However, there are some inconsistencies regarding the role of DEX as osteogenic media supplementation. Therefore, this study verified the influence of DEX culture media supplementation on the osteogenic differentiation, especially the capacity to mineralize the extracellular matrix of stem cells from human exfoliated deciduous teeth (SHED). Five groups were established: G1-SHED + Dulbecco's Modified Eagles' Medium (DMEM) + fetal bovine serum (FBS); G2-SHED + DMEM + FBS + DEX; G3-SHED + DMEM + FBS + ASC + ßGLY; G4-SHED + DMEM + FBS + ASC + ßGLY + DEX; G5-MC3T3-E1 + α Minimal Essential Medium (MEM) + FBS + ASC + ßGLY. DNA content, alkaline phosphatase (ALP) activity, free calcium quantification in the extracellular medium, and extracellular matrix mineralization quantification through staining with von Kossa, alizarin red, and tetracycline were performed on days 7 and 21. Osteogenic media supplemented with ASC and ß-GLY demonstrated similar effects on SHED in the presence or absence of DEX for DNA content (day 21) and capacity to mineralize the extracellular matrix according to alizarin red and tetracycline quantifications (day 21). In addition, the presence of DEX in the osteogenic medium promoted less ALP activity (day 7) and extracellular matrix mineralization according to the von Kossa assay (day 21), and more free calcium quantification at extracellular medium (day 21). In summary, the presence of DEX in the osteogenic media supplementation did not interfere with SHED commitment into mineral matrix depositor cells. We suggest that DEX may be omitted from culture media supplementation for SHED osteogenic differentiation in vitro studies.

Influence of Glycerophosphate Salt Solubility on the Gelation Mechanism of Colloidal Chitosan Systems.

Recently, thermosensitive chitosan systems have attracted the interest of many researchers due to their growing application potential. Nevertheless, the mechanism of the sol-gel phase transition is still being discussed, and the glycerophosphate salt role is ambiguous. The aim of the work is to analyze the possibility of the exclusive use of a non-sodium glycerophosphate salt and to determine its impact on the gelation conditions determined by rheological and turbidimetric measurements as well as the stability of the systems by measuring changes in the Zeta potential value. It was found that ensuring the same proportions of glycerophosphate ions differing in cation to amino groups present in chitosan chains, leads to obtaining systems significantly different in viscoelastic properties and phase transition conditions. It was clearly shown that the systems with the calcium glycerophosphate, the insoluble form of which may constitute additional aggregation nuclei, undergo the gelation the fastest. The use of magnesium glycerophosphate salt delays the gelation due to the heat-induced dissolution of the salt. Thus, it was unequivocally demonstrated that the formulation of the gelation mechanism of thermosensitive chitosan systems based solely on the concentration of glycerophosphate without discussing its type is incorrect.

Biosynthetic Mechanisms and Biological Significance of Glycerol Phosphate-Containing Glycan in Mammals.

Bacteria contain glycerol phosphate (GroP)-containing glycans, which are important constituents of cell-surface glycopolymers such as the teichoic acids of Gram-positive bacterial cell walls. These glycopolymers comprising GroP play crucial roles in bacterial physiology and virulence. Recently, the first identification of a GroP-containing glycan in mammals was reported as a variant form of O-mannosyl glycan on α-dystroglycan (α-DG). However, the biological significance of such GroP modification remains largely unknown. In this review, we provide an overview of this new discovery of GroP-containing glycan in mammals and then outline the recent progress in elucidating the biosynthetic mechanisms of GroP-containing glycans on α-DG. In addition, we discuss the potential biological role of GroP modification along with the challenges and prospects for further research. The progress in this newly identified glycan modification will provide insights into the phylogenetic implications of glycan.

The C-type lectin receptor MGL senses N-acetylgalactosamine on the unique Staphylococcus aureus ST395 wall teichoic acid.

Staphylococcus aureus is a common skin commensal but is also associated with various skin and soft tissue pathologies. Upon invasion, S. aureus is detected by resident innate immune cells through pattern-recognition receptors (PRRs), although a comprehensive understanding of the specific molecular interactions is lacking. Recently, we demonstrated that the PRR langerin (CD207) on epidermal Langerhans cells senses the conserved ß-1,4-linked N-acetylglucosamine (GlcNAc) modification on S. aureus wall teichoic acid (WTA), thereby increasing skin inflammation. Interestingly, the S. aureus ST395 lineage as well as certain species of coagulase-negative staphylococci (CoNS) produce a structurally different WTA molecule, consisting of poly-glycerolphosphate with α-O-N-acetylgalactosamine (GalNAc) residues, which are attached by the glycosyltransferase TagN. Here, we demonstrate that S. aureus ST395 strains interact with the human Macrophage galactose-type lectin (MGL; CD301) receptor, which is expressed by dendritic cells and macrophages in the dermis. MGL bound S. aureus ST395 in a tagN- and GalNAc-dependent manner but did not interact with different tagN-positive CoNS species. However, heterologous expression of Staphylococcus lugdunensis tagN in S. aureus conferred phage infection and MGL binding, confirming the role of this CoNS enzyme as GalNAc-transferase. Functionally, the detection of GalNAc on S. aureus ST395 WTA by human monocyte-derived dendritic cells significantly enhanced cytokine production. Together, our findings highlight differential recognition of S. aureus glycoprofiles by specific human innate receptors, which may affect downstream adaptive immune responses and pathogen clearance.

Evaluation of the Immunomodulatory Effects of All-Trans Retinoic Acid Solid Lipid Nanoparticles and Human Mesenchymal Stem Cells in an A549 Epithelial Cell Line Model.

PURPOSE: To investigate two potential strategies aimed at targeting the inflammatory pathogenesis of COPD: a small molecule, all trans retinoic acid (atRA) and human mesenchymal stem cells (hMSCs). METHODS: atRA was formulated into solid lipid nanoparticles (SLNs) via the emulsification-ultrasonication method, and these SLNs were characterised physicochemically. Assessment of the immunomodulatory effects of atRA-SLNs on A549 cells in vitro was determined using ELISA. hMSCs were suspended in a previously developed methylcellulose, collagen and beta-glycerophosphate hydrogel prior to investigating their immunomodulatory effects in vitro. RESULTS: SLNs provided significant encapsulation of atRA and also sustained its release over 72 h. A549 cells were viable following the addition of atRA SLNs and showed a reduction in IL-6 and IL-8 levels. A549 cells also remained viable following addition of the hMSC/hydrogel formulation - however, this formulation resulted in increased levels of IL-6 and IL-8, indicating a potentially pro-inflammatory effect. CONCLUSION: Both atRA SLNs and hMSCs show potential for modulating the environment in inflammatory disease, though through different mechanisms and leading to different outcomes - despite both being explored as strategies for use in inflammatory disease. atRA shows promise by acting in a directly anti-inflammatory manner, whereas further research into the exact mechanisms and behaviours of hMSCs in inflammatory diseases is required.

Bioactive injectable triple acting thermosensitive hydrogel enriched with nano-hydroxyapatite for bone regeneration: in-vitro characterization, Saos-2 cell line cell viability and osteogenic markers evaluation.

Hydrogels forming in-situ have gained great attention in the area of bone tissue engineering recently, they were also showed to be a good and less invasive alternative to surgically applied ones. The primal focus of this study was to prepare chitosan-glycerol phosphate thermosensitive hydrogel formed in-situ and loaded with risedronate (bone resorption inhibitor) in an easy way with no requirement of complicated processes or large number of equipment. Then we investigated its effectiveness for bone regeneration. In-situ forming hydrogels were prepared using chitosan cross-linked with glycerol phosphate and loaded with risedronate and nano-hydroxyapatite as bone cement. The prepared hydrogels were characterized by analyzing their gelation time at 37 °C, % porosity, swelling index, in-vitro degradation, rheological properties, and in-vitro drug release. Results showed that the in-situ hydrogels prepared using 2.5% (w/v) chitosan cross-linked with 50% (w/v) glycerol phosphate in the ratio (9:1, v/v) reinforced with 20 mg/mL and nano-hydroxyapatite possessed the most sustained drug release profile. This optimized formulation was further evaluated using DSC and FTIR studies, in addition to their morphological properties using scanning electron microscopy. The effect on Saos-2 cell line viability was evaluated also using MTT assay on the optimized hydrogel formulation in addition to their action on cell proliferation using fluorescence microscope. Moreover, calcium deposition on the hydrogel and alkaline phosphatase activity were evaluated. Risedronate-nano-hydroxyapatite loaded hydrogels significantly enhanced the Saos-2 cell proliferation in addition to enhanced alkaline phosphatase activity and calcium deposition. Such results suggest that risedronate-nano-hydroxyapatite loaded hydrogels present great biocompatibility for bone regeneration. Proliferation of cells, as well as deposition of mineral on the hydrogel, was an evidence of the biocompatible nature of the hydrogel. This hydrogel formed in-situ present a good less invasive alternative for bone tissue engineering.

Hexamerization of Geranylgeranylglyceryl Phosphate Synthase Ensures Structural Integrity and Catalytic Activity at High Temperatures.

The cell membranes of all archaea contain ether lipids, and a number of archaea are hyperthermophilic. Consequently, the enzymes that catalyze the synthesis of membrane ether lipids had to adopt to these rough conditions. Interestingly, the enzyme that establishes the first ether bond in these lipids, the geranylgeranylglyceryl phosphate synthase (GGGPS), forms hexamers in many hyperthermophilic archaea, while also dimeric variants of this enzyme exist in other species. We used Methanothermobacter thermautotrophicus GGGPS (mtGGGPS) as a model to elucidate the benefit of hexamerization. We studied the oligomerization interfaces in detail by introducing disturbing mutations and subsequently compared the stability and activity of generated dimeric and monomeric variants with the wild-type enzyme. Differential scanning calorimetry revealed a biphasic denaturation of mtGGGPS. The temperature of the first transition varies and rises with increasing oligomerization state. This first phase of denaturation leads to catalytic inactivation, but CD spectroscopy indicated only minor changes on the secondary structure level. The residual part of the fold is extremely thermostable and denatures in a second phase at temperatures >120 °C. The analysis of another distant native GGGPS enzyme affirms these observations. Molecular dynamics simulations revealed three structural elements close to the substrate binding sites with elevated flexibility. We assume that hexamerization might stabilize these structures, and kinetic studies support this hypothesis for the binding pocket of the substrate glycerol 1-phosphate. Oligomerization might thus positively affect the thermostability-flexibility trade-off in GGGPS by allowing a higher intrinsic flexibility of the individual protomers.

Thermogelling Platform for Baicalin Delivery for Versatile Biomedical Applications.

Baicalin (BG) is a natural glycoside with several promising therapeutic and preventive applications. However, its pharmaceutical potential is compromised by its poor water solubility, complex oral absorption kinetics, and low bioavailability. In this work, BG was incorporated in a series of chitosan (Ch)/glycerophosphate (GP)-based thermosensitive hydrogel formulations to improve its water solubility and control its release profile. Molecular interactions between BG and GP were investigated using Fourier transform infrared spectroscopy (FT-IR), and the ability of GP to enhance the water solubility of BG was studied in different release media. Drug-loaded Ch/GP hydrogels were prepared and characterized for their gelation time, swelling ratio, and rheological properties in addition to surface and internal microstructure. Polyethylene glycol (PEG) 6000 and hydroxypropyl methyl cellulose (HPMC) were incorporated in the formulations at different ratios to study their effect on modulating the sol-gel behavior and the in vitro drug release. In vivo pharmacokinetic (PK) studies were carried out using a rabbit model to study the ability of drug-loaded Ch/GP thermosensitive hydrogels to control the absorption rate and improve the bioavailability of BG. Results showed that the solubility of BG was enhanced in the presence of GP, while the incorporation of PEG and/or HPMC had an impact on gelation time, rheological behavior, and rate of drug release in vitro. PK results obtained following buccal application of drug-loaded Ch/GP thermosensitive hydrogels to rabbits showed that the rate of BG absorption was controlled and the in vivo bioavailability was increased by 330% relative to BG aqueous oral suspension. The proposed Ch/GP thermosensitive hydrogel is an easily modifiable delivery platform that is not only capable of improving the solubility and bioavailability of BG following buccal administration but also can be suited for various local and injectable therapeutic applications.

A novel vehicle for local protein delivery to the inner ear: injectable and biodegradable thermosensitive hydrogel loaded with PLGA nanoparticles.

Delivery of biomacromolecular drugs into the inner ear is challenging, mainly because of their inherent instability as well as physiological and anatomical barriers. Therefore, protein-friendly, hydrogel-based delivery systems following local administration are being developed for inner ear therapy. Herein, biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) containing interferon α-2 b (IFN α-2 b) were loaded in chitosan/glycerophosphate (CS/GP)-based thermosensitive hydrogel for IFN delivery by intratympanic injection. The injectable hydrogel possessed a physiological pH and formed semi-solid gel at 37 °C, with good swelling and deswelling properties. The CS/GP hydrogel could slowly degrade as visualized by scanning electron microscopy (SEM). The presence of NPs in CS/GP gel largely influenced in vitro drug release. In the guinea pig cochlea, a 1.5- to 3-fold increase in the drug exposure time of NPs-CS/GP was found than those of the solution, NPs and IFN-loaded hydrogel. Most importantly, a prolonged residence time was attained without obvious histological changes in the inner ear. This biodegradable, injectable, and thermosensitive NPs-CS/GP system may allow longer delivery of protein drugs to the inner ear, thus may be a potential novel vehicle for inner ear therapy.

Chitosan/ß-glycerophosphate-based microparticles manufactured by laminar jet break-up technology.

This study is about the use of ß-glycerophosphate (ßGP) to modulate the production of chitosan microparticles through a technology of jet break-up. ßGP has been described as capable of producing chitosan gels without additional complexing agents via a thermal transition (inverse gelation). A preliminary assessment on the effect of temperature on the viscosity and gelation of chitosan/ßGP precursors demonstrated that the crosslinking process was too slow to afford microparticle production via jet break-up. Instead, ßGP was used as a solubilizer to provide stable chitosan solution at neutral pH, which allowed the preparation of microparticles through polyelectrolyte complexation (with triphosphate) under physiological conditions, as opposed to the more conventional method of chitosan solubilisation in acids. Here, the key parameters of the microencapsulation process have been optimized, aiming to produce spherical particle of well-defined size and circularity, as well as toroidal microparticles, with a physico-chemical evaluation of the products.

Significance of a Posttranslational Modification of the PilA Protein of Geobacter sulfurreducens for Surface Attachment, Biofilm Formation, and Growth on Insoluble Extracellular Electron Acceptors.

Geobacter sulfurreducens, an anaerobic metal-reducing bacterium, possesses type IV pili. These pili are intrinsic structural elements in biofilm formation and, together with a number of c-type cytochromes, are thought to serve as conductive nanowires enabling long-range electron transfer (ET) to metal oxides and graphite anodes. Here, we report that a posttranslational modification of a nonconserved amino acid residue within the PilA protein, the structural subunit of the type IV pili, is crucial for growth on insoluble extracellular electron acceptors. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of the secreted PilA protein revealed a posttranslational modification of tyrosine-32 with a moiety of a mass consistent with a glycerophosphate group. Mutating this tyrosine into a phenylalanine inhibited cell growth with Fe(III) oxides as the sole electron acceptor. In addition, this amino acid substitution severely diminished biofilm formation on graphite surfaces and impaired current output in microbial fuel cells. These results demonstrate that the capability to attach to insoluble electron acceptors plays a crucial role for the cells' ability to utilize them. The work suggests that glycerophosphate modification of Y32 is a key factor contributing to the surface charge of type IV pili, influencing the adhesion of Geobacter to specific surfaces.IMPORTANCE Type IV pili are bacterial appendages that function in cell adhesion, virulence, twitching motility, and long-range electron transfer (ET) from bacterial cells to insoluble extracellular electron acceptors. The mechanism and role of type IV pili for ET in Geobacter sulfurreducens is still a subject of research. In this study, we identified a posttranslational modification of the major G. sulfurreducens type IV pilin, suggested to be a glycerophosphate moiety. We show that a mutant in which the glycerophosphate-modified tyrosine-32 is replaced with a phenylalanine has reduced abilities for ET and biofilm formation compared with those of the wild type. The results show the importance of the glycerophosphate-modified tyrosine for surface attachment and electron transfer in electrode- or Fe(III)-respiring G. sulfurreducens cells.

Stochastic thermodynamics of a chemical nanomachine: The channeling enzyme tryptophan synthase.

The enzyme tryptophan synthase is characterized by a complex pattern of allosteric interactions that regulate the catalytic activity of its two subunits and opening or closing of their ligand gates. As a single macromolecule, it implements 13 different reaction steps, with an intermediate product directly channeled from one subunit to another. Based on experimental data, a stochastic model for the operation of tryptophan synthase has been earlier constructed [D. Loutchko, D. Gonze, and A. S. Mikhailov, J. Phys. Chem. B 120, 2179 (2016)]. Here, this model is used to consider stochastic thermodynamics of such a chemical nanomachine. The Gibbs energy landscape of the internal molecular states is determined, the production of entropy and its flow within the enzyme are analyzed, and the information exchange between the subunits resulting from allosteric cross-regulations and channeling is discussed.

Fluoride and calcium concentrations in the biofilm fluid after use of fluoridated dentifrices supplemented with polyphosphate salts.

OBJECTIVES: The present study evaluated fluoride (F) and calcium (Ca) concentrations in the biofilm fluid formed in situ under cariogenic challenge after using F dentifrices supplemented or not with sodium trimetaphosphate (TMP) or calcium glycerophosphate (CaGP). METHODS: Volunteers (n = 12) were randomly divided into 5 groups according to the toothpastes used: placebo (without F, CaGP or TMP), 1100 ppm F (1100F) and low-fluoride dentifrice (LFD, 550 ppm F) with no supplementation (550F) or supplemented with 1 % TMP (550F-TMP) or 0.25 % CaGP (550F-CaGP). In each phase, volunteers wore palatal appliances containing 4 bovine enamel blocks. Cariogenic challenge was performed with 30 % sucrose solution, 6 times/day. On the morning of the eigth day, biofilm samples were collected 12 h and 1 h after brushing and cariogenic challenge. F and Ca analyses in the biofilm fluid were performed with the inverted electrode after buffering with TISAB III and using the Arsenazo III method, respectively. Data were submitted to two-way ANOVA (repeated measures) and Student-Newman-Keuls test (p < 0.05). RESULTS: A dose-response relationship was verified between F concentrations in the dentifrices and in the biofilm fluid. Significant differences were observed among placebo, 550F, and 1100F only 1 h after brushing, without statistical differences among 550F, 550F-TMP, and 550F-CaGP. No defined trend was observed among the groups regarding Ca concentrations, with the highest values seen for placebo and 550F-CaGP. CONCLUSION: The anticaries effect of LFDs supplemented with CaGP or TMP cannot be related to an increased availability of F and Ca in the biofilm fluid. CLINICAL SIGNIFICANCE: The better performance of LFDs containing CaGP or TMP shown in previous studies should be attributed to their ability to interact with tooth enamel and with the biofilm, rather to their effect on the biofilm fluid.

Design and optimization of thermosensitive nanoemulsion hydrogel for sustained-release of praziquantel.

OBJECTIVE: This work aimed to develop an alternative sustained-release thermosensitive praziquantel-loaded nanoemulsion (PZQ-NE) hydrogel for better schistosomiasis treatment. SIGNIFICANCE: PZQ-NE-dispersed chitosan/glycerol 2-phosphate disodium/HPMC (NE/CS/ß-GP/HMPC) hydrogel was successfully prepared to improve bioavailability of PZQ. METHODS: Solubility tests and pseudo-ternary phase diagrams were applied to screen optimal oils, surfactants and co-surfactants of NE. The hydrogels were characterized for gelling time, surface exudates, rheological properties and in vitro drug release. Formulation optimization of NE/CS/ß-GP/HMPC hydrogel was conducted by Box-Behnken experimental design combined with response surface methodology. In vitro cytotoxicity of hydrogel was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide method. The sustained-release property of PZQ in NE and optimized hydrogel was evaluated by pharmacokinetic study in rabbits. RESULTS: The formulation of PZQ-NE consisted of mass ratio of 12.5% capryol 90 containing PZQ (160 mg/g), 40% cremophor RH 40/tween 20 and transcutol HP (S/CoS = 2:1), 47.5% deionized water. PZQ releasing from NE/CS/ß-GP/HMPC hydrogels was best fitted to Higuchi model and governed by diffusion. Rheological investigation evidenced the themosensitive gelation of different hydrogel systems and their gel-like character at 37 °C. The optimized hydrogel formulation consisted of HPMC solution (103.69 mg/g), 3.03% (w/v) chitosan and 14.1% (w/v) ß-GP showed no cytotoxicity when the addition of NE was no more than 100 mg/g. Pharmacokinetic parameters indicated that NE/CS/ß-GP/HMPC hydrogel can significantly slow down drug elimination, prolong mean residence time and improve bioavailability of PZQ. CONCLUSIONS: NE/CS/ß-GP/HMPC hydrogel possessed sustained-release property and could be an alternative antischistosomal drug delivery system with improved therapeutic effect.

Tryptophan Synthase Uses an Atypical Mechanism To Achieve Substrate Specificity.

Tryptophan synthase (TrpS) catalyzes the final steps in the biosynthesis of l-tryptophan from l-serine (Ser) and indole-3-glycerol phosphate (IGP). We report that native TrpS can also catalyze a productive reaction with l-threonine (Thr), leading to (2S,3S)-ß-methyltryptophan. Surprisingly, ß-substitution occurs in vitro with a 3.4-fold higher catalytic efficiency for Ser over Thr using saturating indole, despite a >82000-fold preference for Ser in direct competition using IGP. Structural data identify a novel product binding site, and kinetic experiments clarify the atypical mechanism of specificity: Thr binds efficiently but decreases the affinity for indole and disrupts the allosteric signaling that regulates the catalytic cycle.

Age-related changes in retinoic, docosahexaenoic and arachidonic acid modulation in nuclear lipid metabolism.

The aim of this work was to study how age-related changes could modify several enzymatic activities that regulate lipid mediator levels in nuclei from rat cerebellum and how these changes are modulated by all-trans retinoic acid (RA), docosahexaenoic acid (DHA) and arachidonic acid (AA). The higher phosphatidate phosphohydrolase activity and lower diacylglycerol lipase (DAGL) activity observed in aged animals compared with adults could augment diacylglycerol (DAG) availability in the former. Additionally, monoacylglycerol (MAG) availability could be high due to an increase in lysophosphatidate phosphohydrolase (LPAPase) activity and a decrease in monocylglycerol lipase activity. Interestingly, RA, DHA and AA were observed to modulate these enzymatic activities and this modulation was found to change in aged rats. In adult nuclei, whereas RA led to high DAG and MAG production through inhibition of their hydrolytic enzymes, DHA and AA promoted high MAG production by LPAPase and DAGL stimulation. In contrast, in aged nuclei RA caused high MAG generation whereas DHA and AA diminished it through LPAPase activity modulation. These results demonstrate that aging promotes a different nuclear lipid metabolism as well as a different type of non-genomic regulation by RA, DHA and AA, which could be involved in nuclear signaling events.